Felicia D Goodrum
- Interim Associate Department Head, Immunobiology
- Professor, Immunobiology
- Professor, BIO5 Institute
- Professor, Cellular and Molecular Medicine
- Professor, Cancer Biology - GIDP
- Professor, Genetics - GIDP
- Professor, Molecular and Cellular Biology
- Member of the Graduate Faculty
- (520) 626-7468
- TW Keating Bioresearch Bldg., Rm. 425
- Tucson, AZ 85721
- fgoodrum@arizona.edu
Biography
Felicia Goodrum graduated with her Ph.D. from Wake Forest University and then trained as a postdoctoral fellow with Thomas Shenk at Princeton University where she was a Leukemia and Lymphoma Society Fellow and Special Fellow. Dr. Goodrum joined the faculty at the University of Arizona in the Department of Immunobiology and the BIO5 Institute in 2006 and is an Assoicate Professor. Dr. Goodrum is the recipient of the Pew Scholar in Biomedical Sciences Award and the Presidential Award for Early Career Scientists and Engineers. Dr. Goodrum's work throughout her career has focused on complex interaction between viruses and their host and is currently focused on the mechanisms of viral persistence of human cytomegalovirus.
Degrees
- Ph.D. Molecular Genetics
- Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States
- Function of the early region 1B-55kD protein of adenovirus in promoting cell cycle-independent virus replication and mediating mRNA transport
- B.S. Biology
- Virginia Tech, Blacksburg, Virginia, United States
Work Experience
- University of Arizona, College of Medicine, Department of Immunobiology (2013 - Ongoing)
- University of Arizona, College of Medicine, Department of Immunobiology (2006 - 2013)
- Princeton University, Department of Molecular Biology (1999 - 2006)
- Wake Forest University, Department of Microbiology and Immunology (1998 - 1999)
Awards
- Woman of Impact
- University of Arizona, Fall 2023
- ELAM Executive Leadership in Academic Medicine
- ELAM, Summer 2023
- Edith Sayer Auslander Emerging Visionary
- University of Arizona’s Commission on the Status of Women, Fall 2022
- President-elect, President
- American Society for Virology, Summer 2021
- American Academy of Microbiology
- Summer 2019
- Jamie McNew Endowed Lecturer
- University of Minnesota, Spring 2014
- Distinguished Speaker
- Wake Forest University, Spring 2013
- Robert Honess Lecturer
- American Society of Virology, Spring 2012
- Top 25 Reviewers
- Journal of Virology, Spring 2012
- Priscilla A. Schaffer Lecturship
- Priscilla A. Schaffer Fund, Spring 2010
- Kavli Fellow
- National Academy of Sciences, Spring 2009
- Top 20 Reviewers
- Journal of Virology, Spring 2009
- Junior Faculty International Conference Travel Award
- University of Arizona, Spring 2008
- Pew Scholar in Biomedical Sciences
- Pew Charitable Trust, Spring 2008
- Young Investigator Award
- Human Frontiers in Science Program, Spring 2008
- Howard Temin Career Development Award
- National Cancer Institute, Spring 2005
- Leukemia and Lymphoma Society, Special Fellow
- Leukemia and Lymphoma Society, Spring 2003
- Leukemia and Lymphoma Society, Fellow
- Leukemia and Lymphoma Society, Spring 2000
Interests
Teaching
I am interested in helping students written and oral communication skills to enhance the competitive advantage of my students.
Research
Our broad research interests are to understand the molecular underpinnings of viral persistence using herpesviruses as a model system.
Courses
2024-25 Courses
-
Dissertation
IMB 920 (Fall 2024)
2023-24 Courses
-
Dissertation
IMB 920 (Spring 2024) -
Research
IMB 900 (Spring 2024) -
Directed Research
ABBS 792 (Fall 2023) -
Dissertation
IMB 920 (Fall 2023) -
Research
IMB 900 (Fall 2023)
2022-23 Courses
-
Directed Research
MCB 792 (Spring 2023) -
Dissertation
IMB 920 (Spring 2023) -
Honors Independent Study
BIOC 399H (Spring 2023) -
Honors Thesis
BIOC 498H (Spring 2023) -
Research
IMB 900 (Spring 2023) -
Directed Research
MCB 792 (Fall 2022) -
Directed Rsrch
MCB 392 (Fall 2022) -
Dissertation
IMB 920 (Fall 2022) -
Honors Directed Research
BIOC 492H (Fall 2022) -
Honors Thesis
BIOC 498H (Fall 2022) -
Research
IMB 900 (Fall 2022)
2021-22 Courses
-
Directed Research
BIOC 392 (Spring 2022) -
Dissertation
CBIO 920 (Spring 2022) -
Dissertation
IMB 920 (Spring 2022) -
Honors Directed Research
BIOC 392H (Spring 2022) -
Research
IMB 900 (Spring 2022) -
Directed Research
MCB 792 (Fall 2021) -
Dissertation
CBIO 920 (Fall 2021) -
Dissertation
IMB 920 (Fall 2021) -
Honors Independent Study
BIOC 299H (Fall 2021)
2020-21 Courses
-
Directed Research
ACBS 492 (Summer I 2021) -
Directed Research
MCB 792 (Spring 2021) -
Dissertation
CBIO 920 (Spring 2021) -
Dissertation
IMB 920 (Spring 2021) -
Research
IMB 900 (Spring 2021) -
Research Conference
CBIO 695A (Spring 2021) -
Thesis
IMB 910 (Spring 2021) -
Directed Research
MCB 792 (Fall 2020) -
Dissertation
CBIO 920 (Fall 2020) -
Dissertation
IMB 920 (Fall 2020) -
Research
IMB 900 (Fall 2020) -
Research Conference
CBIO 695A (Fall 2020) -
Thesis
IMB 910 (Fall 2020)
2019-20 Courses
-
Research
CBIO 900 (Spring 2020) -
Research
IMB 900 (Spring 2020) -
Research Conference
CBIO 695A (Spring 2020) -
Introduction to Research
MCB 795A (Fall 2019) -
Research
CBIO 900 (Fall 2019) -
Research
IMB 900 (Fall 2019) -
Research Conference
CBIO 695A (Fall 2019)
2018-19 Courses
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Honors Thesis
MIC 498H (Spring 2019) -
IMB- Journal Club
IMB 595A (Spring 2019) -
Research
CBIO 900 (Spring 2019) -
Research
IMB 900 (Spring 2019) -
Research Conference
CBIO 695A (Spring 2019) -
Scientific Grantsmanship
IMB 521 (Spring 2019) -
Honors Thesis
MIC 498H (Fall 2018) -
Introduction to Research
MCB 795A (Fall 2018) -
Research
CBIO 900 (Fall 2018) -
Research
IMB 900 (Fall 2018) -
Research Conference
CBIO 695A (Fall 2018)
2017-18 Courses
-
Dissertation
CMM 920 (Spring 2018) -
Scientific Grantsmanship
IMB 521 (Spring 2018) -
Dissertation
CMM 920 (Fall 2017) -
Introduction to Research
MCB 795A (Fall 2017) -
Research
CMM 900 (Fall 2017)
2016-17 Courses
-
Dissertation
CMM 920 (Spring 2017) -
Introduction to Research
MCB 795A (Spring 2017) -
Dissertation
CMM 920 (Fall 2016) -
Introduction to Research
MCB 795A (Fall 2016) -
Research
CMM 900 (Fall 2016) -
Scientific Grantsmanship
IMB 521 (Fall 2016)
2015-16 Courses
-
Dissertation
CMM 920 (Spring 2016)
Scholarly Contributions
Chapters
- Goodrum Sterling, F. D. (2022). The complex biology of human cytomegalovirus latency and reactivation.. In Advances in Virus Research.
- Goodrum, F., Britt, W., & Mocarski, E. (2022). Cytomegaloviruses. In Field's Virology(pp Cytomegaloviruses). Wolters Kluwer.
- Goodrum Sterling, F. D., Britt, W., & Mocarski, E. (2022). Cytomegalovirus. In Fields Virology: DNA Viruses(pp 389-444). Philadelphia: Wolters Kluwer.More infoPreeminent virology text book.
Journals/Publications
- Rasmussen, A. L., Gronvall, G. K., Lowen, A. C., Goodrum, F., Alwine, J., Andersen, K. G., Anthony, S. J., Baines, J., Banerjee, A., Broadbent, A. J., Brooke, C. B., Campos, S. K., Caposio, P., Casadevall, A., Chan, G. C., Cliffe, A. R., Collins-McMillen, D., Connell, N., Damania, B., , Daugherty, M. D., et al. (2024). Virology-the path forward. Journal of virology, e0179123.More infoIn the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.
- Alwine, J. C., Casadevall, A., Enquist, L. W., Goodrum, F. D., & Imperiale, M. J. (2023). A Critical Analysis of the Evidence for the SARS-CoV-2 Origin Hypotheses. Journal of virology, 97(4), e0036523.More infoWhen humans experience a new, devastating viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), significant challenges arise. How should individuals as well as societies respond to the situation? One of the primary questions concerns the origin of the SARS-CoV-2 virus that infected and was transmitted efficiently among humans, resulting in a pandemic. At first glance, the question appears straightforward to answer. However, the origin of SARS-CoV-2 has been the topic of substantial debate primarily because we do not have access to some relevant data. At least two major hypotheses have been suggested: a natural origin through zoonosis followed by sustained human-to-human spread or the introduction of a natural virus into humans from a laboratory source. Here, we summarize the scientific evidence that informs this debate to provide our fellow scientists and the public with the tools to join the discussion in a constructive and informed manner. Our goal is to dissect the evidence to make it more accessible to those interested in this important problem. The engagement of a broad representation of scientists is critical to ensure that the public and policy-makers can draw on relevant expertise in navigating this controversy.
- Alwine, J. C., Casadevall, A., Enquist, L. W., Goodrum, F. D., & Imperiale, M. J. (2023). A Critical Analysis of the Evidence for the SARS-CoV-2 Origin Hypotheses. mBio, 14(2), e0058323.More infoWhen humans experience a new, devastating viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), significant challenges arise. How should individuals as well as societies respond to the situation? One of the primary questions concerns the origin of the SARS-CoV-2 virus that infected and was transmitted efficiently among humans, resulting in a pandemic. At first glance, the question appears straightforward to answer. However, the origin of SARS-CoV-2 has been the topic of substantial debate primarily because we do not have access to some relevant data. At least two major hypotheses have been suggested: a natural origin through zoonosis followed by sustained human-to-human spread or the introduction of a natural virus into humans from a laboratory source. Here, we summarize the scientific evidence that informs this debate to provide our fellow scientists and the public with the tools to join the discussion in a constructive and informed manner. Our goal is to dissect the evidence to make it more accessible to those interested in this important problem. The engagement of a broad representation of scientists is critical to ensure that the public and policy-makers can draw on relevant expertise in navigating this controversy.
- Alwine, J. C., Casadevall, A., Enquist, L. W., Goodrum, F. D., & Imperiale, M. J. (2023). A Critical Analysis of the Evidence for the SARS-CoV-2 Origin Hypotheses. mSphere, 8(2), e0011923.More infoWhen humans experience a new, devastating viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), significant challenges arise. How should individuals as well as societies respond to the situation? One of the primary questions concerns the origin of the SARS-CoV-2 virus that infected and was transmitted efficiently among humans, resulting in a pandemic. At first glance, the question appears straightforward to answer. However, the origin of SARS-CoV-2 has been the topic of substantial debate primarily because we do not have access to some relevant data. At least two major hypotheses have been suggested: a natural origin through zoonosis followed by sustained human-to-human spread or the introduction of a natural virus into humans from a laboratory source. Here, we summarize the scientific evidence that informs this debate to provide our fellow scientists and the public with the tools to join the discussion in a constructive and informed manner. Our goal is to dissect the evidence to make it more accessible to those interested in this important problem. The engagement of a broad representation of scientists is critical to ensure that the public and policy-makers can draw on relevant expertise in navigating this controversy.
- Domma, A. J., Henderson, L. A., Goodrum, F. D., Moorman, N. J., & Kamil, J. P. (2023). Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins. Journal of virology, 97(10), e0056323.More infoHuman cytomegalovirus (HCMV) requires inactivation of AKT to efficiently replicate, yet how AKT is shut off during HCMV infection has remained unclear. We show that UL38, an HCMV protein that activates mTORC1, is necessary and sufficient to destabilize insulin receptor substrate 1 (IRS1), a model insulin receptor substrate (IRS) protein. Degradation of IRS proteins in settings of excessive mTORC1 activity is an important mechanism for insulin resistance. When IRS proteins are destabilized, PI3K cannot be recruited to growth factor receptor complexes, and hence, AKT membrane recruitment, a rate limiting step in its activation, fails to occur. Despite its penchant for remodeling host cell signaling pathways, our results reveal that HCMV relies upon a cell-intrinsic negative regulatory feedback loop to inactivate AKT. Given that pharmacological inhibition of PI3K/AKT potently induces HCMV reactivation from latency, our findings also imply that the expression of UL38 activity must be tightly regulated within latently infected cells to avoid spontaneous reactivation.
- Goodrum, F., & Schultz-Cherry, S. (2023). -the next generation. Journal of virology, 97(10), e0133723.
- Goodrum, F., Lowen, A. C., Lakdawala, S., Alwine, J., Casadevall, A., Imperiale, M. J., Atwood, W., Avgousti, D., Baines, J., Banfield, B., Banks, L., Bhaduri-McIntosh, S., Bhattacharya, D., Blanco-Melo, D., Bloom, D., Boon, A., Boulant, S., Brandt, C., Broadbent, A., , Brooke, C., et al. (2023). Virology under the Microscope-a Call for Rational Discourse. Journal of virology, 97(2), e0008923.More infoViruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.
- Goodrum, F., Lowen, A. C., Lakdawala, S., Alwine, J., Casadevall, A., Imperiale, M. J., Atwood, W., Avgousti, D., Baines, J., Banfield, B., Banks, L., Bhaduri-McIntosh, S., Bhattacharya, D., Blanco-Melo, D., Bloom, D., Boon, A., Boulant, S., Brandt, C., Broadbent, A., , Brooke, C., et al. (2023). Virology under the Microscope-a Call for Rational Discourse. mBio, 14(1), e0018823.More infoViruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.
- Goodrum, F., Lowen, A. C., Lakdawala, S., Alwine, J., Casadevall, A., Imperiale, M. J., Atwood, W., Avgousti, D., Baines, J., Banfield, B., Banks, L., Bhaduri-McIntosh, S., Bhattacharya, D., Blanco-Melo, D., Bloom, D., Boon, A., Boulant, S., Brandt, C., Broadbent, A., , Brooke, C., et al. (2023). Virology under the Microscope-a Call for Rational Discourse. mSphere, 8(2), e0003423.More infoViruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.
- Lowen, A. C., Casadevall, A., Alwine, J. C., Enquist, L. W., Goodrum, F. D., Imperiale, M. J., & Lakdawala, S. S. (2023). Oversight of Pathogen Research Must Be Carefully Calibrated and Clearly Defined. Journal of virology, 97(3), e0017623.
- Lowen, A. C., Casadevall, A., Alwine, J. C., Enquist, L. W., Goodrum, F. D., Imperiale, M. J., & Lakdawala, S. S. (2023). Oversight of Pathogen Research Must Be Carefully Calibrated and Clearly Defined. mBio, 14(2), e0032323.
- Lowen, A. C., Casadevall, A., Alwine, J. C., Enquist, L. W., Goodrum, F. D., Imperiale, M. J., & Lakdawala, S. S. (2023). Oversight of Pathogen Research Must Be Carefully Calibrated and Clearly Defined. mSphere, 8(2), e0006623.
- Mlera, L., Collins-McMillen, D., Zeltzer, S., Buehler, J. C., Moy, M., Zarrella, K., Caviness, K., Cicchini, L., Tafoya, D. J., & Goodrum, F. (2023). Liver X Receptor-Inducible Host E3 Ligase IDOL Targets a Human Cytomegalovirus Reactivation Determinant. Journal of virology, 97(7), e0075823.More infoLiver X receptor (LXR) signaling broadly restricts virus replication; however, the mechanisms of restriction are poorly defined. Here, we demonstrate that the cellular E3 ligase LXR-inducible degrader of low-density lipoprotein receptor (IDOL) targets the human cytomegalovirus (HMCV) UL136p33 protein for turnover. encodes multiple proteins that differentially impact latency and reactivation. UL136p33 is a determinant of reactivation. UL136p33 is targeted for rapid turnover by the proteasome, and its stabilization by mutation of lysine residues to arginine results in a failure to quiet replication for latency. We show that IDOL targets UL136p33 for turnover but not the stabilized variant. IDOL is highly expressed in undifferentiated hematopoietic cells where HCMV establishes latency but is sharply downregulated upon differentiation, a stimulus for reactivation. We hypothesize that IDOL maintains low levels of UL136p33 for the establishment of latency. Consistent with this hypothesis, knockdown of IDOL impacts viral gene expression in wild-type (WT) HCMV infection but not in infection where UL136p33 has been stabilized. Furthermore, the induction of LXR signaling restricts WT HCMV reactivation from latency but does not affect the replication of a recombinant virus expressing a stabilized variant of UL136p33. This work establishes the UL136p33-IDOL interaction as a key regulator of the bistable switch between latency and reactivation. It further suggests a model whereby a key viral determinant of HCMV reactivation is regulated by a host E3 ligase and acts as a sensor at the tipping point between the decision to maintain the latent state or exit latency for reactivation. Herpesviruses establish lifelong latent infections, which pose an important risk for disease particularly in the immunocompromised. Our work is focused on the betaherpesvirus human cytomegalovirus (HCMV) that latently infects the majority of the population worldwide. Defining the mechanisms by which HCMV establishes latency or reactivates from latency is important for controlling viral disease. Here, we demonstrate that the cellular inducible degrader of low-density lipoprotein receptor (IDOL) targets a HCMV determinant of reactivation for degradation. The instability of this determinant is important for the establishment of latency. This work defines a pivotal virus-host interaction that allows HCMV to sense changes in host biology to navigate decisions to establish latency or to replicate.
- Moy, M. A., Collins-McMillen, D., Crawford, L., Parkins, C., Zeltzer, S., Caviness, K., Zaidi, S. S., Caposio, P., & Goodrum, F. (2023). Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells. Journal of virology, 97(8), e0014823.More infoHuman cytomegalovirus (HCMV) is a beta herpesvirus that persists indefinitely in the human host through a latent infection. The polycistronic gene locus of HCMV encodes genes regulating latency and reactivation. While is pro-latency, restricting virus replication in CD34 hematopoietic progenitor cells (HPCs), overcomes this restriction and is required for reactivation. By contrast, is expressed with later kinetics and encodes multiple proteins with differential roles in latency and reactivation. Like , the largest isoform, UL136p33, is required for reactivation from latency in HPCs; viruses failing to express either protein are unresponsive to reactivation stimuli. Furthermore, UL136p33 is unstable, and its instability is important for the establishment of latency, and sufficient accumulation of UL136p33 is a checkpoint for reactivation. We hypothesized that stabilizing UL136p33 might overcome the requirement of for replication. We generated recombinant viruses lacking that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact the replication of the mutant virus in fibroblasts. However, in the context of infection in HPCs, stabilization of UL136p33 strikingly compensated for the loss of resulting in increased replication in CD34 HPCs and in humanized NOD- IL2Rγ (huNSG) mice. This finding suggests that while is essential for replication in HPCs, it functions largely at steps preceding the accumulation of UL136p33, and that stabilized expression of UL136p33 largely overcomes the requirement for . Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and , whereby may initiate events early in reactivation that drive the accumulation of UL136p33 to a threshold required for productive reactivation. IMPORTANCE Human cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a lifelong latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immunocompromised. Defining viral genes important in the establishment of or reactivation from latency is important to defining the molecular basis of latent and replicative states and in controlling infection and CMV disease. Here we define a genetic relationship between two viral genes in controlling virus reactivation from latency using primary human hematopoietic progenitor cells and humanized mouse models.
- Runstadler, J. A., Lowen, A. C., Kayali, G., Tompkins, S. M., Albrecht, R. A., Fouchier, R. A., Stallknecht, D. E., Lakdawala, S. S., Goodrum, F. D., Casadevall, A., Enquist, L. W., Alwine, J. C., Imperiale, M. J., Schultz-Cherry, S., & Webby, R. J. (2023). Field Research Is Essential to Counter Virological Threats. Journal of virology, 97(5), e0054423.More infoThe interface between humans and wildlife is changing and, with it, the potential for pathogen introduction into humans has increased. Avian influenza is a prominent example, with an ongoing outbreak showing the unprecedented expansion of both geographic and host ranges. Research in the field is essential to understand this and other zoonotic threats. Only by monitoring dynamic viral populations and defining their biology can we gather the information needed to ensure effective pandemic preparation.
- White, T. M., & Goodrum, F. D. (2023). Viruses trick bystander cells into lowering their defences. Nature, 619(7968), 39-40.
- Zarrella, K., Longmire, P., Zeltzer, S., Collins-McMillen, D., Hancock, M., Buehler, J., Reitsma, J. M., Terhune, S. S., Nelson, J. A., & Goodrum, F. (2023). Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection. PLoS pathogens, 19(6), e1011185.More infoInnate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquitinating complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated in infection and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection.
- Zeltzer, S., & Goodrum, F. D. (2022). Host translesion polymerases are required for viral genome integrity. Proc Natl Acad Sci USA, 119(33), e2203203119.
- Zhang, H., & Goodrum Sterling, F. D. (2022). The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication. mBio, e0104222.
- Alwine, J. C., Enquist, L. W., Dermody, T. S., & Goodrum, F. (2021). What Is the Price of Science?. mBio, 12(2).More infoThe peer-reviewed scientific literature is the bedrock of science. However, scientific publishing is undergoing dramatic changes, which include the expansion of open access, an increased number of for-profit publication houses, and ready availability of preprint manuscripts that have not been peer reviewed. In this opinion article, we discuss the inequities and concerns that these changes have wrought.
- Crawford, L. B., Hancock, M. H., Struthers, H. M., Streblow, D. N., Yurochko, A. D., Caposio, P., Goodrum, F. D., & Nelson, J. A. (2021). CD34 Hematopoietic Progenitor Cell Subsets Exhibit Differential Ability To Maintain Human Cytomegalovirus Latency and Persistence. Journal of virology, 95(3).More infoIn human cytomegalovirus (HCMV)-seropositive patients, CD34 hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34 HPCs to investigate mechanisms of viral latency. However, analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34 HPCs from different donors, availability of cells, and low frequency of reactivation. In addition, multiple progenitor cell types express surface CD34, and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions In this study, we generated CD34 progenitor cells from two different embryonic stem cell (ESC) lines, WA01 and WA09, to circumvent limitations associated with primary CD34 HPCs. HCMV infection of CD34 HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34 HPCs. Analysis of HCMV-infected primary or ESC-derived CD34 HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38 CD90 and CD38 CD90 HPCs but persistently infected CD38 CD90 cells to produce infectious virus. These results indicate that ESC-derived CD34 HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34 cells. Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34 hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however, these cells are a heterogeneous pool with donor-to-donor variation in functional, genetic, and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states, which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation.
- Lee, B. J., Min, C. K., Hancock, M., Streblow, D. N., Caposio, P., Goodrum, F. D., & Yurochko, A. D. (2021). Human Cytomegalovirus Host Interactions: EGFR and Host Cell Signaling Is a Point of Convergence Between Viral Infection and Functional Changes in Infected Cells. Frontiers in microbiology, 12, 660901.More infoViruses have evolved diverse strategies to manipulate cellular signaling pathways in order to promote infection and/or persistence. Human cytomegalovirus (HCMV) possesses a number of unique properties that allow the virus to alter cellular events required for infection of a diverse array of host cell types and long-term persistence. Of specific importance is infection of bone marrow derived and myeloid lineage cells, such as peripheral blood monocytes and CD34 hematopoietic progenitor cells (HPCs) because of their essential role in dissemination of the virus and for the establishment of latency. Viral induced signaling through the Epidermal Growth Factor Receptor (EGFR) and other receptors such as integrins are key control points for viral-induced cellular changes and productive and latent infection in host organ systems. This review will explore the current understanding of HCMV strategies utilized to hijack cellular signaling pathways, such as EGFR, to promote the wide-spread dissemination and the classic life-long herpesvirus persistence.
- Momtaz, S., Molina, B., Mlera, L., Goodrum, F., & Wilson, J. M. (2021). Cell type-specific biogenesis of novel vesicles containing viral products in human cytomegalovirus infection. Journal of virology.More infoHuman cytomegalovirus (HCMV), while highly restricted for the human species, infects an diverse array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies (MVBs) that incorporate viral products, including dense bodies (DBs) and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in primary human microvascular endothelial cells (HMVECs) were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that UL32-GFP containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. However, UL32-GFP containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non- canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV, which may reflect distinct pathways for virus egress.Human cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life-long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with age-related pathology. HCMV infects many cells in the human host and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact how progeny virus is trafficked out of infected cells.
- Peppenelli, M., Buehler, J., & Goodrum, F. (2021). Human Hematopoietic Long-Term Culture (hLTC) for Human Cytomegalovirus Latency and Reactivation. Methods in molecular biology (Clifton, N.J.), 2244, 83-101.More infoOf the many research challenges posed by the study of human cytomegalovirus (HCMV) latency, one of the most notable is the requirement for the use of primary hematopoietic cell culture. Culturing hematopoietic progenitor subpopulations requires that consideration be given to maintaining their physiological relevance. We describe a long-standing primary CD34+ hematopoietic progenitor cell (HPC) system as an in vitro model to study HCMV latent infection. Key aspects of the model include infection of primary human CD34+ HPCs prior to ex vivo expansion, a long-term culture with a stromal cell support designed to maintain the ability of stem cells to support hematopoietic reconstitution, and an assay to quantify infectious centers produced prior to and following a reactivation stimulus. Importantly, this system has been used to identify a number of viral determinants of latency or reactivation and findings have been recapitulated in vivo using a humanized mouse model for HCMV latency. Therefore, this system offers a powerful approach to defining virus-host interactions and mechanisms important for HCMV latency and reactivation.
- Yun, T. J., Igarashi, S., Zhao, H., Perez, O. A., Pereira, M. R., Zorn, E., Shen, Y., Goodrum, F., Rahman, A., Sims, P. A., Farber, D. L., & Reizis, B. (2021). Human plasmacytoid dendritic cells mount a distinct antiviral response to virus-infected cells. Science immunology, 6(58).More infoPlasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, human cytomegalovirus (CMV). Human pDCs produced high amounts of type I interferon (IFN-I) when incubated with live CMV-infected fibroblasts but not with free CMV; the response involved integrin-mediated adhesion, transfer of DNA-containing virions to pDCs, and the recognition of DNA through TLR9. Compared with transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of IFN-I and IFN-III, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged, and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells critical for CMV control. Last, patients with CMV viremia harbored phenotypically activated pDCs and increased circulating IFN-I and IFN-III. Thus, recognition of live infected cells is a mechanism of virus detection by pDCs that elicits a unique antiviral immune response.
- Chaturvedi, S., Klein, J., Vardi, N., Bolovan-Fritts, C., Wolf, M., Du, K., Mlera, L., Calvert, M., Moorman, N. J., Goodrum, F., Huang, B., & Weinberger, L. S. (2020). A molecular mechanism for probabilistic bet hedging and its role in viral latency. Proceedings of the National Academy of Sciences of the United States of America, 117(29), 17240-17248.More infoProbabilistic bet hedging, a strategy to maximize fitness in unpredictable environments by matching phenotypic variability to environmental variability, is theorized to account for the evolution of various fate-specification decisions, including viral latency. However, the molecular mechanisms underlying bet hedging remain unclear. Here, we report that large variability in protein abundance within individual herpesvirus virion particles enables probabilistic bet hedging between viral replication and latency. Superresolution imaging of individual virions of the human herpesvirus cytomegalovirus (CMV) showed that virion-to-virion levels of pp71 tegument protein-the major viral transactivator protein-exhibit extreme variability. This super-Poissonian tegument variability promoted alternate replicative strategies: high virion pp71 levels enhance viral replicative fitness but, strikingly, impede silencing, whereas low virion pp71 levels reduce fitness but promote silencing. Overall, the results indicate that stochastic tegument packaging provides a mechanism enabling probabilistic bet hedging between viral replication and latency.
- Collins-McMillen, D., Kamil, J., Moorman, N., & Goodrum, F. (2020). Control of Immediate Early Gene Expression for Human Cytomegalovirus Reactivation. Frontiers in cellular and infection microbiology, 10, 476.More infoHuman cytomegalovirus (HCMV) is a beta herpesvirus that persists for life in the majority of the world's population. The persistence of HCMV in the human population is due to the exquisite ability of herpesviruses to establish a latent infection that evades elimination by the host immune response. How the virus moves into and out of the latent state has been an intense area of research focus and debate. The prevailing paradigm is that the major immediate early promoter (MIEP), which drives robust expression of the major immediate early (MIE) transactivators, is epigenetically silenced during the establishment of latency, and must be reactivated for the virus to exit latency and re-enter productive replication. While it is clear that the MIEP is silenced by the association of repressive chromatin remodeling factors and histone marks, the mechanisms by which HCMV de-represses MIE gene expression for reactivation are less well understood. We have identified alternative promoter elements within the MIE locus that drive a second or delayed phase of MIE gene expression during productive infection. In the context of reactivation in THP-1 macrophages and primary CD34+ human progenitor cells, MIE transcripts are predominantly derived from initiation at these alternative promoters. Here we review the mechanisms by which alternative viral promoters might tailor the control of viral gene expression and the corresponding pattern of infection to specific cell types. Alternative promoter control of the HCMV MIE locus increases versatility in the system and allows the virus to tightly repress viral gene expression for latency but retain the ability to sense and respond to cell type-specific host cues for reactivation of replication.
- Crawford, L. B., Tempel, R., Streblow, D. N., Yurochko, A. D., Goodrum, F. D., Nelson, J. A., & Caposio, P. (2020). Human Cytomegalovirus Infection Suppresses CD34 Progenitor Cell Engraftment in Humanized Mice. Microorganisms, 8(4).More infoHuman cytomegalovirus (HCMV) infection is a serious complication in hematopoietic stem cell transplant (HSCT) recipients due to virus-induced myelosuppression and impairment of stem cell engraftment. Despite the clear clinical link between myelosuppression and HCMV infection, little is known about the mechanism(s) by which the virus inhibits normal hematopoiesis because of the strict species specificity and the lack of surrogate animal models. In this study, we developed a novel humanized mouse model system that recapitulates the HCMV-mediated engraftment failure after hematopoietic cell transplantation. We observed significant alterations in the hematopoietic populations in peripheral lymphoid tissues following engraftment of a subset of HCMV CD34 hematopoietic progenitor cells (HPCs) within the transplant, suggesting that a small proportion of HCMV-infected CD34 HPCs can profoundly affect HPC differentiation in the bone marrow microenvironment. This model will be instrumental to gain insight into the fundamental mechanisms of HCMV myelosuppression after HSCT and provides a platform to assess novel treatment strategies.
- Hale, A. E., Collins-McMillen, D., Lenarcic, E. M., Igarashi, S., Kamil, J. P., Goodrum, F., & Moorman, N. J. (2020). FOXO transcription factors activate alternative major immediate early promoters to induce human cytomegalovirus reactivation. Proceedings of the National Academy of Sciences of the United States of America, 117(31), 18764-18770.More infoHuman progenitor cells (HPCs) support human cytomegalovirus (HCMV) latency, and their differentiation along the myeloid lineage triggers cellular cues that drive reactivation. A key step during HCMV reactivation in latently infected HPCs is reexpression of viral major immediate early (MIE) genes. We recently determined that the major immediate early promoter (MIEP), which is primarily responsible for MIE gene expression during lytic replication, remains silent during reactivation. Instead, alternative promoters in the MIE locus are induced by reactivation stimuli. Here, we find that forkhead family (FOXO) transcription factors are critical for activation of alternative MIE promoters during HCMV reactivation, as mutating FOXO binding sites in alternative MIE promoters decreased HCMV IE gene expression upon reactivation and significantly decreased the production of infectious virus from latently infected primary CD34 HPCs. These findings establish a mechanistic link by which infected cells sense environmental cues to regulate latency and reactivation, and emphasize the role of contextual activation of alternative MIE promoters as the primary drivers of reactivation.
- Hancock, M. H., Mitchell, J., Goodrum, F. D., & Nelson, J. A. (2020). Human Cytomegalovirus miR-US5-2 Downregulation of GAB1 Regulates Cellular Proliferation and Expression through Modulation of Epidermal Growth Factor Receptor Signaling Pathways. mSphere, 5(4).More infoRegulation of epidermal growth factor (EGF) receptor (EGFR) signaling is critical for the replication of human cytomegalovirus (HCMV) as well as latency and reactivation in CD34 hematopoietic progenitor cells. HCMV microRNAs (miRNAs) provide a means to modulate the signaling activated by EGF through targeting components of the EGFR signaling pathways. Here, we demonstrate that HCMV miR-US5-2 directly downregulates the critical EGFR adaptor protein GAB1 that mediates activation and sustained signaling through the phosphatidylinositol 3-kinase (PI3K) and MEK/extracellular signal-regulated kinase (ERK) pathways and cellular proliferation in response to EGF. Expression of HCMV UL138 is regulated by the transcription factor early growth response gene 1 (EGR1) downstream of EGFR-induced MEK/ERK signaling. We show that by targeting GAB1 and attenuating MEK/ERK signaling, miR-US5-2 indirectly regulates EGR1 and UL138 expression, which implicates the miRNA in critical regulation of HCMV latency. Human cytomegalovirus (HCMV) causes significant disease in immunocompromised individuals, including transplant patients. HCMV establishes latency in hematopoietic stem cells in the bone marrow. The mechanisms governing latency and reactivation of viral replication are complex and not fully understood. HCMV-encoded miRNAs are small regulatory RNAs that reduce protein expression. In this study, we found that the HCMV miRNA miR-US5-2 targets the epidermal growth factor receptor (EGFR) adaptor protein GAB1 which directly affects downstream cellular signaling pathways activated by EGF. Consequently, miR-US5-2 blocks the EGF-mediated proliferation of human fibroblasts. Early growth response gene 1 (EGR1) is a transcription factor activated by EGFR signaling that regulates expression of HCMV UL138. We show that miR-US5-2 regulates UL138 expression through GAB1-mediated downregulation of the signaling pathways that lead to EGR1 expression. These data suggest that miR-US5-2, through downregulation of GAB1, could play a critical role during reactivation from latency by reducing proliferation and UL138 expression.
- Mlera, L., Moy, M., Maness, K., Tran, L. N., & Goodrum, F. D. (2020). The Role of the Human Cytomegalovirus Gene Locus in Latency and Reactivation. Viruses, 12(7).More infoHuman cytomegalovirus (HCMV) latency, the means by which the virus persists indefinitely in an infected individual, is a major frontier of current research efforts in the field. Towards developing a comprehensive understanding of HCMV latency and its reactivation from latency, viral determinants of latency and reactivation and their host interactions that govern the latent state and reactivation from latency have been identified. The polycistronic locus encodes determinants of both latency and reactivation. In this review, we survey the model systems used to investigate latency and new findings from these systems. Particular focus is given to the roles of the UL133, UL135, UL136 and UL138 proteins in regulating viral latency and how their known host interactions contribute to regulating host signaling pathways towards the establishment of or exit from latency. Understanding the mechanisms underlying viral latency and reactivation is important in developing strategies to block reactivation and prevent CMV disease in immunocompromised individuals, such as transplant patients.
- Rak, M. A., Buehler, J., Zeltzer, S., Reitsma, J., Molina, B., Terhune, S., & Goodrum, F. (2020). Erratum for Rak et al., "Human Cytomegalovirus UL135 Interacts with Host Adaptor Proteins To Regulate Epidermal Growth Factor Receptor and Reactivation from Latency". Journal of virology, 94(7).
- Buehler, J., Carpenter, E., Zeltzer, S., Igarashi, S., Rak, M., Mikell, I., Nelson, J. A., & Goodrum, F. (2019). Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency. PLoS pathogens, 15(11), e1008037.More infoSustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of alpha and beta herpesvirus latency. We have previously shown that the beta-herpesvirus, human cytomegalovirus (CMV), regulates epidermal growth factor receptor (EGFR), upstream of PI3K, to control states of latency and reactivation. How signaling downstream of EGFR is regulated and how this impacts CMV infection and latency is not fully understood. We demonstrate that CMV downregulates EGFR early in the productive infection, which blunts the activation of EGFR and its downstream pathways in response to stimuli. However, CMV infection sustains basal levels of EGFR and downstream pathway activity in the context of latency in CD34+ hematopoietic progenitor cells (HPCs). Inhibition of MEK/ERK, STAT or PI3K/AKT pathways downstream of EGFR increases viral reactivation from latently infected CD34+ HPCs, defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription important to latency. Indeed, EGF-stimulation increased expression of the UL138 latency gene, but not immediate early or early viral genes, suggesting that EGFR signaling promotes latent gene expression. The early growth response-1 (EGR1) transcription factor is induced downstream of EGFR signaling through the MEK/ERK pathway and is important for the maintenance of hematopoietic stemness. We demonstrate that EGR1 binds the viral genome upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevents the establishment of latency in CD34+ HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 gene expression and suppression of replication for latency. By this mechanism, the virus has hardwired itself into host cell biology to sense and respond to changes in homeostatic host cell signaling.
- Collins-McMillen, D., Rak, M., Buehler, J. C., Igarashi-Hayes, S., Kamil, J. P., Moorman, N. J., & Goodrum, F. (2019). Alternative promoters drive human cytomegalovirus reactivation from latency. Proceedings of the National Academy of Sciences of the United States of America, 116(35), 17492-17497.More infoReactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP has poor activity in HPCs, it is unclear how viral transactivators are expressed during reactivation. It has been presumed that viral gene expression is reinitiated via de-repression of the MIEP. We demonstrate that immediate early transcripts arising from reactivation originate predominantly from alternative promoters within the canonical major immediate early locus. Disruption of these intronic promoters results in striking defects in re-expression of viral genes and viral genome replication in the THP-1 latency model. Furthermore, we show that these promoters are necessary for efficient reactivation in primary CD34 HPCs. Our findings shift the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.
- Mikell, I., Crawford, L. B., Hancock, M. H., Mitchell, J., Buehler, J., Goodrum, F., & Nelson, J. A. (2019). HCMV miR-US22 down-regulation of EGR-1 regulates CD34+ hematopoietic progenitor cell proliferation and viral reactivation. PLoS pathogens, 15(11), e1007854.More infoReactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.
- Collins-McMillen, D., Buehler, J., Peppenelli, M., & Goodrum, F. (2018). Molecular Determinants and the Regulation of Human Cytomegalovirus Latency and Reactivation. Viruses, 10(8).More infoHuman cytomegalovirus (HCMV) is a beta herpesvirus that establishes a life-long persistence in the host, like all herpesviruses, by way of a latent infection. During latency, viral genomes are maintained in a quieted state. Virus replication can be reactivated from latency in response to changes in cellular signaling caused by stress or differentiation. The past decade has brought great insights into the molecular basis of HCMV latency. Here, we review the complex persistence of HCMV with consideration of latent reservoirs, viral determinants and their host interactions, and host signaling and the control of cellular and viral gene expression that contributes to the establishment of and reactivation from latency.
- Goodrum, F., Goodrum, F., McWeeney, S., & McWeeney, S. (2018). A Single-Cell Approach to the Elusive Latent Human Cytomegalovirus Transcriptome. mBio, 9(3).More infoHerpesvirus latency has been difficult to understand molecularly due to low levels of viral genomes and gene expression. In the case of the betaherpesvirus human cytomegalovirus (HCMV), this is further complicated by the heterogeneity inherent to hematopoietic subpopulations harboring genomes and, as a consequence, the various patterns of infection that simultaneously exist in a host, ranging from latent to lytic. Single-cell RNA sequencing (scRNA-seq) provides tremendous potential in measuring the gene expression profiles of heterogeneous cell populations for a wide range of applications, including in studies of cancer, immunology, and infectious disease. A recent study by Shnayder et al. (mBio 9:e00013-18, 2018, https://doi.org/10.1128/mBio.00013-18) utilized scRNA-seq to define transcriptomal characteristics of HCMV latency. They conclude that latency-associated gene expression is similar to the late lytic viral program but at lower levels of expression. The study highlights the numerous challenges, from the definition of latency to the analysis of scRNA-seq, that exist in defining a latent transcriptome.
- Rak, M. A., Buehler, J., Zeltzer, S., Reitsma, J., Molina, B., Terhune, S., & Goodrum, F. (2018). Human Cytomegalovirus UL135 Interacts with Host Adaptor Proteins To Regulate Epidermal Growth Factor Receptor and Reactivation from Latency. Journal of virology, 92(20).More infoHuman cytomegalovirus, HCMV, is a betaherpesvirus that establishes a lifelong latent infection in its host that is marked by recurrent episodes of reactivation. The molecular mechanisms by which the virus and host regulate entry into and exit from latency remain poorly understood. We have previously reported that is critical for reactivation, functioning in part by overcoming suppressive effects of the latency determinant We have demonstrated a role for in diminishing cell surface levels and targeting epidermal growth factor receptor (EGFR) for turnover. The attenuation of EGFR signaling promotes HCMV reactivation in combination with cellular differentiation. In this study, we sought to define the mechanisms by which functions in regulating EGFR turnover and viral reactivation. Screens to identify proteins interacting with pUL135 identified two host adaptor proteins, CIN85 and Abi-1, with overlapping activities in regulating EGFR levels in the cell. We mapped the amino acids in pUL135 necessary for interaction with Abi-1 and CIN85 and generated recombinant viruses expressing variants of pUL135 that do not interact with CIN85 or Abi-1. These recombinant viruses replicate in fibroblasts but are defective for reactivation in an experimental model for latency using primary CD34 hematopoietic progenitor cells (HPCs). These variants have altered trafficking of EGFR and are defective in targeting EGFR for turnover. These studies demonstrate a requirement for pUL135 interactions with Abi-1 and CIN85 for regulation of EGFR and mechanistically link the regulation of EGFR to reactivation. Human cytomegalovirus (HCMV) establishes a lifelong latent infection in the human host. While the infection is typically asymptomatic in healthy individuals, HCMV infection poses life-threatening disease risk in immunocompromised individuals and is the leading cause of birth defects. Understanding how HCMV controls the lifelong latent infection and reactivation of replication from latency is critical to developing strategies to control HCMV disease. Here, we identify the host factors targeted by a viral protein that is required for reactivation. We define the importance of this virus-host interaction in reactivation from latency, providing new insights into the molecular underpinnings of HCMV latency and reactivation.
- Zeltzer, S., Zeltzer, C. A., Igarashi, S., Wilson, J., Donaldson, J. G., & Goodrum, F. (2018). Virus Control of Trafficking from Sorting Endosomes. mBio, 9(4).More infoThe maintenance of cell surface proteins is critical to the ability of a cell to sense and respond to information in its environment. As such, modulation of cell surface composition and receptor trafficking is a potentially important target of control in virus infection. Sorting endosomes (SEs) are control stations regulating the recycling or degradation of internalized plasma membrane proteins. Here we report that human cytomegalovirus (HCMV), a ubiquitous betaherpesvirus, alters the fate of internalized clathrin-independent endocytosis (CIE) cargo proteins, retaining them in virally reprogrammed SEs. We show that the small G protein ARF6 (ADP ribosylation factor 6), a regulator of CIE trafficking, is highly associated with SE membranes relative to uninfected cells. Combined with the observation of accumulated CIE cargo at the SE, these results suggest that infection diminishes the egress of ARF6 and its cargo from the SE. Expression of ubiquitin-specific protease 6 (USP6), also known as TRE17, was sufficient to restore ARF6 and some ARF6 cargo trafficking to the cell surface in infected cells. The USP activity of TRE17 was required to rescue both ARF6 and associated cargo from SE retention in infection. The finding that TRE17 expression does not rescue the trafficking of all CIE cargos retained at SEs in infection suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic microdomains that are subject to alternative sorting fates. Cells maintain their surface composition, take up nutrients, and respond to their environment through the internalization and recycling of cargo at the cell surface through endocytic trafficking pathways. During infection with human cytomegalovirus (HCMV), host endocytic membranes are reorganized into a juxtanuclear structure associated with viral assembly and egress. Less appreciated is the effect of this reorganization on the trafficking of host proteins through the endocytic pathway. We show that HCMV retains internalized cargo and the effector of clathrin-independent endocytosis at sorting endosomes. The retention of some cargo, but not all, was reversed by overexpression of a ubiquitin-specific protease, TRE17. Our results demonstrate that HCMV induces profound reprogramming of endocytic trafficking and influences cargo sorting decisions. Further, our work suggests the presence of a novel ubiquitin-regulated checkpoint for the recycling of cargo from sorting endosome. These findings have important implications for host signaling and immune pathways in the context of HCMV infection.
- Cheng, S., Caviness, K., Buehler, J., Smithey, M., Nikolich-Žugich, J., & Goodrum, F. (2017). Transcriptome-wide characterization of human cytomegalovirus in natural infection and experimental latency. Proceedings of the National Academy of Sciences of the United States of America, 114(49), E10586-E10595.More infoThe transcriptional program associated with herpesvirus latency and the viral genes regulating entry into and exit from latency are poorly understood and controversial. Here, we developed and validated a targeted enrichment platform and conducted large-scale transcriptome analyses of human cytomegalovirus (HCMV) infection. We used both an experimental hematopoietic cell model of latency and cells from naturally infected, healthy human subjects (clinical) to define the breadth of viral genes expressed. The viral transcriptome derived from experimental infection was highly correlated with that from clinical infection, validating our experimental latency model. These transcriptomes revealed a broader profile of gene expression during infection in hematopoietic cells than previously appreciated. Further, using recombinant viruses that establish a nonreactivating, latent-like or a replicative infection in CD34+ hematopoietic progenitor cells, we defined classes of low to moderately expressed genes that are differentially regulated in latent vs. replicative states of infection. Most of these genes have yet to be studied in depth. By contrast, genes that were highly expressed, were expressed similarly in both latent and replicative infection. From these findings, a model emerges whereby low or moderately expressed genes may have the greatest impact on regulating the switch between viral latency and replication. The core set of viral genes expressed in natural infection and differentially regulated depending on the pattern of infection provides insight into the HCMV transcriptome associated with latency in the host and a resource for investigating virus-host interactions underlying persistence.
- Collins-McMillen, D., & Goodrum, F. D. (2017). The loss of binary: Pushing the herpesvirus latency paradigm. Current clinical microbiology reports, 4(3), 124-131.More infoHerpesvirus latency has been viewed as a binary state where replication is either on or off. During latency, gene expression is thought to be restricted to non-coding RNAs or very few proteins so that the virus avoids detection by the immune system. However, a number of recent studies across herpesvirus families call into question the existence of a binary switch for latency, and suggest that latency is far more dynamic than originally presumed. These studies are the focus of this review.
- Gordon, C. L., Miron, M., Thome, J. J., Matsuoka, N., Weiner, J., Rak, M. A., Igarashi, S., Granot, T., Lerner, H., Goodrum, F., & Farber, D. L. (2017). Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection. The Journal of experimental medicine, 214(3), 651-667.More infoT cell responses to viruses are initiated and maintained in tissue sites; however, knowledge of human antiviral T cells is largely derived from blood. Cytomegalovirus (CMV) persists in most humans, requires T cell immunity to control, yet tissue immune responses remain undefined. Here, we investigated human CMV-specific T cells, virus persistence and CMV-associated T cell homeostasis in blood, lymphoid, mucosal and secretory tissues of 44 CMV seropositive and 28 seronegative donors. CMV-specific T cells were maintained in distinct distribution patterns, highest in blood, bone marrow (BM), or lymph nodes (LN), with the frequency and function in blood distinct from tissues. CMV genomes were detected predominantly in lung and also in spleen, BM, blood and LN. High frequencies of activated CMV-specific T cells were found in blood and BM samples with low virus detection, whereas in lung, CMV-specific T cells were present along with detectable virus. In LNs, CMV-specific T cells exhibited quiescent phenotypes independent of virus. Overall, T cell differentiation was enhanced in sites of viral persistence with age. Together, our results suggest tissue T cell reservoirs for CMV control shaped by both viral and tissue-intrinsic factors, with global effects on homeostasis of tissue T cells over the lifespan.
- Kim, J. H., Collins-mcmillen, D., Buehler, J. C., Goodrum, F. D., & Yurochko, A. D. (2017). Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34+ Human Progenitor Cells.. Journal of virology, 91(5). doi:10.1128/jvi.01206-16More infoThe establishment of human cytomegalovirus (HCMV) latency and persistence relies on the successful infection of hematopoietic cells, which serve as sites of viral persistence and contribute to viral spread. Here, using blocking antibodies and pharmacological inhibitors, we document that HCMV activation of the epidermal growth factor receptor (EGFR) and downstream phosphatidylinositol 3-kinase (PI3K) mediates viral entry into CD34+ human progenitor cells (HPCs), resulting in distinct cellular trafficking and nuclear translocation of the virus compared to that in other immune cells, such as we have documented in monocytes. We argue that the EGFR allows HCMV to regulate the cellular functions of these replication-restricted cells via its signaling activity following viral binding. In addition to regulating HCMV entry/trafficking, EGFR signaling may also shape the early steps required for the successful establishment of viral latency in CD34+ cells, as pharmacological inhibition of EGFR increases the transcription of lytic IE1/IE2 mRNA while curbing the expression of latency-associated UL138 mRNA. EGFR signaling following infection of CD34+ HPCs may also contribute to changes in hematopoietic potential, as treatment with the EGFR kinase (EGFRK) inhibitor AG1478 alters the expression of the cellular hematopoietic cytokine interleukin 12 (IL-12) in HCMV-infected cells but not in mock-infected cells. These findings, along with our previous work with monocytes, suggest that EGFR likely serves as an important determinant of HCMV tropism for select subsets of hematopoietic cells. Moreover, our new data suggest that EGFR is a key receptor for efficient viral entry and that the ensuing signaling regulates important early events required for successful infection of CD34+ HPCs by HCMV.IMPORTANCE HCMV establishes lifelong persistence within the majority of the human population without causing overt pathogenesis in healthy individuals. Despite this, reactivation of HCMV from its latent reservoir in the bone marrow causes significant morbidity and mortality in immunologically compromised individuals, such as bone marrow and solid organ transplant patients. Lifelong persistent infection has also been linked with the development of various cardiovascular diseases in otherwise healthy individuals. Current HCMV therapeutics target lytic replication, but not the latent viral reservoir; thus, an understanding of the molecular basis for viral latency and persistence is paramount to controlling or eliminating HCMV infection. Here, we show that the viral signalosome activated by HCMV binding to its entry receptor, EGFR, in CD34+ HPCs initiates early events necessary for successful latent infection of this cell type. EGFR and associated signaling players may therefore represent promising targets for mitigating HCMV persistence.
- Leng, S. X., Kamil, J., Purdy, J. G., Lemmermann, N. A., Reddehase, M. J., & Goodrum, F. D. (2017). Recent advances in CMV tropism, latency, and diagnosis during aging. GeroScience, 39(3), 251-259.More infoHuman cytomegalovirus (CMV) is one of the largest viruses known to cause human diseases. Chronic CMV infection, as defined by anti-CMV IgG serology, increases with age and is highly prevalent in older adults. It has complex biology with significant immunologic and health consequences. This article aims to summarize research findings presented at the 6th International Workshop on CMV and Immunosenescence that relate to advances in the areas of CMV tropism, latency, CMV manipulation of cell metabolism, and T cell memory inflation, as well as novel diagnostic evaluation and translational research of chronic CMV infection in older adults. Information summarized here represents the current state of knowledge in these important fields. Investigators have also identified a number of areas that deserve further and more in-depth investigation, including building more precise parallels between mouse CMV (mCMV) and human CMV (HCMV) research. It is hoped that this article will also stimulate engaging discussion on strategies and direction to advance the science to the next level.
- Nikolich-Zugich, J., Goodrum, F., Knox, K., & Smithey, M. J. (2017). Known unknowns: how might the persistent herpesvirome shape immunity and aging?. Current opinion in immunology, 48, 23-30.More infoThe microbial community that colonizes all living organisms is gaining appreciation for its contributions to both physiologic and pathogenic processes. The virome, a subset of the overall microbiome, large and diverse, including viruses that persistently inhabit host cells, endogenous viral elements genomically or epigenomically integrated into cells, and viruses that infect the other (bacterial, protozoan, fungal, and archaeal) microbiome phylla. These viruses live in the organism for its life, and therefore are to be considered part of the aging process experienced by the organism. This review considers the impact of the persistent latent virome on immune aging. Specific attention will be devoted to the role of herpesviruses, and within them, the cytomegalovirus, as the key modulators of immune aging.
- Buehler, J., Zeltzer, S., Reitsma, J., Petrucelli, A., Umashankar, M., Rak, M., Zagallo, P., Schroeder, J., Terhune, S., & Goodrum, F. (2016). Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication. PLoS pathogens, 12(5), e1005655.More infoHerpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous β-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling.
- Caviness, K., Bughio, F., Crawford, L. B., Streblow, D. N., Nelson, J. A., Caposio, P., & Goodrum, F. (2016). Complex Interplay of the UL136 Isoforms Balances Cytomegalovirus Replication and Latency. mBio, 7(2), e01986.More infoHuman cytomegalovirus (HCMV), a betaherpesvirus, persists indefinitely in the human host through poorly understood mechanisms. The UL136 gene is carried within a genetic locus important to HCMV latency termed the UL133/8 locus, which also carries UL133, UL135, and UL138. Previously, we demonstrated that UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and, likely, translation initiation mechanisms. We previously showed that the UL136 isoforms are largely dispensable for virus infection in fibroblasts, a model for productive virus replication. In our current work, UL136 has emerged as a complex regulator of HCMV infection in multiple contexts of infection relevant to HCMV persistence: in an endothelial cell (EC) model of chronic infection, in a CD34(+) hematopoietic progenitor cell (HPC) model of latency, and in an in vivo NOD-scid IL2Rγc (null) humanized (huNSG) mouse model for latency. The 33- and 26-kDa isoforms promote replication, while the 23- and 19-kDa isoforms suppress replication in ECs, in CD34(+) HPCs, and in huNSG mice. The role of the 25-kDa isoform is context dependent and influences the activity of the other isoforms. These isoforms localize throughout the secretory pathway, and loss of the 33- and 26-kDa UL136 isoforms results in virus maturation defects in ECs. This work reveals an intriguing functional interplay between protein isoforms that impacts virus replication, latency, and dissemination, contributing to the overall role of the UL133/8 locus in HCMV infection.
- Goodrum, F. (2016). Human Cytomegalovirus Latency: Approaching the Gordian Knot. Annual Reviews in Virology, 333-357.More infoHerpesviruses have evolved exquisite virus-host interactions that co-opt or evade a number of host pathways to enable the viruses to persist. Persistence of human cytomegalovirus (CMV), the prototypical betaherpesvirus, is particularly complex in the host organism. Depending on host physiology and the cell types infected, CMV persistence comprises latent, chronic, and productive states that may occur concurrently. Viral latency is a central strategy by which herpesviruses ensure their lifelong persistence. Although much remains to be defined about the virus-host interactions important to CMV latency, it is clear that checkpoints composed of viral and cellular factors exist to either maintain a latent state or initiate productive replication in response to host cues. CMV offers a rich platform for defining the virus-host interactions and understanding the host biology important to viral latency. This review describes current understanding of the virus-host interactions that contribute to viral latency and reactivation.
- Kim, J. H., Collins-McMillen, D., Buehler, J. C., Goodrum, F. D., & Yurochko, A. D. (2016). HCMV requires EGFR signaling to enter and initiate the early steps in the establishment of latency in CD34+ human progenitor cells. Journal of virology.More infoThe establishment of human cytomegalovirus (HCMV) latency and persistence relies on the successful infection of hematopoietic cells, which serve as sites of viral persistence and contribute to viral spread. Here, using blocking antibodies and pharmacological inhibitors, we document that HCMV activation of the epidermal growth factor receptor (EGFR) and downstream phosphatidylinositol-3-kinase (PI (3)K) mediates viral entry into CD34+ human progenitor cells (HPCs), resulting in distinct cellular trafficking and nuclear translocation of the virus compared to other immune cells, such as we have documented in monocytes. We argue that the EGFR allows HCMV to regulate the cellular functions of these replication-restricted cells via its signaling activity following viral binding. In addition to regulating HCMV entry/trafficking, EGFR signaling may also shape the early steps required for the successful establishment of viral latency in CD34+ cells, as pharmacological inhibition of EGFR increases the transcription of lytic IE1/IE2 mRNA, while curbing the expression of latency-associated UL138 mRNA. EGFR signaling following infection of CD34+ HPCs may also contribute to changes in hematopoietic potential, as treatment with the EGFRK inhibitor AG1478 alters the expression of the cellular hematopoietic cytokine IL-12 in HCMV-infected, but not in mock-infected cells. These findings, along with our previous work in monocytes, suggest that EGFR likely serves as an important determinant of HCMV tropism for select subsets of hematopoietic cells. Moreover, our new data suggest that EGFR is a key receptor for efficient viral entry and that the ensuing signaling regulates important early events required for successful infection of CD34+ HPCs by HCMV.
- Lee, S. H., Caviness, K., Albright, E. R., Lee, J., Gelbmann, C. B., Rak, M., Goodrum, F., & Kalejta, R. F. (2016). Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency. Journal of virology, 90(20), 9483-94.More infoThe UL133-138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes proteins required for latency and reactivation in CD34(+) hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo- and hetero-interactions and localize within secretory membranes. One of these genes, UL136 gene, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, the UL138 gene, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino-terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a subcellular localization similar to that of pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and pUL138-S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells in which HCMV latency is studied. The long form appears to be more potent at silencing IE transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency.
- Bughio, F., Umashankar, M., Wilson, J., & Goodrum, F. (2015). Human Cytomegalovirus UL135 and UL136 Genes Are Required for Postentry Tropism in Endothelial Cells. Journal of virology, 89(13), 6536-50.More infoEndothelial cells (ECs) are a critical target of viruses, and infection of the endothelium represents a defining point in viral pathogenesis. Human cytomegalovirus (HCMV), the prototypical betaherpesvirus, encodes proteins specialized for entry into ECs and delivery of the genome to the nuclei of ECs. Virus strains competent to enter ECs replicate with differing efficiencies, suggesting that the virus encodes genes for postentry tropism in ECs. We previously reported a specific requirement for the UL133/8 locus of HCMV for replication in ECs. The UL133/8 locus harbors four genes: UL133, UL135, UL136, and UL138. In this study, we find that while UL133 and UL138 are dispensable for replication in ECs, both UL135 and UL136 are important. These genes are not required for virus entry or the expression of viral genes. The phenotypes associated with disruption of either gene reflect phenotypes observed for the UL133/8NULL virus, which lacks the entire UL133/8 locus, but are largely distinct from one another. Viruses lacking UL135 fail to properly envelop capsids in the cytoplasm, produce fewer dense bodies (DB) than the wild-type (WT) virus, and are unable to incorporate viral products into multivesicular bodies (MVB). Viruses lacking UL136 also fail to properly envelop virions and produce larger dense bodies than the WT virus. Our results indicate roles for the UL135 and UL136 proteins in commandeering host membrane-trafficking pathways for virus maturation. UL135 and UL136 represent the first HCMV genes crucial for early- to late-stage tropism in ECs.
- Goodrum, F., & Bughio, F. (2015). Viral infection at the endothelium. Oncotarget, 6(29), 26541-2.
- Caviness, K., Cicchini, L., Rak, M., Umashankar, M., & Goodrum, F. (2014). Complex expression of the UL136 gene of human cytomegalovirus results in multiple protein isoforms with unique roles in replication. Journal of virology, 88(24), 14412-25.More infoHuman cytomegalovirus (HCMV) is a complex DNA virus with a 230-kb genome encoding 170 and up to 750 proteins. The upper limit of this coding capacity suggests the evolution of complex mechanisms to substantially increase the coding potential from the 230-kb genome. Our work examines the complexity of one gene, UL136, encoded within the ULb' region of the genome that is lost during serial passage of HCMV in cultured fibroblasts. UL136 is expressed as five protein isoforms. We mapped these isoforms and demonstrate that they originate from both a complex transcriptional profile and, possibly, the usage of multiple translation initiation sites. Intriguingly, the pUL136 isoforms exhibited distinct subcellular distributions with varying association with the Golgi apparatus. The subcellular localization of membrane-bound isoforms of UL136 differed between when they were expressed exogenously and when they were expressed in the context of viral infection, suggesting that the trafficking of these isoforms is mediated by infection-specific factors. While UL136, like most ULb' genes, was dispensable for replication in fibroblasts, the soluble 23- and 19-kDa isoforms suppressed virus replication. In CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro, disruption of the 23- and 19-kDa isoforms resulted in increased replication and a loss of the latency phenotype, similar to the effects of the UL138 latency determinant encoded within the same genetic locus. Our work suggests a complex interplay between the UL136 isoforms which balances viral replication in multiple cell types and likely contributes to the cell type-dependent phenotypes of the UL133/8 locus and the outcome of HCMV infection.
- Goodrum, F., Schultz-cherry, S., Tsai, B., Sullivan, C. S., & Pfeiffer, J. K. (2014). Welcome from the Gems Associate Editors. Journal of Virology, 88(10), 5207-5207. doi:10.1128/jvi.00474-14More infoWelcome to Gems! We were honored to be invited to serve as the first Associate Editors of this exciting new platform for the Journal of Virology . Our goals are to highlight the “News and Views” in virology including developments from new investigators and our established colleagues, topics considered controversial or cutting-edge, new tools and resources, and opinions on areas important to the virology community as a few examples.
- Goodrum, F., Sobonya, R. E., Knox, K. S., Sobonya, R. E., Sam, A., Ricciotti, R. W., Knox, K. S., & Goodrum, F. (2014). Medical image of the week: CMV cytopathic effect. Southwest Journal of Pulmonary and Critical Care, 9(6), 341-342. doi:10.13175/swjpcc161-14
- Li, G., Rak, M., Nguyen, C. C., Umashankar, M., Goodrum, F. D., & Kamil, J. P. (2014). An epistatic relationship between the viral protein kinase UL97 and the UL133-UL138 latency locus during the human cytomegalovirus lytic cycle. Journal of virology, 88(11), 6047-60.More infoWe report that UL133-UL138 (UL133/8), a transcriptional unit within the ULb' region (ULb') of the human cytomegalovirus (HCMV) genome, and UL97, a viral protein kinase encoded by HCMV, play epistatic roles in facilitating progression of the viral lytic cycle. In studies with HCMV strain TB40/E, pharmacological blockade or genetic ablation of UL97 significantly reduced the levels of mRNA and protein for IE2 and viral early and early-late genes during a second wave of viral gene expression that commenced at between 24 and 48 h postinfection. These effects were accompanied by significant defects in viral DNA synthesis and viral replication. Interestingly, deletion of UL133/8 likewise caused significant defects in viral DNA synthesis, viral gene expression, and viral replication, which were not exacerbated upon UL97 inhibition. When UL133/8 was restored to HCMV laboratory strain AD169, which otherwise lacks the locus, the resulting recombinant virus replicated similarly to the parental virus. However, during UL97 inhibitor treatment, the virus in which UL133/8 was restored showed significantly exacerbated defects in viral DNA synthesis, viral gene expression, and production of infectious progeny virus, thus recapitulating the differences between wild-type TB40/E and its UL133/8-null derivative. Phenotypic evaluation of mutants null for specific open reading frames within UL133/8 revealed a role for UL135 in promoting viral gene expression, viral DNA synthesis, and viral replication, which depended on UL97. Taken together, our findings suggest that UL97 and UL135 play interdependent roles in promoting the progression of a second phase of the viral lytic cycle and that these roles are crucial for efficient viral replication.
- Sansoni, P., Vescovini, R., Fagnoni, F. F., Akbar, A., Arens, R., Chiu, Y., Cičin-Šain, L., Dechanet-Merville, J., Derhovanessian, E., Ferrando-Martinez, S., Franceschi, C., Frasca, D., Fulöp, T., Furman, D., Gkrania-Klotsas, E., Goodrum, F., Grubeck-Loebenstein, B., Hurme, M., Kern, F., , Lilleri, D., et al. (2014). New advances in CMV and immunosenescence. Experimental gerontology, 55, 54-62.More infoImmunosenescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterized by impaired protective immunity and decreased efficacy of vaccines. An increasing number of immunological, clinical and epidemiological studies suggest that persistent Cytomegalovirus (CMV) infection is associated with accelerated aging of the immune system and with several age-related diseases. However, current evidence on whether and how human CMV (HCMV) infection is implicated in immunosenescence and in age-related diseases remains incomplete and many aspects of CMV involvement in immune aging remain controversial. The attendees of the 4th International Workshop on "CMV & Immunosenescence", held in Parma, Italy, 25-27th March, 2013, presented and discussed data related to these open questions, which are reported in this commentary.
- Umashankar, M., & Goodrum, F. (2014). Hematopoietic long-term culture (hLTC) for human cytomegalovirus latency and reactivation. Methods in molecular biology (Clifton, N.J.), 1119, 99-112.More infoOf the many research challenges posed by human cytomegalovirus latency, perhaps the most notable is the requirement for primary hematopoietic cell culture. Culturing hematopoietic subpopulations while maintaining physiological relevance must be given utmost consideration. We describe a long-standing primary CD34(+) hematopoietic progenitor cell (HPCs) system as an experimental model to study human cytomegalovirus (HCMV) latency and reactivation. Key aspects of our model include infection of primary human CD34(+) HPCs prior to ex vivo expansion, maintenance of undifferentiated cells in a long-term culture with a stromal cell support, and an assay to quantitate infectious centers produced prior to and following a reactivation stimulus. Our method offers a unique way to quantitatively assess HCMV latency and reactivation to study the contribution of viral and host genes in latency and reactivation.
- Umashankar, M., Rak, M., Bughio, F., Zagallo, P., Caviness, K., & Goodrum, F. D. (2014). Antagonistic determinants controlling replicative and latent states of human cytomegalovirus infection. Journal of virology, 88(11), 5987-6002.More infoThe mechanisms by which viruses persist and particularly those by which viruses actively contribute to their own latency have been elusive. Here we report the existence of opposing functions encoded by genes within a polycistronic locus of the human cytomegalovirus (HCMV) genome that regulate cell type-dependent viral fates: replication and latency. The locus, referred to as the UL133-UL138 (UL133/8) locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. As part of the ULb' region of the genome, the UL133/8 locus is lost upon serial passage of clinical strains of HCMV in cultured fibroblasts and is therefore considered dispensable for replication in this context. Strikingly, we could not reconstitute infection in permissive fibroblasts from bacterial artificial chromosome clones of the HCMV genome where UL135 alone was disrupted. The loss of UL135 resulted in complex phenotypes and could ultimately be overcome by infection at high multiplicities. The requirement for UL135 but not the entire locus led us to hypothesize that another gene in this locus suppressed virus replication in the absence of UL135. The defect associated with the loss of UL135 was largely rescued by the additional disruption of the UL138 latency determinant, indicating a requirement for UL135 for virus replication when UL138 is expressed. In the CD34(+) hematopoietic progenitor model of latency, viruses lacking only UL135 were defective for viral genome amplification and reactivation. Taken together, these data indicate that UL135 and UL138 comprise a molecular switch whereby UL135 is required to overcome UL138-mediated suppression of virus replication to balance states of latency and reactivation.
- Bughio, F., Elliott, D. A., & Goodrum, F. (2013). An endothelial cell-specific requirement for the UL133-UL138 locus of human cytomegalovirus for efficient virus maturation. Journal of virology, 87(6), 3062-75.More infoHuman cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in a varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well defined, the molecular determinants regulating postentry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb' region of the HCMV genome as important for the latent infection in CD34(+) hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or fusion-inducing factor X (FIX) HCMV strains. ECs infected with a virus lacking the entire locus (UL133-UL138(NULL) virus) complete the immediate-early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138(NULL) virus exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138(NULL) virus-infected ECs, Golgi stacks were disrupted, and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138(NULL) virus-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138(NULL) virus, suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type-dependent, postentry tropism determinant required for viral maturation.
- Zalckvar, E., Paulus, C., Tillo, D., Asbach-Nitzsche, A., Lubling, Y., Winterling, C., Strieder, N., Mücke, K., Goodrum, F., Segal, E., & Nevels, M. (2013). Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein. Proceedings of the National Academy of Sciences of the United States of America, 110(32), 13126-31.More infoHuman CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function.
- Goodrum, F., Caviness, K., & Zagallo, P. (2012). Human cytomegalovirus persistence. Cellular microbiology, 14(5), 644-55.More infoViral persistence is the rule following infection with all herpesviruses. The β-herpesvirus, human cytomegalovirus (HCMV), persists through chronic and latent states of infection. Both of these states of infection contribute to HCMV persistence and to the high HCMV seroprevalence worldwide. The chronic infection is poorly defined molecularly, but clinically manifests as low-level virus shedding over extended periods of time and often in the absence of symptoms. Latency requires long-term maintenance of viral genomes in a reversibly quiescent state in the immunocompetent host. In this review, we focus on recent advances in the biology of HCMV persistence, particularly with respect to the latent mode of persistence. Latently infected individuals harbour HCMV genomes in haematopoietic cells and maintain large subsets of HCMV-specific T-cells. In the last few years, impressive advances have been made in understanding virus-host interactions important to HCMV infection, many of which will profoundly impact HCMV persistence. We discuss these advances and their known or potential impact on viral latency. As herpesviruses are met with similar challenges in achieving latency and often employ conserved strategies to persist, we discuss current and future directions of HCMV persistence in the context of the greater body of knowledge regarding α- and γ-herpesviruses persistence.
- Petrucelli, A., Umashankar, M., Zagallo, P., Rak, M., & Goodrum, F. (2012). Interactions between proteins encoded within the human cytomegalovirus UL133-UL138 locus. Journal of virology, 86(16), 8653-62.More infoWe previously described a novel genetic locus within the ULb' region of the human cytomegalovirus (HCMV) genome that, while dispensable for replication in fibroblasts, suppresses replication in hematopoietic progenitors and augments replication in endothelial cells. This locus, referred to as the UL133-UL138 locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. In this work, we have mapped the interactions among these proteins. An analysis of all pairwise interactions during transient expression revealed a robust interaction between pUL133 and pUL138. Potential interactions between pUL136 and both pUL133 and pUL138 were also revealed. In addition, each of the UL133-UL138 locus proteins self-associated, suggesting a potential to form higher-order homomeric complexes. As both pUL133 and pUL138 function in promoting viral latency in CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro, we further focused on this interaction. pUL133 and pUL138 are the predominant complex detected when all proteins are expressed together and require no other proteins in the locus for their association. During infection, the interaction between pUL133 and pUL138 or pUL136 can be detected. A recombinant virus that fails to express both pUL133 and pUL138 exhibited a latency phenotype similar to that of viruses that fail to express either pUL133 or pUL138, indicating that these proteins function cooperatively in latency and do not have independent functions that additively contribute to HCMV latency. These studies identify protein interactions among proteins encoded by the UL133-UL138 locus and demonstrate an important interaction impacting the outcome of HCMV infection.
- Umashankar, M., Petrucelli, A., Cicchini, L., Caposio, P., Kreklywich, C. N., Rak, M., Bughio, F., Goldman, D. C., Hamlin, K. L., Nelson, J. A., Fleming, W. H., Streblow, D. N., & Goodrum, F. (2011). A novel human cytomegalovirus locus modulates cell type-specific outcomes of infection. PLoS pathogens, 7(12), e1002444.More infoClinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb' that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb'-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb' sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγ(c) (null)-humanized mouse model. The UL133-UL138(NULL) virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations.
- Grainger, L., Cicchini, L., Rak, M., Petrucelli, A., Fitzgerald, K. D., Semler, B. L., & Goodrum, F. (2010). Stress-inducible alternative translation initiation of human cytomegalovirus latency protein pUL138. Journal of virology, 84(18), 9472-86.More infoWe have previously characterized a 21-kDa protein encoded by UL138 (pUL138) as a viral factor inherent to low-passage strains of human cytomegalovirus (HCMV) that is required for latent infection in vitro. pUL138 is encoded on 3.6-, 2.7-, and 1.4-kb 3' coterminal transcripts that are produced during productive and latent infections. pUL138 is encoded at the 3' end of each transcript and is preceded by an extensive 5' sequence (approximately 0.5 to 2.5 kb) containing several putative open reading frames (ORFs). We determined that three putative ORFs upstream of UL138 (UL133, UL135, and UL136) encode proteins. The UL138 transcripts are polycistronic, such that each transcript expresses pUL138 in addition to the most-5' ORF. The upstream coding sequences (CDS) present a significant challenge for the translation of pUL138 in mammalian cells. We hypothesized that sequences 5' of UL138 mediate translation initiation of pUL138 by alternative strategies. Accordingly, a 663-nucloetide (nt) sequence overlapping the UL136 CDS supported expression of a downstream cistron in a bicistronic reporter system. We did not detect cryptic promoter activity or RNA splicing events that could account for downstream cistron expression. These data are consistent with the sequence element functioning as an internal ribosome entry site (IRES). Interestingly, pUL138 expression from the 3.6- and 2.7-kb transcripts was induced by serum stress, which concomitantly inhibited normal cap-dependent translation. Our work suggests that an alternative and stress-inducible strategy of translation initiation ensures expression of pUL138 under a variety of cellular contexts. The UL138 polycistronic transcripts serve to coordinate the expression of multiple proteins, including a viral determinant of HCMV latency.
- Tey, S., Goodrum, F., & Khanna, R. (2010). CD8+ T-cell recognition of human cytomegalovirus latency-associated determinant pUL138. The Journal of general virology, 91(Pt 8), 2040-8.More infoRecent studies have shown that long-term persistence of human cytomegalovirus (HCMV) in mononuclear cells of myeloid lineage is dependent on the UL138 open reading frame, which promotes latent infection. Although T-cell recognition of protein antigens from all stages of lytic HCMV infection is well established, it is not clear whether proteins expressed during latent HCMV infection can also be recognized. This study conducted an analysis of T-cell response towards proteins associated with HCMV latency. Ex vivo analysis of T cells from healthy virus carriers revealed a dominant CD8(+) T-cell response to the latency-associated pUL138 protein, which recognized a non-canonical 13 aa epitope in association with HLA-B*3501. These pUL138-specific T cells displayed a range of memory phenotypes that were in general less differentiated than that previously described in T cells specific for HCMV lytic antigens. Antigen-presentation assays revealed that endogenous pUL138 could be presented efficiently by HCMV-infected cells. However, T-cell recognition of pUL138 was dependent on newly synthesized protein, with little presentation from stable, long-lived protein. These data demonstrate that T cells targeting latency-associated protein products exist, although HCMV may limit the presentation of latent proteins, thereby restricting T-cell recognition of latently infected cells.
- Petrucelli, A., Rak, M., Grainger, L., & Goodrum, F. (2009). Characterization of a novel Golgi apparatus-localized latency determinant encoded by human cytomegalovirus. Journal of virology, 83(11), 5615-29.More infoHuman cytomegalovirus (HCMV) exists indefinitely in infected individuals by a yet poorly characterized latent infection in hematopoietic cells. We previously demonstrated a requirement for the putative UL138 open reading frame (ORF) in promoting a latent infection in CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro. In our present study, we have identified two coterminal transcripts of 2.7 and 3.6 kb and a 21-kilodalton (kDa) protein (pUL138) that are derived from the UL138 locus with early-late gene kinetics during productive infection. The UL138 transcripts and protein are detected in both fibroblasts and HPCs. A recombinant virus, FIX-UL138(STOP), that synthesizes the UL138 transcripts but not the protein exhibited a partial loss-of-latency phenotype in HPCs, similar to the phenotype observed for the UL138-null recombinant virus. This finding suggests that the UL138 protein is required for latency, but it does not exclude the possibility that the UL138 transcripts or other ORFs also contribute to latency. The mechanisms by which pUL138 contributes to latency remain unknown. While the 86- and 72-kDa immediate-early proteins were not detected in HPCs infected with HCMV in vitro, pUL138 did not function directly to suppress expression from the major immediate-early promoter in reporter assays. Interestingly, pUL138 localizes to the Golgi apparatus in infected cells but is not incorporated into virus particles. The localization of pUL138 to the Golgi apparatus suggests that pUL138 contributes to HCMV latency by a novel mechanism. pUL138 is the first HCMV protein demonstrated to promote an infection with the hallmarks of latency in CD34(+) HPCs.
- Thomas, M. A., Broughton, R. S., Goodrum, F. D., & Ornelles, D. A. (2009). E4orf1 limits the oncolytic potential of the E1B-55K deletion mutant adenovirus. Journal of virology, 83(6), 2406-16.More infoClinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G(1) phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G(1) restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3'-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.
- Goodrum, F., Reeves, M., Sinclair, J., High, K., & Shenk, T. (2007). Human cytomegalovirus sequences expressed in latently infected individuals promote a latent infection in vitro. Blood, 110(3), 937-45.More infoLatency enables human cytomegalovirus (HCMV) to persist in the hematopoietic cells of infected individuals indefinitely and prevents clearance of the pathogen. Despite its critical importance to the viral infectious cycle, viral mechanisms that contribute to latency have not been identified. We compared the ability of low-passage clinical and laboratory-adapted strains of HCMV to establish a latent infection in primary human CD34(+) cells. The low-passage strains, Toledo and FIX, established an infection with the hallmarks of latency, whereas the laboratory strains, AD169 and Towne, replicated producing progeny virus. We hypothesized that ULb' region of the genome, which is unique to low-passage strains, may encode a latency-promoting activity. We created and analyzed recombinant viruses lacking segments or individual open reading frames (ORFs) in the ULb' region. One 5-kb segment, and more specifically the UL138 ORF, was required for HCMV to establish and/or maintain a latent infection in hematopoietic progenitor cells infected in vitro. This is the first functional demonstration of a virus-coded sequence required for HCMV latency. Importantly, UL138 RNA was expressed in CD34(+) cells and monocytes from HCMV-seropositive, healthy individuals. UL138 might be a target for antivirals against latent virus.
- Ornelles, D. A., Broughton-Shepard, R. N., & Goodrum, F. D. (2007). Analysis of adenovirus infections in synchronized cells. Methods in molecular medicine, 131, 83-101.More infoAdenoviruses (Ads) are small DNA tumor viruses that have played a pivotal role in understanding eukaryotic cell biology and viral oncogenesis. Among other cellular pathways, Ad usurps cell cycle progression following infection. Likewise, progression of the viral infection is influenced by the host cell cycle. We describe here methods developed for synchronizing dividing cell populations and for analysis of cell cycle synchrony by flow cytometry. Furthermore, three methods used to evaluate the outcome of Ad infection in synchronized cell populations are described. These include two assays for infectious centers and an assay for analyzing production of progeny virus by transmission electron microscopy. These methods have been used to demonstrate that Ads that fail to direct synthesis of the E1B 55-kDa or E4orf6 proteins replicate most effectively upon infecting cells in S phase.
- Goodrum, F., Jordan, C. T., Terhune, S. S., High, K., & Shenk, T. (2004). Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations. Blood, 104(3), 687-95.More infoThe cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.
- Goodrum, F. D., Jordan, C. T., High, K., & Shenk, T. (2002). Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: a model for latency. Proceedings of the National Academy of Sciences of the United States of America, 99(25), 16255-60.More infoHuman cytomegalovirus (HCMV) resides latently in hematopoietic cells of the bone marrow. Although viral genomes can be found in CD14+ monocytes and CD34+ progenitor cells, the primary reservoir for latent cytomegalovirus is unknown. We analyzed human hematopoietic subpopulations infected in vitro with a recombinant virus that expresses a green fluorescent protein marker gene. Although many hematopoietic cell subsets were infected in vitro, CD14+ monocytes and various CD34+ subpopulations were infected with the greatest efficiency. We have developed an in vitro system in which to study HCMV infection and latency in CD34+ cells cultured with irradiated stromal cells. Marker gene expression was substantially reduced by 4 days postinfection, and infectious virus was not made during the culture period. However, viral DNA sequences were maintained in infected CD34+ cells for >20 days in culture, and, importantly, virus replication could be reactivated by coculture with human fibroblasts. Using an HCMV gene array, we examined HCMV gene expression in CD34+ cells. The pattern of viral gene expression was distinct from that observed during productive or nonproductive infections. Some of these expressed viral genes may function in latency and are targets for further analysis. Altered gene expression in hematopoietic progenitors may be indicative of the nature and outcome of HCMV infection.
- Goodrum, F. D., & Ornelles, D. A. (1999). Roles for the E4 orf6, orf3, and E1B 55-kilodalton proteins in cell cycle-independent adenovirus replication. Journal of virology, 73(9), 7474-88.More infoAdenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.
- Goodrum, F. D., & Ornelles, D. A. (1998). p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lytic infection. Journal of virology, 72(12), 9479-90.More infoThe ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G1. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39 degreesC, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39 degreesC. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.
- Goodrum, F. D., & Ornelles, D. A. (1997). The early region 1B 55-kilodalton oncoprotein of adenovirus relieves growth restrictions imposed on viral replication by the cell cycle. Journal of virology, 71(1), 548-61.More infoThe E1B 55-kDa oncoprotein of adenovirus enables the virus to overcome restrictions imposed on viral replication by the cell cycle. Approximately 20% of HeLa cells infected with an E1B 55-kDa mutant adenovirus produced virus when evaluated by electron microscopy or by assays for infectious centers. By contrast, all HeLa cells infected with a wild-type adenovirus produced virus. The yield of E1B mutant virus from randomly cycling HeLa cells correlated with the fraction of cells in S phase at the time of infection. In synchronously growing HeLa cells, approximately 75% of the cells infected during S phase with the E1B mutant virus produced virus, whereas only 10% of the cells infected during G1 produced virus. The yield of E1B mutant virus from HeLa cells infected during S phase was sevenfold greater than that of cells infected during G1 and threefold greater than that of cells infected during asynchronous growth. Cells infected during S phase with the E1B mutant virus exhibited severe cytopathic effects, whereas cells infected with the E1B mutant virus during G1 exhibited a mild cytopathic effect. Viral DNA synthesis appeared independent of the cell cycle because equivalent amounts of viral DNA were synthesized in cells infected with either wild-type or E1B mutant virus. The inability of the E1B mutant virus to replicate was not mediated by the status of p53. These results define a novel property of the large tumor antigen of adenovirus in relieving growth restrictions imposed on viral replication by the cell cycle.
- Goodrum, F. D., Shenk, T., & Ornelles, D. A. (1996). Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells. Journal of virology, 70(9), 6323-35.More infoThe localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B proteins was determined in the absence of other adenovirus proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B protein colocalized in the nucleus with the E4 protein. A complex of the E4 and E1B proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 protein failed to direct the E1B protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 protein to direct the nuclear localization of the E1B protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with protein synthesis inhibited, a portion of the E4 protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B protein colocalized with the E4 protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 proteins of adenovirus.
Presentations
- Goodrum Sterling, F. D. (2021, April). HCMV regulation of Host Signaling. Pathogenic Human Viruses. Duke University: Duke University.
- Goodrum Sterling, F. D. (2021, July). Debunking Disinformation. American Society for Virology-Communications Workshop. Virtual.More infoPresented on presentation on writing opinion editorials on scientific topics for a lay audience.
- Goodrum Sterling, F. D. (2021, July). Human ctyomegalovirus parasitizes host polymerases. 45th International Herpesvirus Workshop. virtual: International Herpesvirus Workshop.
- Goodrum Sterling, F. D. (2021, July). Human cytomegalovirus UL136 isoforms. Shaping the Host Cell Proteome Satellite. Virtual: American Society of Virology Annual Conference.
- Goodrum Sterling, F. D. (2020, December). Cytomegalovirus- Making the Switch. Invited Seminar in Series. Philadelphia, PA: University of Pennsylvania.
- Goodrum Sterling, F. D. (2020, January). Human Cytomegalovirus Latency and Pathogenesis. Infectious Disease Grand Rounds. Tucson, AZ: University of Arizona.
- Goodrum Sterling, F. D. (2020, July). Human Cytomegalovirus latency and reactiavtions-making the switch. Invited seminar in seminar series. Hannover, Germany: Hannover Medical School.
- Goodrum Sterling, F. D. (2020, March). The New Coronavirus Pandemic: What is COVID-19?. Molecular and Cellular Biology Seminar Series. Tucson, AZ: Molecular and Cellular Biology.
- Goodrum Sterling, F. D. (2020, October). Scientific Activism. Invited Seminar. Chapel Hill, NC: University of North Carolina, Department of Microbiology and Immunology.
- Goodrum Sterling, F. D. (2020, September). Scientific Activism. Invited Seminar. New York, New York: Columbia University Medical School.
- Goodrum Sterling, F. D. (2019, April). Keynote-CMV Latency and Persistence. 7th International Congenital CMV Coference & 17th International CMV Workshop. Birmingham, AL.More infoKeynote talk
- Goodrum Sterling, F. D. (2019, February). Making the switch-CMV latency and reactivation. Department of Microbiology and Molecular Genetics Seminar Series. University of California-Irvine.
- Goodrum Sterling, F. D. (2019, May 2019). Making the switch--CMV latency and reactivation. Virology Student Invited Seminar SpeakerUniversity of Maryland-Baltimore.
- Goodrum Sterling, F. D. (2019, September). Making the switch- CMV latency and reactivation. Department of Molecular Genetics and MicrobiologyUniversity of Florida.
- Goodrum Sterling, F. D. (2018, April). Human Cytomegalovirus Control of Host Trafficking and Signaling. Research Seminar Series. Charlottesville, VA: University of Virginia.
- Goodrum Sterling, F. D. (2018, December). Human Cytomegalovirus Control of Latency and Reactivation. Research Seminar Series. Phoenix, AZ: Arizona State University.
- Goodrum Sterling, F. D. (2018, March). Human Cytomegalovirus Control of Host Trafficking and Signaling. Research Seminar Series. Winston-Salem, NC: Wake Forest University School of Medicine.
- Goodrum Sterling, F. D. (2018, November). Human Cytomegalovirus Control of Host Trafficking and Signaling. Research Seminar Series. Detroit, MI: Wayne State University School of Medicine.
- Goodrum Sterling, F. D. (2018, November). Human Cytomegalovirus Control of Host Trafficking and Signaling. Research Seminar Series. Reno, NV: University of Nevada, Reno.
- Goodrum Sterling, F. D. (2018, October). Human Cytomegalovirus Control of Host Trafficking and Signaling. Research Seminar Series. Philadelphia, PA: Temple University.
- Goodrum Sterling, F. D. (2018, October). Human Cytomegalovirus Control of Trafficking and Signaling for Latency. Research Seminar Series. Tucson, AZ: School of Animal and Comparative Biomedical Science, University of Arizona.
- Goodrum Sterling, F. D. (2018, September). Cytomegalovirus: Agign, Cardiovascular Disease and Cancer. Cytomegalovirus Infection: Advancing Strategies for Prevention and Treatment. Rockwille, MD: National Institutes of Health.
- Goodrum Sterling, F. D. (2017, April). Viral control of host signaling to promote latency. Invited Seminar-UI-Chicago. Chicago, IL: University of Illinois at Chicago, College of Medicine.
- Goodrum Sterling, F. D. (2017, April). Viral modulation of host trafficking and MVB biogenesis. Invited Seminar- Penn State. Hershey, Pennsylvania: Pennsylvania State University, College of Medicine.
- Goodrum Sterling, F. D. (2017, December/Winter). Cytomegalovirus Control of Host Trafficking and Signaling. Invited Seminar-Northwestern. Chicago, IL: Northwestern University Feinberg School of Medicine.
- Goodrum Sterling, F. D. (2017, July/Summer). Human Cytomegalovirus Latency: Control of Host Trafficking and Signaling. 42nd International Herpesvirus Workshop. Ghent, Belgium.More infoKeynote lecture
- Goodrum Sterling, F. D. (2017, May). Viral Control of Host Trafficking. Gordon Research Conference on Viruses and Cells. Il Ciocco, Italy: Gordon Research Conference.
- Goodrum Sterling, F. D. (2017, November/Fall). Human cytomegalovirus control of host trafficking. Invited seminar- UC Irvine. Irvine, CA: University of California, Irvine.
- Goodrum Sterling, F. D. (2016, April). Regulating Host Trafficking and Signaling for Viral Persistence. Cellular & Molecular Medicine/Molecular & Cellular Biology Joint Seminar Series. Tucson, AZ: Departments of CMM and MCB.
- Goodrum Sterling, F. D. (2016, January). Cytomegalovirus control of host trafficking and signaling. Invited Seminar. Boston, MA: Harvard University, Harvard Medical School, Department of Microbiology and Immunology.
- Goodrum Sterling, F. D. (2016, July). Human Cyotmegalovirus Persistence. P01 Symposium- Northwestern. Chicago, IL: Northwestern University.
- Goodrum Sterling, F. D. (2016, July). Viral control of host trafficking and signaling. 41st International Herpesvirus Workshop. Madison, WI: IHW.
- Goodrum Sterling, F. D. (2016, June). Complex roles of the UL136 gene of human cytomegalovirus in replication and latency. American Society of Virology 35th Annual Meeting. Balcksburg, VA: American Society of Virology.
- Goodrum Sterling, F. D. (2016, June). Virus control of host trafficking and signaling. Invited Seminar-Dartmouth. Hanover, NH: Dartmouth College, Geisel School of Medicine, Department of Microbiology and Immunology.
- Goodrum Sterling, F. D. (2016, November). Opposing regulation of the EGF Receptor: A molecular switch controlling cytomegalovirus latency and reactivation. 6th International Workshop on CMV and Immunosenescence. Tucson, AZ.
- Goodrum Sterling, F. D. (2016, September). Virus control of host trafficking and signaling. Invited Seminar- UPenn. Philadelphia, PA: University of Pennsylvania, Perelman School of Medicine, Department of Microbiology.
- Goodrum Sterling, F. D. (2015, April). Virus-Host Interaction Contributing to Cytomegalovirus Persistence. Invited Seminar. Ann Arbor, MI: Department of Microbiology and Immunology.
- Goodrum Sterling, F. D. (2015, December). Cytomegalovirus Persistence. Invited Seminar, Pediatric Infectious Disease Research Conference. Nashville, TN: Vanderbilt University.
- Goodrum Sterling, F. D. (2015, February). Viral Regulation of EGFR for Latency. Invited Seminar. Irvine, CA: University of California, Irvine, Department of Microbiology and Molecular Genetics.
- Goodrum Sterling, F. D. (2015, Februray). Promoter Switching and Reactivation from Latency. MicroLunch. Tucson, AZ: University of Arizona.
- Goodrum, F. D. (2015, July). Hijacking of cellular trafficking pathways for viral persistence. FASEB Microbial Pathogenesis: Mechanisms of Infectious Disease. Keystone, CO: FASEB.More infoInvited Speaker
- Goodrum Sterling, F. D. (2014, January). Questions of Viral Persistence: Switches and Gatekeepers. Invited Seminar, College of Medicine-Phoenix, Biomedical Sciences. Phoenix, AZ: College of Medicine-Phoenix, Biomedical Sciences.
- Goodrum Sterling, F. D. (2014, March). Human Cytomegalovirus Persistence. Jamie McNew Endowed Lectureship, Invited Speaker. Minneapolis, MN: University of Minnesota, Amplatz Children's Hospital.
- Goodrum Sterling, F. D. (2014, May). Biphasic Regulation of HCMV Gene Expression in Latency and Lytic Cycle Progression. MicroLunch. Tucson, AZ: University of Arizona.
- Goodrum Sterling, F. D. (2014, May). Human Cytomegalovirus Regulation of EGFR for Latency. Graduate Student Invited Seminar. Syracuse, NY: SUNY Upstate Medical University, Department of Microbiology and Immunology.
- Goodrum Sterling, F. D. (2014, October). Complexity of HCMV gene expression and its differential effects on virus replication. 3rd Annual ASM Conference on Viral Manipulation of Nuclear Processes. Washington, DC: American Society of Microbiology.
- Goodrum Sterling, F. D. (2014, October). Viral Regulation of EGFR for Latency. Plenary Speaker, 14th Annual Symposium on Virology. Lincoln, NE: University of Nebraska, Nebraska Center for Virology.
- Goodrum Sterling, F. D. (2013, April). Viral Determinants of Human Cytomegalovirus Latency. Post-doctoral Association Invited Speaker. Chapel Hill, NC: University of North Carolina, Chapel Hill, Department of Microbiology and Immunology.
- Goodrum Sterling, F. D. (2013, February). The Multifunctional UL135 Protein of Human Cytomegalovirus. MicroLunch. Tucson, AZ: University of Arizona.
- Goodrum Sterling, F. D. (2013, July). Endothelium Tropism Determinants of Human Cytomegalovirus. 38th International Herpesvirus Workshop. Grand Rapids, MI: International Herpesvirus Workshop.
- Goodrum Sterling, F. D. (2013, October). The Issue of Viral Persistence. Invited Seminar, Department of Pharmacology and Toxicology. Tucson, AZ: Department of Pharmacology and Toxicology.
- Goodrum Sterling, F. D. (2013, Septemeber). The Human Cytomegalovirus UL135/UL138 Switch. MicroLunch. Tucson, AZ: University of Arizona.
- Goodrum Sterling, F. D. (2017, Februrary). Viral Determinants of Cytomegalovirus Latency. Invited Seminar. St. Louis, MO: Washington University in St. Louis, School of Medicine, Department of Pathology and Immunobiology.
- Goodrum, F. D. (2013, March). The Question of Persistence. 4th International Workshop on CMV and Immunosenescence. Parma, Italy.
- Goodrum Sterling, F. D. (2012, April). Human Cytomegalovirus Latency. Invited Seminar. Winston-Salem, NC: Wake Forest University School of Medicine.
- Goodrum Sterling, F. D. (2012, December). Host-virus interactions underlying human cytomegalovirus latency. Invited Seminar. Shreveport, LA: Louisiana State University Health Sciences Center, Department of Microbiology and Immunology.
- Goodrum Sterling, F. D. (2012, March). Determinants of Virus Fate. Frontiers in Immunobiology and Immunopathogenesis Symposium. Tucson, AZ: Department of Immunobiology, University of Arizona.
- Goodrum Sterling, F. D. (2012, March). Human Cytomegalovirus Latency. Invited Seminar. Cleveland, OH: Cleveland Clinic, Department of Molecular Biology.
- Goodrum Sterling, F. D. (2012, September). The Multifunctional UL135 Protein of Human Cytomegalovirus. Immunobiology Seminar Series. Tucson, AZ: Department of Immunobiology, University of Arizona.
- Goodrum, F. D. (2012, July). Viral Mechanisms of Human Cytomegalovirus Persistence. American Society of Virology. Madison, WI: American Society of Virology.
- Goodrum, F. D. (2012, November). How many sites of latency are there?. 14th International CMV/Beta Herpesvirus Workshop. San Francisco, CA: International CMV/Beta Herpesvirus Workshop.More infopanel member
- Goodrum, F. D. (2012, November). Novel HCMV Determinants Controlling Replication in Endothelial Cells. 14th International CMV/Beta Herpesvirus Workshop. San Francisco, CA: CMV/Beta Herpesvirus Workshop.
- Goodrum Sterling, F. D. (2011, May). Viral Control of Translation. Gordon Conference on Viruses and Cells. Il Ciocco, Italy: Gordon Research Conferences.
- Goodrum, F. D. (2011, October). Viruses and the Stress Response. 2nd Manipulation of Nuclear Processes by DNA Viruses. Santa Fe, NM.
- Goodrum Sterling, F. D. (2010, July). Questions of Human Cytomegalovirus Coexistence. Inaugural Priscilla A. Schaffer Lectureship. Salt Lake City, UT: International Herpesvirus Workshop.
- Goodrum Sterling, F. D. (2010, November). Viral Determinants of Coexistence. 22nd Annual Kavli Frontiers of Science Symposium. Irvine, CA: National Academy of Sciences.
Poster Presentations
- Goodrum, F. D. (2015, July). Endothelium Tropism Determinants of Human Cytomegalovirus. 40th International Herpesvirus Workshop. Boise, ID: International Herpesvirus Workshop.
Others
- Goodrum Sterling, F. D., & Pottinger, H. L. (2019, February). Everyone Has a Part to Play in Ending Vaccine Hesitancy. The Health Care Blog. https://thehealthcareblog.com/blog/2019/02/12/everyone-has-a-part-to-play-in-ending-vaccine-hesitancy/
- Goodrum Sterling, F. D., & Pottinger, H. L. (2019, March). Vaccine exemptions create problems for states. The Hill. https://thehill.com/opinion/healthcare/435211-vaccine-exemptions-create-problems-for-states