Samuel K Campos

Interests

Research

Virology, Papillomavirus, Cell Biology, Molecular Biology, Biochemistry, Protein Structure, Structural Biology, Protein-Membrane Interactions

Courses

Scholarly Contributions

Journals/Publications

• Li, S., Williamson, Z. L., Christofferson, M. A., Jeevanandam, A., & Campos, S. K. (2024). A Peptide Derived from Sorting Nexin 1 Inhibits HPV16 Entry, Retrograde Trafficking, and L2 Membrane Spanning. bioRxiv : the preprint server for biology.
  More info

  High risk human papillomavirus (HPV) infection is responsible for 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires the viral genome (vDNA) to gain access to nuclei of basal keratinocytes of epithelium. After virion endocytosis, the minor capsid protein L2 dictates the subcellular retrograde trafficking and nuclear localization of the vDNA during mitosis. Prior work identified a cell-permeable peptide termed SNX1.3, derived from the BAR domain of sorting nexin 1 (SNX1), that potently blocks the retrograde and nuclear trafficking of EGFR in triple negative breast cancer cells. Given the importance of EGFR and retrograde trafficking pathways in HPV16 infection, we set forth to study the effects of SNX1.3 within this context. SNX1.3 inhibited HPV16 infection by both delaying virion endocytosis, as well as potently blocking virion retrograde trafficking and Golgi localization. SNX1.3 had no effect on cell proliferation, nor did it affect post-Golgi trafficking of HPV16. Looking more directly at L2 function, SNX1.3 was found to impair membrane spanning of the minor capsid protein. Future work will focus on mechanistic studies of SNX1.3 inhibition, and the role of EGFR signaling and SNX1- mediated endosomal tubulation, cargo sorting, and retrograde trafficking in HPV infection.
• Li, S., Williamson, Z. L., Christofferson, M. A., Jeevanandam, A., & Campos, S. K. (2024). A peptide derived from sorting nexin 1 inhibits HPV16 entry, retrograde trafficking, and L2 membrane spanning. Tumour virus research, 18, 200287.
  More info

  High risk human papillomavirus (HPV) infection is responsible for 99 % of cervical cancers and 5 % of all human cancers worldwide. HPV infection requires the viral genome (vDNA) to gain access to nuclei of basal keratinocytes of epithelium. After virion endocytosis, the minor capsid protein L2 dictates the subcellular retrograde trafficking and nuclear localization of the vDNA during mitosis. Prior work identified a cell-permeable peptide termed SNX1.3, derived from the BAR domain of sorting nexin 1 (SNX1), that potently blocks the retrograde and nuclear trafficking of EGFR in triple negative breast cancer cells. Given the importance of EGFR and retrograde trafficking pathways in HPV16 infection, we set forth to study the effects of SNX1.3 within this context. SNX1.3 inhibited HPV16 infection by both delaying virion endocytosis, as well as potently blocking virion retrograde trafficking and Golgi localization. SNX1.3 had no effect on cell proliferation, nor did it affect post-Golgi trafficking of HPV16. Looking more directly at L2 function, SNX1.3 was found to impair membrane spanning of the minor capsid protein. Future work will focus on mechanistic studies of SNX1.3 inhibition, and the role of EGFR signaling and SNX1-mediated endosomal tubulation, cargo sorting, and retrograde trafficking in HPV infection.
• 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). Correction for Rasmussen et al., "Virology-the path forward". Journal of virology, 98(3), e0007424.
• 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, 98(1), e0179123.
  More info

  In 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.
• Rizzato, M., Mao, F., Chardon, F., Lai, K. Y., Villalonga-Planells, R., Drexler, H. C., Pesenti, M. E., Fiskin, M., Roos, N., King, K. M., Li, S., Gamez, E. R., Greune, L., Dersch, P., Simon, C., Masson, M., Van Doorslaer, K., Campos, S. K., & Schelhaas, M. (2023). Master mitotic kinases regulate viral genome delivery during papillomavirus cell entry. Nature communications, 14(1), 355.
  More info

  Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2's chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.
• Campos, S. K. (2021). Antihypertensive drug treatment and susceptibility to SARS-CoV-2 infection in human PSC-derived cardiomyocytes and primary endothelial cells.. Stem cell reports.
  More info

  The pathogenicity of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been attributed to its ability to enter through the membrane-bound angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, it has been heavily speculated that angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) therapy may modulate SARS-CoV-2 infection. In this study, exposure of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and human endothelial cells (hECs) to SARS-CoV-2 identified significant differences in protein coding genes involved in immunity, viral response, and cardiomyocyte/endothelial structure. Specifically, transcriptome changes were identified in the tumor necrosis factor (TNF), interferon α/β, and mitogen-activated protein kinase (MAPK) (hPSC-CMs) as well as nuclear factor kappa-B (NF-κB) (hECs) signaling pathways. However, pre-treatment of hPSC-CMs or hECs with two widely prescribed antihypertensive medications, losartan and lisinopril, did not affect the susceptibility of either cell type to SARS-CoV-2 infection. These findings demonstrate the toxic effects of SARS-CoV-2 in hPSC-CMs/hECs and, taken together with newly emerging multicenter trials, suggest that antihypertensive drug treatment alone does not alter SARS-CoV-2 infection.
• Campos, S. K. (2021). Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals. Cell Reports.
• Campos, S. K. (2021). Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals.. Cell reports.
  More info

  Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.
• Campos, S. K. (2021). Novel Compounds Targeting Neuropilin Receptor 1 with Potential To Interfere with SARS-CoV-2 Virus Entry.. ACS chemical neuroscience.
  More info

  Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 Spike protein interferes with pain signaling. Here, we report confirmed hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physicochemical properties. Using ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Further, two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.
• Campos, S. K. (2021). Paradoxical effects of cigarette smoke and COPD on SARS-CoV-2 infection and disease.. BMC pulmonary medicine.
  More info

  BackgroundHow cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection and severity is controversial. We investigated the effects of COPD and CS on the expression of SARS-CoV-2 entry receptor ACE2 in vivo in COPD patients and controls and in CS-exposed mice, and the effects of CS on SARS-CoV-2 infection in human bronchial epithelial cells in vitro.MethodsWe quantified: (1) pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and/or TMPRSS2 mRNA levels by RT-qPCR in two independent human cohorts; and (2) pulmonary ACE2 protein levels by immunostaining and ELISA in C57BL/6 WT mice exposed to air or CS for up to 6 months. The effects of CS exposure on SARS-CoV-2 infection were evaluated after in vitro infection of Calu-3 cells and differentiated human bronchial epithelial cells (HBECs), respectively.ResultsACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus controls but similar in central airways. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice. CS treatment decreased viral replication in Calu-3 cells, as determined by immunofluorescence staining for replicative double-stranded RNA (dsRNA) and western blot for viral N protein. Acute CS exposure decreased in vitro SARS-CoV-2 replication in HBECs, as determined by plaque assay and RT-qPCR.ConclusionsACE2 levels were decreased in both bronchial and alveolar epithelial cells from COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-exposure potently inhibited SARS-CoV-2 replication in vitro. These findings urge to investigate further the controversial effects of CS and COPD on SARS-CoV-2 infection.
• Campos, S. K. (2021). The long and winding road: human papillomavirus entry and subcellular trafficking.. Current opinion in virology.
  More info

  Human papillomaviruses (HPVs) infect and replicate in differentiating mucosal and cutaneous epithelium. Most HPV infections are asymptomatic or cause transient benign neoplasia. However, persistent infections by oncogenic HPV types can progress to cancer. During infectious entry into host keratinocytes, HPV particles interact with many host proteins, beginning with major capsid protein L1 binding to cellular heparan sulfate and a series of enzymatic capsid modifications that promote infectious cellular entry. After utilizing the endosomal pathway to uncoat the viral genome (vDNA), the minor capsid protein L2/vDNA complex is retrograde trafficked to the Golgi, and thereafter, to the nucleus where viral transcription initiates. Post-Golgi trafficking is dependent on mitosis, with L2-dependent tethering of vDNA to mitotic chromosomes before accumulation at nuclear substructures in G1. This review summarizes the current knowledge of the HPV entry pathway, the role of cellular proteins in this process, and notes many gaps in our understanding.
• Campos, S. K., Diamond, M. S., Thorne, C. A., Crowe, J. E., Doranz, B. J., Li, S., Davidson, E., Williams, J. K., Trivette, A., Myers, L., Day, S., Rodriguez, J., Sutton, R. E., Binshtein, E., Cabel, C. R., Winkler, E. S., Suryadevara, N., Zost, S. J., Gilchuk, P., & Chen, E. C. (2021). Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals. Cold Spring Harbor Laboratory - bioRxiv. doi:10.1101/2021.05.02.442326
  More info

  ABSTRACT Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo ), and others recognizing non-RBD epitopes that bound the heptad repeat 1 region of the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.
• Chen, E. C., Gilchuk, P., Zost, S. J., Suryadevara, N., Winkler, E. S., Cabel, C. R., Binshtein, E., Chen, R. E., Sutton, R. E., Rodriguez, J., Day, S., Myers, L., Trivette, A., Williams, J. K., Davidson, E., Li, S., Doranz, B. J., Campos, S. K., Carnahan, R. H., , Thorne, C. A., et al. (2021). Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals. Cell reports, 36(8), 109604.
  More info

  Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.
• Iwanski, J., Kazmouz, S. G., Li, S., Stansfield, B., Salem, T. T., Perez-Miller, S., Kazui, T., Jena, L., Uhrlaub, J. L., Lick, S., Nikolich-Žugich, J., Konhilas, J. P., Gregorio, C. C., Khanna, M., Campos, S. K., & Churko, J. M. (2021). Antihypertensive drug treatment and susceptibility to SARS-CoV-2 infection in human PSC-derived cardiomyocytes and primary endothelial cells. Stem cell reports, 16(10), 2459-2472.
  More info

  The pathogenicity of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been attributed to its ability to enter through the membrane-bound angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, it has been heavily speculated that angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) therapy may modulate SARS-CoV-2 infection. In this study, exposure of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and human endothelial cells (hECs) to SARS-CoV-2 identified significant differences in protein coding genes involved in immunity, viral response, and cardiomyocyte/endothelial structure. Specifically, transcriptome changes were identified in the tumor necrosis factor (TNF), interferon α/β, and mitogen-activated protein kinase (MAPK) (hPSC-CMs) as well as nuclear factor kappa-B (NF-κB) (hECs) signaling pathways. However, pre-treatment of hPSC-CMs or hECs with two widely prescribed antihypertensive medications, losartan and lisinopril, did not affect the susceptibility of either cell type to SARS-CoV-2 infection. These findings demonstrate the toxic effects of SARS-CoV-2 in hPSC-CMs/hECs and, taken together with newly emerging multicenter trials, suggest that antihypertensive drug treatment alone does not alter SARS-CoV-2 infection.
• Ozbun, M. A., & Campos, S. K. (2021). The long and winding road: human papillomavirus entry and subcellular trafficking. Current opinion in virology, 50, 76-86.
  More info

  Human papillomaviruses (HPVs) infect and replicate in differentiating mucosal and cutaneous epithelium. Most HPV infections are asymptomatic or cause transient benign neoplasia. However, persistent infections by oncogenic HPV types can progress to cancer. During infectious entry into host keratinocytes, HPV particles interact with many host proteins, beginning with major capsid protein L1 binding to cellular heparan sulfate and a series of enzymatic capsid modifications that promote infectious cellular entry. After utilizing the endosomal pathway to uncoat the viral genome (vDNA), the minor capsid protein L2/vDNA complex is retrograde trafficked to the Golgi, and thereafter, to the nucleus where viral transcription initiates. Post-Golgi trafficking is dependent on mitosis, with L2-dependent tethering of vDNA to mitotic chromosomes before accumulation at nuclear substructures in G1. This review summarizes the current knowledge of the HPV entry pathway, the role of cellular proteins in this process, and notes many gaps in our understanding.
• Thorne, C. A., Perez-miller, S., Patek, M., Moutal, A., Khanna, R., Duran, P., Campos, S. K., & Cabel, C. R. (2021). Novel Compounds Targeting Neuropilin Receptor 1 with Potential To Interfere with SARS-CoV-2 Virus Entry.. ACS chemical neuroscience, 12(8), 1299-1312. doi:10.1021/acschemneuro.0c00619
  More info

  Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 Spike protein interferes with pain signaling. Here, we report confirmed hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physicochemical properties. Using ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Further, two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.
• Tomchaney, M., Contoli, M., Mayo, J., Baraldo, S., Li, S., Cabel, C. R., Bull, D. A., Lick, S., Malo, J., Knoper, S., Kim, S. S., Tram, J., Rojas-Quintero, J., Kraft, M., Ledford, J. G., Tesfaigzi, Y., Martinez, F. D., Thorne, C. A., Kheradmand, F., , Campos, S. K., et al. (2021). Paradoxical effects of cigarette smoke and COPD on SARS-CoV-2 infection and disease. BMC pulmonary medicine, 21(1), 275.
  More info

  How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection and severity is controversial. We investigated the effects of COPD and CS on the expression of SARS-CoV-2 entry receptor ACE2 in vivo in COPD patients and controls and in CS-exposed mice, and the effects of CS on SARS-CoV-2 infection in human bronchial epithelial cells in vitro.
• Campos, S. K. (2020). Attenuation of cGAS/STING activity during mitosis. Life Science Alliance.
• Campos, S. K. (2020). Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease. bioRxiv.
• Campos, S. K. (2020). Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection. PLOS Pathogens.
• Campos, S. K., Li, S., Gamez, E. R., & Uhlorn, B. L. (2020). Attenuation of cGAS/STING Activity During Mitosis. bioRxiv. doi:10.1101/2019.12.19.883090
• Li, S., Campos, S. K., Doorslaer, K. V., Bratton, S. M., Jackson, R. L., & Uhlorn, B. L. (2020). Vesicular Trafficking Permits Evasion of cGAS/STING Surveillance During Initial Human Papillomavirus Infection. bioRxiv. doi:10.1101/2020.03.29.014118
  More info

  Abstract Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection. Importance Persistent infection is the main risk factor for all HPV-associated cancers. However, cellular innate immune pathways exist to detect and limit viral infections. The cGAS/STING pathway senses cytosolic DNA to initiate antiviral IFN responses. Such responses would likely be detrimental towards the establishment of persistent HPV infections. We therefore hypothesize that HPV evades cGAS/STING detection via its unique vesicular trafficking mechanism. Here, we show that indeed HPV is a stealthy virus, capable of infecting keratinocytes with minimal activation of the cGAS/STING pathway. Such evasion is dependent on HPV’s vesicular trafficking, as perturbation of vesicular integrity during infection results in sensing of virions.
• Malo, J., Campos, S. K., Polverino, F., Papi, A., Kheradmand, F., Thorne, C. A., Martinez, F. D., Tesfaigzi, Y., Ledford, J. G., Kraft, M., Rojas, J., Tram, J., Kim, S. S., Knoper, S., Lick, S. D., Bull, D. A., Cabel, C. R., Shuaizhi, L., Baraldo, S., , Mayo, J., et al. (2020). Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease. Cold Spring Harbor Laboratory - bioRxiv. doi:10.1101/2020.12.07.413252
  More info

  How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells.In Cohort 1, ACE2-positive cells were quantified by immunostaining in FFPE sections from both central and peripheral airways. In Cohort 2, we quantified pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and TMPRSS2 mRNA levels by RT-qPCR. In C57BL/6 WT mice exposed to air or CS for up to 6 months, pulmonary ACE2 protein levels were quantified by triple immunofluorescence staining and ELISA. The effects of CS exposure on SARS-CoV-2 infection were evaluated after 72hr in vitro infection of Calu-3 cells. After SARS-CoV-2 infection, the cells were fixed for IF staining with dsRNA-specific J2 monoclonal Ab, and cell lysates were harvested for WB of viral nucleocapsid (N) protein. Supernatants (SN) and cytoplasmic lysates were obtained to measure ACE2 levels by ELISA.In both human cohorts, ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus both smoker and NS controls, but similar in central airways. TMPRSS2 levels were similar across groups. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice exposed to 3 and 6 months of CS. In Calu3 cells in vitro, CS-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72hr.ACE2 levels were decreased in both bronchial and alveolar epithelial cells from uninfected COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-treatment did not affect ACE2 levels but potently inhibited SARS-CoV-2 replication in this in vitro model. These findings urge to further investigate the controversial effects of CS and COPD on SARS-CoV2 infection.
• Moutal, A., Campos, S. K., Khanna, R., Thorne, C. A., Cabel, C. R., Patek, M., & Perez-Miller, S. (2020). In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein. Cold Spring Harbor Laboratory - bioRxiv. doi:10.1101/2020.09.22.308783
  More info

  Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 spike protein interferes with pain signaling. Here, we report hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physico-chemical properties. Using an ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that almost all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.
• Perez-Miller, S., Patek, M., Moutal, A., Cabel, C. R., Thorne, C. A., Campos, S. K., & Khanna, R. (2020). In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein. bioRxiv : the preprint server for biology.
  More info

  Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 spike protein interferes with pain signaling. Here, we report hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physico-chemical properties. Using an ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that almost all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.
• Tomchaney, M., Contoli, M., Mayo, J., Baraldo, S., Shuaizhi, L., Cabel, C. R., Bull, D. A., Lick, S., Malo, J., Knoper, S., Kim, S. S., Tram, J., Rojas-Quintero, J., Kraft, M., Ledford, J., Tesfaigzi, Y., Martinez, F. D., Thorne, C. A., Kheradmand, F., , Campos, S. K., et al. (2020). Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease. bioRxiv : the preprint server for biology.
  More info

  How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells.
• Uhlorn, B. L., Gamez, E. R., Li, S., & Campos, S. K. (2020). Attenuation of cGAS/STING activity during mitosis. Life science alliance, 3(9).
  More info

  The innate immune system recognizes cytosolic DNA associated with microbial infections and cellular stress via the cGAS/STING pathway, leading to activation of phospho-IRF3 and downstream IFN-I and senescence responses. To prevent hyperactivation, cGAS/STING is presumed to be nonresponsive to chromosomal self-DNA during open mitosis, although specific regulatory mechanisms are lacking. Given a role for the Golgi in STING activation, we investigated the state of the cGAS/STING pathway in interphase cells with artificially vesiculated Golgi and in cells arrested in mitosis. We find that whereas cGAS activity is impaired through interaction with mitotic chromosomes, Golgi integrity has little effect on the enzyme's production of cGAMP. In contrast, STING activation in response to either foreign DNA (cGAS-dependent) or exogenous cGAMP is impaired by a vesiculated Golgi. Overall, our data suggest a secondary means for cells to limit potentially harmful cGAS/STING responses during open mitosis via natural Golgi vesiculation.
• Uhlorn, B. L., Jackson, R., Li, S., Bratton, S. M., Van Doorslaer, K., & Campos, S. K. (2020). Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection. PLoS pathogens, 16(11), e1009028.
  More info

  Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.
• Campos, S. K. (2019). Glutathione contributes to efficient post-Golgi trafficking of incoming HPV16 genome. PLOS ONE.
• Campos, S. K., Williams, S. J., Bronnimann, M. P., & Li, S. (2019). Glutathione is Required for Efficient Post-Golgi Trafficking of Incoming HPV16 Genome. bioRxiv. doi:10.1101/655357
  More info

  Abstract Human papillomavirus (HPV) is the most common sexually transmitted pathogen in the United States, causing 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires transport of the viral genome (vDNA) into the nucleus of basal keratinocytes. During this process, minor capsid protein L2 facilitates subcellular retrograde trafficking of the vDNA from endosomes to the Golgi, and accumulation at host chromosomes during mitosis for nuclear retention and localization during interphase. Here we investigated the relationship between cytosolic GSH and HPV16 infection. siRNA knockdown of GSH biosynthetic enzymes results in a partial decrease of HPV16 infection. Likewise, infection of HPV16 in GSH depleted keratinocytes is inefficient, an effect that was not seen with adenoviral vectors. Analysis of trafficking revealed no defects in cellular binding, entry, furin cleavage of L2, or retrograde trafficking of HPV16, but GSH depletion hindered post-Golgi trafficking and translocation, decreasing nuclear accumulation of vDNA. Although precise mechanisms have yet to be defined, this work suggests that GSH is required for a specific post-Golgi trafficking step in HPV16 infection.
• Forte, B. L., Campos, S. K., & Bratton, S. M. (2019). Abstract 2540: Mitotic Golgi vesiculation enables human papillomavirus to evade cGAS/STING. Cancer Research, 79, 2540-2540. doi:10.1158/1538-7445.am2019-2540
  More info

  Oncogenic human papillomaviruses (HPVs) infect and replicate in differentiating mucosal epithelium, causing 5% of cancers worldwide and essentially all cervical cancers. During infection, HPV must traffic viral genome (vDNA) to the nuclei of basal keratinocytes. Minor capsid protein L2 facilitates intracellular transport of vDNA to the Golgi; upon mitosis, the L2-vDNA complex penetrates limiting membranes and localizes to mitotic chromosomes, ensuring infection of both daughter cells. Cytosolic DNA sensor cGAS recognizes cytosolic dsDNA and produces second messenger cGAMP; cGAMP causes activation and relocalization of ER-resident STING to the Golgi, where it recruits TBK1 to phosphorylate and activate IRF3, initiating a type-I IFN response. The cGAS/STING pathway is assumed, though never demonstrated, to be inactive during mitosis to avoid detecting self-DNA. Since the Golgi is a platform for STING/TBK1/IRF3 recruitment and activation, we hypothesize natural Golgi dispersal deactivates cGAS/STING during mitosis. Further, we hypothesize HPV has evolved to traffic to and translocate from the mitotic Golgi as an immunoevasive tactic to avoid detection by cGAS/STING during mitosis. HaCaTs, a human keratinocyte line, were transfected with DNA or infected with HPV pseudovirions and analyzed for cGAS/STING activation. DNA transfection resulted in IRF3 phosphorylation and nuclear translocation, and STING translocation to the Golgi, indicating cGAS/STING activity. Strikingly, chemical disruption of the Golgi ribbon potently blocked IRF3 activation in response to foreign DNA, suggesting Golgi morphology might modulate cGAS/STING activity during mitosis. In accordance, DNA-dependent IRF3 phosphorylation was transiently reduced in synchronized mitotic cells, but chemical impairment of mitotic Golgi vesiculation enabled cGAS/STING activation, even without foreign DNA transfection. Further, HPV infection resulted in minimal IRF3 phosphorylation, indicating HPV can efficiently evade detection during initial infection. To determine if HPV’s unique trafficking enables evasion, we used cationic liposomes to permit premature virion translocation across limiting membranes. Such treatment renders a non-infectious, translocation-defective mutant HPV infectious, yet susceptible to cGAS/STING sensing. Overall, the cGAS/STING pathway may be inactivated by mitotic Golgi dispersal, allowing HPV to evade detection during mitosis. Citation Format: Brittany L. Forte, Shauna M. Bratton, Samuel K. Campos. Mitotic Golgi vesiculation enables human papillomavirus to evade cGAS/STING [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2540.
• Li, S., Bronnimann, M. P., Williams, S. J., & Campos, S. K. (2019). Glutathione contributes to efficient post-Golgi trafficking of incoming HPV16 genome. PloS one, 14(11), e0225496.
  More info

  Human papillomavirus (HPV) is the most common sexually transmitted pathogen in the United States, causing 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires transport of the viral genome (vDNA) into the nucleus of basal keratinocytes. During this process, minor capsid protein L2 facilitates subcellular retrograde trafficking of the vDNA from endosomes to the Golgi, and accumulation at host chromosomes during mitosis for nuclear retention and localization during interphase. Here we investigated the relationship between cellular glutathione (GSH) and HPV16 infection. siRNA knockdown of GSH biosynthetic enzymes results in a partial decrease of HPV16 infection. Likewise, infection of HPV16 in GSH depleted keratinocytes is inefficient, an effect that was not seen with adenoviral vectors. Analysis of trafficking revealed no defects in cellular binding, entry, furin cleavage of L2, or retrograde trafficking of HPV16, but GSH depletion hindered post-Golgi trafficking and translocation, decreasing nuclear accumulation of vDNA. Although precise mechanisms have yet to be defined, this work suggests that GSH is required for a specific post-Golgi trafficking step in HPV16 infection.
• Uhlorn, B. L., Gamez, E. R., & Campos, S. K. (2019). Attenuation of cGAS/STING During Mitosis. bioRxiv. doi:doi: https://doi.org/10.1101/2019.12.19.883090
• Aydin, I., Villalonga-Planells, R., Greune, L., Bronnimann, M. P., Calton, C. M., Becker, M., Lai, K. Y., Campos, S. K., Schmidt, M. A., & Schelhaas, M. (2017). A central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry. PLoS pathogens, 13(5), e1006308.
  More info

  Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE) or L2(IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei.
• Calton, C. M., Bronnimann, M. P., Manson, A. R., Li, S., Chapman, J. A., Suarez-Berumen, M., Williamson, T. R., Molugu, S. K., Bernal, R. A., & Campos, S. K. (2017). Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis. PLoS pathogens, 13(5), e1006200.
  More info

  The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes.
• Campos, S. K. (2017). A central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry.. PLoS pathogens.
  More info

  Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE) or L2(IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei.
• Campos, S. K. (2017). Subcellular Trafficking of the Papillomavirus Genome during Initial Infection: The Remarkable Abilities of Minor Capsid Protein L2. Viruses, 9(12).
  More info

  Since 2012, our understanding of human papillomavirus (HPV) subcellular trafficking has undergone a drastic paradigm shift. Work from multiple laboratories has revealed that HPV has evolved a unique means to deliver its viral genome (vDNA) to the cell nucleus, relying on myriad host cell proteins and processes. The major breakthrough finding from these recent endeavors has been the realization of L2-dependent utilization of cellular sorting factors for the retrograde transport of vDNA away from degradative endo/lysosomal compartments to the Golgi, prior to mitosis-dependent nuclear accumulation of L2/vDNA. An overview of current models of HPV entry, subcellular trafficking, and the role of L2 during initial infection is provided below, highlighting unresolved questions and gaps in knowledge.
• Campos, S. K. (2017). Subcellular Trafficking of the Papillomavirus Genome during Initial Infection: The Remarkable Abilities of Minor Capsid Protein L2.. Viruses.
  More info

  Since 2012, our understanding of human papillomavirus (HPV) subcellular trafficking has undergone a drastic paradigm shift. Work from multiple laboratories has revealed that HPV has evolved a unique means to deliver its viral genome (vDNA) to the cell nucleus, relying on myriad host cell proteins and processes. The major breakthrough finding from these recent endeavors has been the realization of L2-dependent utilization of cellular sorting factors for the retrograde transport of vDNA away from degradative endo/lysosomal compartments to the Golgi, prior to mitosis-dependent nuclear accumulation of L2/vDNA. An overview of current models of HPV entry, subcellular trafficking, and the role of L2 during initial infection is provided below, highlighting unresolved questions and gaps in knowledge.
• Campos, S. K. (2017). Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis. PLOS Pathogens.
• Bronnimann, M. P., Calton, C. M., Chiquette, S. F., Li, S., Lu, M., Chapman, J. A., Bratton, K. N., Schlegel, A. M., & Campos, S. K. (2016). Furin Cleavage of L2 During Papillomavirus Infection: Minimal Dependence on Cyclophilins. Journal of virology.
  More info

  Despite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during Human Papillomavirus Type 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N-terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N-terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurs on both the HaCaT cell surface and ECM. Contrary to current models, experiments on the involvement of cyclophilins revealed little if any role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12 as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues reveal that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage.
• Campos, S. K. (2016). Furin Cleavage of L2 during Papillomavirus Infection: Minimal Dependence on Cyclophilins..
• Campos, S. K. (2016). PhiXing-it, displaying foreign peptides on bacteriophage ΦX174..
• Fane, B. A., Christakos, K. J., Chapman, J. A., & Campos, S. K. (2016). PhiXing-it, displaying foreign peptides on bacteriophage ΦX174.. Virology, 488, 242-8. doi:10.1016/j.virol.2015.11.021
  More info

  Although bacteriophage φX174 is easy to propagate and genetically tractable, it is use as a peptide display platform has not been explored. One region within the φX174 major spike protein G tolerated 13 of 16 assayed insertions, ranging from 10 to 75 amino acids. The recombinant proteins were functional and incorporated into infectious virions. In the folded protein, the peptides would be icosahedrally displayed within loops that extend from the protein׳s β-barrel core. The well-honed genetics of φX174 allowed permissive insertions to be quickly identified by the cellular phenotypes associated with cloned gene expression. The cloned genes were easily transferred from plasmids to phage genomes via recombination rescue. Direct ELISA validated several recombinant virions for epitope display. Some insertions conferred a temperature-sensitive (ts) protein folding defect, which was suppressed by global suppressors in protein G, located too far away from the insertion to directly alter peptide display.
• Christakos, K. J., Chapman, J. A., Fane, B. A., & Campos, S. K. (2015). PhiXing-it, displaying foreign peptides on bacteriophage ΦX174. Virology, 488, 242-248.
  More info

  Although bacteriophage φX174 is easy to propagate and genetically tractable, it is use as a peptide display platform has not been explored. One region within the φX174 major spike protein G tolerated 13 of 16 assayed insertions, ranging from 10 to 75 amino acids. The recombinant proteins were functional and incorporated into infectious virions. In the folded protein, the peptides would be icosahedrally displayed within loops that extend from the protein׳s β-barrel core. The well-honed genetics of φX174 allowed permissive insertions to be quickly identified by the cellular phenotypes associated with cloned gene expression. The cloned genes were easily transferred from plasmids to phage genomes via recombination rescue. Direct ELISA validated several recombinant virions for epitope display. Some insertions conferred a temperature-sensitive (ts) protein folding defect, which was suppressed by global suppressors in protein G, located too far away from the insertion to directly alter peptide display.
• Campos, S. K. (2014). New structural model of adenoviral cement proteins is not yet concrete..
• Campos, S. K. (2014). New structural model of adenoviral cement proteins is not yet concrete.. Proceedings of the National Academy of Sciences of the United States of America, 111(43), E4542-3. doi:10.1073/pnas.1415364111
  More info

  A recent reinterpretation of the refined crystal structure of human adenovirus type 5 (HAdV5) (1) has resulted in the reassignment of minor capsid/cement proteins IX and IIIa. The revised model differs substantially from the prior one, based on recent cryo-EM studies (2). Which model is correct? The answer may affect the assignment of proteins V, VI, and VIII and will affect our understanding of Ad structure and function.
• Campos, S. K. (2013). A transmembrane domain and GxxxG motifs within L2 are essential for papillomavirus infection..
• Campos, S. K. (2013). Human papillomavirus type 16 does not require cathepsin L or B for infection..
• Campos, S. K., Calton, C. M., Schlegel, A. M., & Chapman, J. A. (2013). Human papillomavirus type 16 does not require cathepsin L or B for infection. Journal of General Virology, 94(8), 1865-1869. doi:10.1099/vir.0.053694-0
• Campos, S. K., Park, C. K., Bronnimann, M. P., & Chapman, J. A. (2013). A Transmembrane Domain and GxxxG Motifs within L2 Are Essential for Papillomavirus Infection. Journal of Virology, 87(1), 464-473. doi:10.1128/jvi.01539-12
• Campos, S., Bronnimann, M. P., Chapman, J. A., Park, C. K., & Campos, S. K. (2013). A transmembrane domain and GxxxG motifs within L2 are essential for papillomavirus infection. Journal of virology, 87(1).
  More info

  During cellular invasion, human papillomavirus type 16 (HPV16) must transfer its viral genome (vDNA) across the endosomal membrane prior to its accumulation at nuclear PML bodies for the establishment of infection. After cellular uptake, the capsid likely undergoes pH-dependent disassembly within the endo-/lysosomal compartment, thereby exposing hidden domains in L2 that facilitate membrane penetration of L2/vDNA complexes. In an effort to identify regions of L2 that might physically interact with membranes, we have subjected the L2 sequence to multiple transmembrane (TM) domain prediction algorithms. Here, we describe a conserved TM domain within L2 (residues 45 to 67) and investigate its role in HPV16 infection. In vitro, the predicted TM domain adopts an alpha-helical structure in lipid environments and can function as a real TM domain, although not as efficiently as the bona fide TM domain of PDGFR. An L2 double point mutant renders the TM domain nonfunctional and blocks HPV16 infection by preventing endosomal translocation of vDNA. The TM domain contains three highly conserved GxxxG motifs. These motifs can facilitate homotypic and heterotypic interactions between TM helices, activities that may be important for vDNA translocation. Disruption of some of these GxxxG motifs resulted in noninfectious viruses, indicating a critical role in infection. Using a ToxR-based homo-oligomerization assay, we show a propensity for this TM domain to self-associate in a GxxxG-dependent manner. These data suggest an important role for the self-associating L2 TM domain and the conserved GxxxG motifs in the transfer of vDNA across the endo-/lysosomal membrane.
• Campos, S., Calton, C. M., Schlegel, A. M., Chapman, J. A., & Campos, S. K. (2013). Human papillomavirus type 16 does not require cathepsin L or B for infection. The Journal of general virology, 94(Pt 8).
  More info

  Cathepsin L (CatL) and cathepsin B (CatB) are lysosomal proteases that many viruses utilize for capsid disassembly. We tested whether CatL and CatB are required for infection by human papillomavirus type 16 (HPV16). CatL- and CatB-deficient mouse embryonic fibroblasts had higher levels of infection when compared with wild-type cells. Similar results were obtained in HaCaT keratinocytes treated with CatL- or CatB-specific small interfering RNA. Thus, CatL and CatB are not required for HPV16 infection but instead appear to restrict infection.
• Bergant Marušič, M., Ozbun, M. A., Campos, S. K., Myers, M. P., & Banks, L. (2012). Human papillomavirus L2 facilitates viral escape from late endosomes via sorting nexin 17. Traffic (Copenhagen, Denmark), 13(3), 455-67.
  More info

  The human papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection, but the molecular mechanisms underlying its mode of action remain obscure. Using a proteomic approach, we have identified the adaptor protein, sorting nexin 17 (SNX17) as a strong interacting partner of HPV L2. This interaction occurs through a highly conserved SNX17 consensus binding motif, which is present in the majority of HPV L2 proteins analysed. Using mutants of L2 defective for SNX17 interaction, or siRNA ablation of SNX17 expression, we demonstrate that the interaction between L2 and SNX17 is essential for viral infection. Furthermore, loss of the L2-SNX17 interaction results in enhanced turnover of the L2 protein and decreased stability of the viral capsids, and concomitantly, there is a dramatic decrease in the efficiency with which viral genomes transit to the nucleus. Indeed, using a range of endosomal and lysosomal markers, we show that capsids defective in their capacity to bind SNX17 transit much more rapidly to the lysosomal compartment. These results demonstrate that the L2-SNX17 interaction is essential for viral infection and facilitates the escape of the L2-DNA complex from the late endosomal/lysosomal compartments.
• Campos, S. K. (2012). Human papillomavirus L2 facilitates viral escape from late endosomes via sorting nexin 17..
• Campos, S. K. (2012). No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection..
• Campos, S. K. (2012). Opposing effects of bacitracin on human papillomavirus type 16 infection: enhancement of binding and entry and inhibition of endosomal penetration..
• Campos, S. K., Bergant Marušič, M., Ozbun, M. A., Myers, M. P., & Banks, L. (2012). Human Papillomavirus L2 Facilitates Viral Escape from Late Endosomes via Sorting Nexin 17: HPVL2 and SNX17. Traffic, 13(3), 455-467. doi:10.1111/j.1600-0854.2011.01320.x
• Campos, S. K., Chapman, J. A., & Schlegel, A. M. (2012). A Yeast‐Two Hybrid Screen Against HPV16 Capsid Proteins Indicates Dynactin 6 is a Potential Cellular Factor Involved in HPV16 Infection. The FASEB Journal. doi:10.1096/fasebj.26.1_supplement.800.4
  More info

  Human papillomaviruses (HPVs) infect genital and mucosal epithelium and include strains carrying different levels of risk for the host upon infection, including high-risk strains that are potentially oncogenic such as HPV16. Little is known about the molecular events of HPV host cell invasion. HPV16 particles consist of two structural proteins: the major capsid protein L1 and the minor capsid protein L2. The goal of this project is to identify host cell proteins that interact with the HPV capsid proteins. A high stringency yeast two-hybrid screen was performed to identify possible interactions and yielded >1000 colonies for the L2 bait and no colonies for the L1 bait. L2 clones were analyzed by PCR and sequencing to identify the interacting proteins, which included two clones encoding full-length dynactin-6. Mapping of the interaction site using the yeast two-hybrid screen indicates a probable interaction site within residues 99–171 of dynactin 6. These results indicate that dynactin-6 is a host cell factor involved in HPV16 infection through interaction with the L2 capsid protein. Current work seeks to confirm this interaction and determine the specific role this interaction plays in HPV16 infection. Support for this research comes from the Bio5 Institute (U. of Arizona) and the American Cancer Society, Research Scholar Grant, RSG-117469.
• Campos, S. K., Chapman, J. A., Deymier, M. J., Bronnimann, M. P., & Ozbun, M. A. (2012). Opposing Effects of Bacitracin on Human Papillomavirus Type 16 Infection: Enhancement of Binding and Entry and Inhibition of Endosomal Penetration. Journal of Virology, 86(8), 4169-4181. doi:10.1128/jvi.05493-11
• Campos, S. K., Chapman, J. A., Deymier, M. J., Bronnimann, M. P., & Ozbun, M. A. (2012). Opposing effects of bacitracin on human papillomavirus type 16 infection: enhancement of binding and entry and inhibition of endosomal penetration. Journal of virology, 86(8), 4169-81.
  More info

  Cell invasion by human papillomavirus type 16 (HPV16) is a complex process relying on multiple host cell factors. Here we describe an investigation into the role of cellular protein disulfide isomerases (PDIs) by studying the effects of the commonly used PDI inhibitor bacitracin on HPV16 infection. Bacitracin caused an unusual time-dependent opposing effect on viral infection. Enhanced cellular binding and entry were observed at early times of infection, while inhibition was observed at later times postentry. Bacitracin was rapidly taken up by host cells and colocalized with HPV16 at late times of infection. Bacitracin had no deleterious effect on HPV16 entry, capsid disassembly, exposure of L1/L2 epitopes, or lysosomal trafficking but caused a stark inhibition of L2/viral DNA (vDNA) endosomal penetration and accumulation at nuclear PML bodies. γ-Secretase has recently been implicated in the endosomal penetration of L2/vDNA, but bacitracin had no effect on γ-secretase activity, indicating that blockage of this step occurs through a γ-secretase-independent mechanism. Transient treatment with the reductant β-mercaptoethanol (β-ME) was able to partially rescue the virus from bacitracin, suggesting the involvement of a cellular reductase activity in HPV16 infection. Small interfering RNA (siRNA) knockdown of cellular PDI and the related PDI family members ERp57 and ERp72 reveals a potential role for PDI and ERp72 in HPV infection.
• Frietze, K. M., Campos, S. K., & Kajon, A. E. (2012). No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection. BMC research notes, 5, 429.
  More info

  Subspecies B1 human adenoviruses (HAdV-B1) are prevalent respiratory pathogens. Compared to their species C (HAdV-C) counterparts, relatively little work has been devoted to the characterization of their unique molecular biology. The early region 3 (E3) transcription unit is an interesting target for future efforts because of its species-specific diversity in genetic content among adenoviruses. This diversity is particularly significant for the subset of E3-encoded products that are membrane glycoproteins and may account for the distinct pathobiology of the different human adenovirus species. In order to understand the role of HAdV-B-specific genes in viral pathogenesis, we initiated the characterization of unique E3 genes. As a continuation of our efforts to define the function encoded in the highly polymorphic ORF E3-10.9K and testing the hypothesis that the E3-10.9K protein orthologs with a hydrophobic domain contribute to the efficient release of viral progeny, we generated HAdV-3 mutant viruses unable to express E3-10.9K ortholog E3-9K and examined their ability to grow, disseminate, and egress in cell culture.
• Campos, S. K. (2010). Open reading frame E3-10.9K of subspecies B1 human adenoviruses encodes a family of late orthologous proteins that vary in their predicted structural features and subcellular localization..
• Campos, S. K., Frietze, K. M., & Kajon, A. E. (2010). Open Reading Frame E3-10.9K of Subspecies B1 Human Adenoviruses Encodes a Family of Late Orthologous Proteins That Vary in Their Predicted Structural Features and Subcellular Localization. Journal of Virology, 84(21), 11310-11322. doi:10.1128/jvi.00512-10
• Frietze, K. M., Campos, S. K., & Kajon, A. E. (2010). Open reading frame E3-10.9K of subspecies B1 human adenoviruses encodes a family of late orthologous proteins that vary in their predicted structural features and subcellular localization. Journal of virology, 84(21), 11310-22.
  More info

  Subspecies B1 human adenoviruses (HAdV-B1s) are important causative agents of acute respiratory disease, but the molecular bases of their distinct pathobiology are still poorly understood. Marked differences in genetic content between HAdV-B1s and the well-characterized HAdV-Cs that may contribute to distinct pathogenic properties map to the E3 region. Between the highly conserved E3-19K and E3-10.4K/RIDα open reading frames (ORFs), and in the same location as the HAdV-C ADP/E3-11.6K ORF, HAdV-B1s carry ORFs E3-20.1K and E3-20.5K and a polymorphic third ORF, designated E3-10.9K, that varies in the size of its predicted product among HAdV-B1 serotypes and genomic variants. As an initial effort to define the function of the E3-10.9K ORF, we carried out a biochemical characterization of E3-10.9K-encoded orthologous proteins and investigated their expression in infected cells. Sequence-based predictions suggested that E3-10.9K orthologs with a hydrophobic domain are integral membrane proteins. Ectopically expressed, C-terminally tagged (with enhanced green fluorescent protein [EGFP]) E3-10.9K and E3-9K localized primarily to the plasma membrane, while E3-7.7K localized primarily to a juxtanuclear compartment that could not be identified. EGFP fusion proteins with a hydrophobic domain were N and O glycosylated. EGFP-tagged E3-4.8K, which lacked the hydrophobic domain, displayed diffuse cellular localization similar to that of the EGFP control. E3-10.9K transcripts from the major late promoter were detected at late time points postinfection. A C-terminally hemagglutinin-tagged version of E3-9K was detected by immunoprecipitation at late times postinfection in the membrane fraction of mutant virus-infected cells. These data suggest a role for ORF E3-10.9K-encoded proteins at late stages of HAdV-B1 replication, with potentially important functional implications for the documented ORF polymorphism.
• Campos, S. K. (2009). Two highly conserved cysteine residues in HPV16 L2 form an intramolecular disulfide bond and are critical for infectivity in human keratinocytes..
• Campos, S. K., & Ozbun, M. A. (2009). Two highly conserved cysteine residues in HPV16 L2 form an intramolecular disulfide bond and are critical for infectivity in human keratinocytes. PloS one, 4(2), e4463.
  More info

  Minor capsid protein L2 performs an indispensable but uncharacterized role in human papillomavirus infections. A neutralizing B cell epitope has recently been mapped to the N-terminus of HPV16 L2, residues 17-36, and exposure of this region of L2 has been implicated in translocation of incoming virions from the endo/lysosomal compartment to the cellular cytoplasm. Here we examine the redox state of Cys22 and Cys28 two highly conserved cysteines located within this epitope. We also investigate the infectivity of virions containing L2 single and double cysteine point mutants.
• Campos, S. K. (2008). Caveolin-1-dependent infectious entry of human papillomavirus type 31 in human keratinocytes proceeds to the endosomal pathway for pH-dependent uncoating..
• Smith, J. L., Campos, S. K., Wandinger-Ness, A., & Ozbun, M. A. (2008). Caveolin-1-dependent infectious entry of human papillomavirus type 31 in human keratinocytes proceeds to the endosomal pathway for pH-dependent uncoating. Journal of virology, 82(19), 9505-12.
  More info

  High-risk human papillomaviruses (HPVs) are small nonenveloped DNA viruses with a strict tropism for squamous epithelium. The viruses are causative agents of cervical cancer and some head and neck cancers, but their differentiation-dependent life cycles have made them difficult to study in simple cell culture. Thus, many aspects of early HPV infection remain mysterious. We recently showed the high-risk HPV type 31 (HPV31) enters its natural host cell type via caveola-dependent endocytosis, a distinct mechanism from that of the closely related HPV16 (Smith et al., J. Virol. 81:9922-9931, 2007). Here, we determined the downstream trafficking events after caveolar entry of HPV31 into human keratinocytes. After initial plasma membrane binding, HPV31 associates with caveolin-1 and transiently localizes to the caveosome before trafficking to the early endosome and proceeding through the endosomal pathway. Caveosome-to-endosome transport was found to be Rab5 GTPase dependent. Although HPV31 capsids were observed in the lysosome, Rab7 GTPase was dispensable for HPV31 infection, suggesting that viral genomes escape from the endosomal pathway prior to Rab7-mediated capsid transport. Consistent with this, the acidic pH encountered by HPV31 within the early endosomal pathway induces a conformational change in the capsid resulting in increased DNase susceptibility of the viral genome, which likely aids in uncoating and/or endosomal escape. The entry and trafficking route of HPV31 into human keratinocytes represents a unique viral pathway by which the virions use caveolar entry to eventually access a low-pH site that appears to facilitate endosomal escape of genomes.
• Campos, S. K. (2007). Current advances and future challenges in Adenoviral vector biology and targeting..
• Campos, S. K. (2007). Human papillomavirus type 31 uses a caveolin 1- and dynamin 2-mediated entry pathway for infection of human keratinocytes..
• Campos, S. K. (2007). The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy..
• Campos, S. K., & Barry, M. A. (2007). Current advances and future challenges in Adenoviral vector biology and targeting. Current gene therapy, 7(3), 189-204.
  More info

  Gene delivery vectors based on Adenoviral (Ad) vectors have enormous potential for the treatment of both hereditary and acquired disease. Detailed structural analysis of the Ad virion, combined with functional studies has broadened our knowledge of the structure/function relationships between Ad vectors and host cells/tissues and substantial achievement has been made towards a thorough understanding of the biology of Ad vectors. The widespread use of Ad vectors for clinical gene therapy is compromised by their inherent immunogenicity. The generation of safer and more effective Ad vectors, targeted to the site of disease, has therefore become a great ambition in the field of Ad vector development. This review provides a synopsis of the structure/function relationships between Ad vectors and host systems and summarizes the many innovative approaches towards achieving Ad vector targeting.
• Smith, J. L., Campos, S. K., & Ozbun, M. A. (2007). Human papillomavirus type 31 uses a caveolin 1- and dynamin 2-mediated entry pathway for infection of human keratinocytes. Journal of virology, 81(18), 9922-31.
  More info

  Papillomaviruses are species-specific and epitheliotropic DNA viruses that cause tumors in their natural hosts. Certain infections with genital human papillomavirus (HPV) types are causally related to cervical cancer development. Most papillomaviruses are thought to infect cells via a clathrin-dependent pathway, yet no studies have determined the entry route in permissive host epithelial cells. Employing fluorescently labeled and native virions, we tested the effects of dominant-negative and biochemical inhibitors of cellular endocytosis pathways. Infections of human keratinocytes, a natural host cell type for HPVs, were assessed visually and by infectious entry assays. We found that HPV type 31 (HPV31) entry and initiation of early infection events require both caveolin 1 and dynamin 2 and occur independently of clathrin-mediated endocytosis. Treatment with chlorpromazine and filipin had opposing effects on HPV31 and HPV16 infection. HPV31 entry was remarkably slow, with a half-time of approximately 14 h, whereas the entry half-time of HPV16 was 4 h. Consistent with a caveola-mediated entry pathway for HPV31, the virions associated with detergent-resistant lipid rafts. During a 16-h microscopic tracking of HPV31 and HPV16 virions, no colocalization of the two viral types was observed. These data suggest that HPV31 and HPV16 virions use distinct routes for host epithelial cell entry.
• Wu, Y., Campos, S. K., Lopez, G. P., Ozbun, M. A., Sklar, L. A., & Buranda, T. (2007). The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy. Analytical biochemistry, 364(2), 180-92.
  More info

  The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.
• Campos, S. K. (2006). Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting..
• Campos, S. K. (2006). Cryoelectron microscopy of protein IX-modified adenoviruses suggests a new position for the C terminus of protein IX..
• Campos, S. K., & Barry, M. A. (2006). Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting. Virology, 349(2), 453-62.
  More info

  The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon.
• Marsh, M. P., Campos, S. K., Baker, M. L., Chen, C. Y., Chiu, W., & Barry, M. A. (2006). Cryoelectron microscopy of protein IX-modified adenoviruses suggests a new position for the C terminus of protein IX. Journal of virology, 80(23), 11881-6.
  More info

  Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.
• Campos, S. K., & Barry, M. A. (2005). 29. Redirection of Adenoviral Tropism through Capsomers Other Than Fiber Is Generally Inefficient and Dependent on Ligand-Receptor Biology. Molecular Therapy, 11, S12. doi:10.1016/j.ymthe.2005.06.031
  More info

  We have generated a panel of metabolically biotinylated adenoviral vectors based on the genetic insertion of a biotin acceptor peptide (BAP) into the fiber, protein IX, and hexon capsomers of the Ad5 virion. Here we demonstrate that avidin-based redirection of Ad transduction through biotinylated ligand:receptor interactions is greatly dependent on the nature of the BAP-modified capsomer. While transduction of Ad-Fiber-BAP can generally be targeted through a wide range of ligand:receptor pairs, Ad-IX-BAP and Ad-Hexon-BAP are surprisingly inefficient as targeting platforms, despite having a higher display of biotins than Ad-Fiber-BAP. We reason this difference is due to the nature of the interactions between viral capsomers and cell surface receptors. Ad5 fiber, the natural cell-binding capsomer, has evolved to bind very tightly to cell surface CAR. Upon endosomal uptake fibers then dissociate from the capsid, releasing the virion from the receptor and enabling trafficking to continue. We believe that targeting Ad through IX or hexon capsomers results in the entrapment of virions on cellular receptors thereby leading to very inefficient transduction. Indeed, preliminary evidence suggests that protein IX stays tightly associated with the virion and does not dissociate at early stages of vector trafficking like fiber. We reason that targeted transduction through IX or hexon may be feasible with ligands that naturally release from their receptors upon endocytosis, resulting in release of the incoming virions. These initial results highlight the importance of knowledge of capsomer, ligand, and receptor biology when engineering new tropism into viral capsids and may be of great importance for vectors like AAV, which do not shed capsomers as they traffic. Current progress on the analysis of the differences in trafficking between the different BAP-modified vectors will be presented.
• Campos, S. K. (2004). Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector..
• Campos, S. K. (2004). Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination..
• Campos, S. K., & Barry, M. A. (2004). Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination. Human gene therapy, 15(11), 1125-30.
  More info

  There are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means. Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome. We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes.
• Campos, S. K., Parrott, M. B., & Barry, M. A. (2004). Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector. Molecular therapy : the journal of the American Society of Gene Therapy, 9(6), 942-54.
  More info

  While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.
• Parrott, M. B., March, M., Chiu, W., Campos, S. K., & Barry, M. A. (2004). 1017. Metabolically Biotinylated Viruses for Vector Targeting, Virus Purification, and Capsid Imaging. Molecular Therapy, 9, S390. doi:10.1016/j.ymthe.2004.06.962
  More info

  While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have demonstrated utility as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high affinity tetrameric avidin (Kd = 10-15 M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (Kd = 10-7 M). In this report, a second metabolically-biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors. Finally, we have recently used pIX-BAP as a structural tag to image the location of pIX on adenoviral virions by cryo-EM. By this approach, we demonstrate that previous reports of pIX's capsid localization were incorrect and that tis has implications for the use of pIX for vector targeting. Therefore, metabolic biotinylation has utility for vector targeting, purification, ligand-receptor screening, and capsid characterization.
• Barry, M. A., Campos, S. K., Ghosh, D., Adams, K. E., Mok, H., Mercier, G. T., & Parrott, M. B. (2003). Biotinylated gene therapy vectors. Expert opinion on biological therapy, 3(6), 925-40.
  More info

  The avidin-biotin system is a fundamental technology in biomedicine for immunolocalisation, imaging, nucleic acid blotting and protein labelling. This technology has recently been adapted for use in gene therapy vector applications to add proteins or cell-targeting ligands to non-viral and viral vectors. Two biotinylation technologies are being used in these applications: chemical biotinylation and metabolic biotinylation. In chemical biotinylation, reactive alkylating agents couple biotin to proteins by random covalent attachment to amino acid side chains. In metabolic biotinylation, proteins are genetically engineered with a biotin acceptor peptide (BAP), such that they are covalently biotinylated by cellular biotin ligases during viral vector production. Both technologies show promise for cell-targeting in vitro and in vivo, and for ligand screening applications. Metabolic biotinylation has the added feature of allowing viruses, vectors and vaccines to be produced from cells already biotinylated, thereby allowing them to purified by affinity chromatography on monomeric avidin columns.
• Campos, S. K. (2003). Biotinylated gene therapy vectors..
• Campos, S. K. (2003). Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification..
• Campos, S. K., Ghosh, D., Adams, K. E., Parrott, M. B., & Barry, M. A. (2003). Metabolic biotinylation of proteins and viral vectors for purification from mammalian cells. Abstracts of Papers American Chemical Society.
• Parrott, M. B., Adams, K. E., Mercier, G. T., Mok, H., Campos, S. K., & Barry, M. A. (2003). Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification. Molecular therapy : the journal of the American Society of Gene Therapy, 8(4), 688-700.
  More info

  Development of cell-targeting vectors is an important focus for gene therapy. While some ligands can be genetically inserted into virus capsid proteins for cell targeting, for many ligands, this approach can disrupt either ligand function or vector function. To address this problem for adenovirus type 5 vectors, the fiber capsid protein was genetically fused to a biotin acceptor peptide (BAP). Adenovirus particles bearing this BAP were metabolically biotinylated during vector production by the endogenous biotin ligase in 293 cells to produce covalently biotinylated virions. The resulting biotinylated vector could be retargeted to new receptors by conjugation to biotinylated antibodies using tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). Finally, this vector was used as a ligand screening platform for dendritic cells in which a variety of structurally diverse protein, carbohydrate, and nucleic acid ligands were easily added to the vector using the biotin-avidin interaction. This work demonstrates the utility of metabolically biotinylated viruses for ligand screening, vector targeting, and virus purification applications.

Presentations

• Campos, S. K. (2023, June).

  “Runnin’ Down a Dream or Everyday is a Winding Road- Research Trajectories & Papillomavirus Trafficking”

  . 2023 Professional Development & BIPOC Seminar Series (Virtual). virtual: National Summer Undergraduate Research Project (NSURP).
  More info

  research and professional development seminar
• Campos, S. K. (2023, June). Slipping Across Membranes- Lessons from a Sneaky Capsid Protein. American Society for Virology 42nd Annual Meeting. Athens, GA: American Society for Virology.
  More info

  State-of-the-Art lecture, invited plenary talk
• Campos, S. K. (2023, March). “Go Your Own Way - Research Trajectories & Papillomavirus Trafficking”. Spring 2023 Omicron Seminar Series. virtual: California State University Chico, Dept. of Biological Sciences.
  More info

  Departmental Seminar
• Campos, S. K. (2023, October). “Mechanisms and Consequences of Papillomavirus Subcellular Trafficking”. Cancer Biology GIDP Seminar. UA campus: Arizona Cancer Center (AZCC), Cancer Biology GIDP.
  More info

  departmental seminar
• Campos, S. K. (2022, September). "Easing off the cGAS- HPV’s Strategies Towards a Persistent Lifestyle?". 2022 Seminar Series. Ann Arbor: University of Michigan, Dept. of Microbiology & Immunology.
• Campos, S. K., & Tobey, I. (2022, July). "HPV Inhibition of the cGAS/STING Pathway". DNA Tumor Virus Meeting. Cambridge, UK.
• Campos, S. K. (2021, July). Mechanisms and Consequences of HPV Trafficking- the Role of Minor Capsid Protein L2. University Hospital of Tubingen, Dept. of Medical Virology, Departmental Seminar. Tubingen, Germany (Virtual): University Hospital of Tubingen, Dept. of Medical Virology.
• Campos, S. K. (2021, July). The Long and Winding Road- Research & Papillomavirus Trafficking. NSURP 2021 BIPOC Seminar Series. Virtual: National Summer Undergraduate Research Project (NSURP).
• Campos, S. K. (2021, June). A Short Amphipathic Helix in Human Papillomavirus Minor Capsid Protein L2 is Essential for Subcellular Trafficking of Viral DNA. American Society for Virology 40th Annual Meeting. Virtual: American Society for Virology.
• Campos, S. K. (2021, November). HPV16 Pseudovirus Infection is Blocked by a Peptide Derived from Sorting Nexin-1. 34th International Papillomavirus Conference. Virtual: International Papillomavirus Society (IPVS).
• Campos, S. K. (2021, October). Interplay of HPV and the cGAS/STING Pathway- Strategies Towards a Persistent Lifestyle?. Ohio State University, Dept. of Microbial Infection and Immunity 2021 Lecture Seminar Series “Current Topics in Virus-Host Interactions. Ohio State University, Colombus, OH (Virtual): Ohio State University (Virtual), Dept. of Microbial Infection and Immunity.
• Campos, S. K. (2020, August). "Slick Moves by HPV to Avoid the STING of IFN". University of New Mexico, Dept. of Molecular Genetics & Microbiology, Annual Departmental Retreat. Albuquerque, NM (online via Zoom): University of New Mexico, Dept. of Molecular Genetics & Microbiology.
  More info

  Invited Speaker, University of New Mexico, Dept. of Molecular Genetics & Microbiology, Annual Departmental Retreat
• Campos, S. K. (2020, March). "How Does the Papillomavirus Minor Capsid Protein Insert into Membranes". 15th Annual Frontiers in Immunobiology and Immunopathogenesis Symposium. Tucson, AZ: UA, Department of Immunobiology.
  More info

  Invited Talk, Annual Symposium
• Campos, S. K. (2019, Spring). Taking the STING out of persistent HPV infections. Departmental Seminar, Microbiology & Immunology, U. of Iowa. Iowa City: University of Iowa, Department of Microbiology & Immunology.
• Campos, S. K., & Li, S. (2019, Summer). NMR Structural Studies of Papillomavirus L2 Reveals an Essential Amphipathic Helix. ICGEB DNA Tumour Virus Meeting. Trieste, Italy: ICGEB.
• Uhlorn, B. L., & Campos, S. K. (2019, Spring). Human Papillomavirus Exploits Mitotic Golgi Vesiculation to Evade Innate Immune Detection. 14th Annual Frontiers in Immunobiology and Immunopathogenesis Symposium. Tucson, AZ: Department of Immunobiology, U. of Arizona.
• Campos, S. K. (2018, August). Taking the STING out of Persistent HPV Infections. Arizona Cancer Center (AZCC), Cancer Biology Program Seminar. UA-COM, Tucson: UACC, CBIO-GIDP.
• Campos, S. K., & Forte, B. L. (2018, July). Sneaking Past cGAS/STING: Pathway Regulation by Mitotic Golgi Dispersal. DNA Tumor Virus Meeting. Madison, WI.
• Campos, S. K., & Li, S. (2018, July). Furin-Dependent Oligomerization of L2. DNA Tumor Virus Meeting. Madison, WI.
• Riggs, M. W., Vedantam, G., Campos, S. K., Viswanathan, V. K., Riggs, M. W., Vedantam, G., Campos, S. K., & Viswanathan, V. K. (2018, May). Dissection of the Contributions of Enteropathogenic Escherichia Coli Secreted Effector Protein ESPZ to Virulence. Digestive Diseases Week. Washington DC: American Gastroenterological Association.
  More info

  Invited oral presentation for DDW conference
• Campos, S. K. (2017, June). Translocation of the incoming papillomavirus L2/vDNA complex across the limiting membrane requires mitosis. American Society for Virology Annual Meeting. Madison, WI: American Society for Virology.
• Campos, S. K. (2017, November). Evasion of cGAS/STING sensing during initial HPV infection via a unique virion trafficking pathway. 1st Annual Joint Virology Symposium. Tempe, AZ: ASU/Biodesign Institute.
• Campos, S. K. (2017, September). Progress Towards Understanding the Remarkable Abilities of the Papillomavirus Minor Capsid Protein. UA Immunobiology departmental Tenure Talk/Microlunch. Tucson, AZ: UA Dept. of Immunobiology.
• Campos, S. K. (2017, September). Subversion of cGAS/STING surveillance through a unique trafficking pathway. Invited Seminar, Virginia Tech Dept. of Biological Sciences. Blacksburg, VA: Virginia Polytechnic University & State University, Dept. of Biological Sciences.
• Campos, S. K., & Forte, B. L. (2017, April). Human papillomavirus evades innate immune system detection by the cGAS/STING pathway during mitosis-dependent infection. American Society for Microbiology AZ/Southern NV 56th Annual Regional Meeting. Tucson, AZ: American Society for Microbiology.
• Campos, S. K., & Forte, B. L. (2017, December). Papillomavirus subversion of cGAS/STING sensing via unique mitosis- dependent subcellular trafficking of viral genome. 4th ASM Conference on Viral Manipulation of Nuclear Processes. Charleston, SC: American Society for Microbiology.
• Campos, S. K., & Forte, B. L. (2017, June). Human Papillomavirus may exploit Golgi vesiculation to evade the cGAS/STING pathway during mitosis-dependent infection. American Society for Virology Annual Meeting. Madison, WI: American Society for Virology.
• Campos, S. K. (2016, July 20). Spanning and Penetration of the Limiting Membrane by the L2 Capsid Protein. DNA Tumor Virus Meeting. Montreal, Quebec.
• Bronnimann, M. P., Williamson, T., Berumen, M., Calton, C. M., & Campos, S. K. (2015, July). Furin Cleavage Facilitates the Cytoplasmic Translocation and Oligomerization of the Human Papillomavirus Minor Capsid Protein L2. ICGEB DNA Tumor Virus Meeting. Trieste, Italy: International Centre for Genetic Engineering and Biotechnology.
  More info

  Poster Presentation
• Calton, C. M., Bronnimann, M. P., Chapman, J. A., Berumen, M., Williamson, T., Manson, A., & Campos, S. K. (2015, July). A Role for Mitosis in the Membrane Translocation of the HPV16 L2/Genome Complex. ICGEB DNA Tumor Virus Meeting. Trieste, Italy: International Centre for Genetic Engineering and Biotechnology.
  More info

  Oral Presentatiobn
• Campos, S. K. (2015, October). Furin cleavage has conserved roles for human papillomavirus type 16 and bacterial toxin entry pathways. Invited Seminar, UTEP Dept. of Chemistry and Biochemistry. El Paso, TX: University of Texas at El Paso, Department of Chemistry and Biochemistry.

Poster Presentations

• Campos, S. K. (2017, April). Mitosis is required for penetration of the limiting membrane during human papillomavirus infection. American Society for Microbiology Southern AZ/NV Regional Meeting. Tucson, AZ: American Society for Microbiology.

Others

• Campos, S. K. (2020, March). Vesicular Trafficking Permits Evasion of cGAS/STING Surveillance During Initial Human Papillomavirus Infection. https://doi.org/10.1101/2020.03.29.014118
• Campos, S. K. (2020, September). In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein. https://doi.org/10.1101/2020.09.22.308783
• Campos, S. K. (2019, December). Attenuation of cGAS/STING Activity During Mitosis. https://doi.org/10.1101/2019.12.19.883090
• Campos, S. K. (2014). New structural model of adenoviral cement proteins is not yet concrete. Proceedings of the National Academy of Sciences of the United States of America.