John G Purdy

Interests

Research

All viruses hijack host cell machinery to facilitate their replication. Producing infectious viral progeny relies on host metabolic pathways to provide energy and building blocks such as nucleotides, amino acids, and lipids. I investigate the molecular remodeling of metabolic pathways by viruses with the overall goal of dissecting the complex virus-host metabolism interactions to guide the development of novel antiviral therapies.

Courses

Scholarly Contributions

Chapters

• Alwine, J. C., & Purdy, J. G. (2023). Metabolism and Viral Infections. In Fields Virology.

Journals/Publications

• Rasmussen, A. L., Gronvall, G. K., Lowen, A. C., Goodrum, F., Alwine, J., Andersen, K. G., Anthony, S. J., Baines, J., Banerjee, A., Broadbent, A. J., Brooke, C. B., Campos, S. K., Caposio, P., Casadevall, A., Chan, G. C., Cliffe, A. R., Collins-McMillen, D., Connell, N., Damania, B., , Daugherty, M. D., et al. (2024). Virology-the path forward. Journal of virology, 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.
• Ganji, R., Paulo, J. A., Xi, Y., Kline, I., Zhu, J., Clemen, C. S., Weihl, C. C., Purdy, J. G., Gygi, S. P., & Raman, M. (2023). The p97-UBXD8 complex regulates ER-Mitochondria contact sites by altering membrane lipid saturation and composition. Nature communications, 14(1), 638.
  More info

  The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-Mitochondria contact sites (ERMCS) is a platform for critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the impact of altered contacts on lipid metabolism remains poorly understood. We show that the p97 AAA-ATPase and its adaptor ubiquitin-X domain adaptor 8 (UBXD8) regulate ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in membrane lipid saturation upon UBXD8 deletion. Loss of p97-UBXD8 increased membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts can be rescued by unsaturated fatty acids or overexpression of SCD1. We find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. We propose that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation.
• Purdy, J., Griffante, G., Hewelt-Belka, W., Albano, C., Gugliesi, F., Pasquero, S., Castillo Pacheco, S. F., Bajetto, G., Porporato, P. E., Mina, E., Vallino, M., Krapp, C., Jakobsen, M. R., von Einem, J., Landolfo, S., Dell’Oste, V., & Biolatti, M. (2022). IFI16 Impacts Metabolic Reprogramming during Human Cytomegalovirus Infection. mBio, 13(3). doi:10.1128/mbio.00435-22
• Purdy, J. G., Slepian, M. J., Bluestein, D., Shariff, J., Saavedra, S. S., Teran Arce, P. F., Roka-Moiia, Y., Wise, L. M., & Sweedo, A. (2021). Shear-Mediated Platelet Activation is Accompanied by Unique Alterations of Platelet Lipid Profile. Cellular and Molecular BioEngineering, 14, 597–612. doi:DOI: 10.1007/s12195-021-00692-x
  More info

  RATIONALE: Platelet activation by mechanical means such as shear stress, is a vital driver of thrombotic risk in implantable blood-contacting devices. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical – shear mediated platelet activation.OBJECTIVE: To determine if shear-activation of platelets induced lipidome changes that differ from those associated with biochemically-mediated platelet activation.METHODS AND RESULTS: We performed high-resolution lipidomic analysis onpurified platelets from four healthy human donors. For each donor, we compared the lipidome of platelets that were non-activated or activated by shear, ADP, or thrombin treatment. We found that shear activation altered cell-associated lipids and led to the release of lipids into the extracellular environment. Shear-activated platelets released 21 phospholipids and sphingomyelins at levels statistically higher than platelets activated by biochemical stimulation. Many of the released phospholipids contained anarachidonic acid tail or were phosphatidylserine lipids, which have procoagulant properties.CONCLUSIONS: Shear-mediated activation of platelets alters the basal plateletlipidome. Further, these alterations differ and are unique in comparison to the lipidome of biochemically activated platelets. Our findings suggest that lipids released by shear activated platelets may contribute to altered thrombosis in patients with implanted cardiovascular therapeutic devices.
• Sweedo, A., Wise, L. M., Roka-Moiia, Y., Arce, F. T., Saavedra, S. S., Sheriff, J., Bluestein, D., Slepian, M. J., & Purdy, J. G. (2021). Shear-Mediated Platelet Activation is Accompanied by Unique Alterations in Platelet Release of Lipids. Cellular and molecular bioengineering, 14(6), 597-612.
  More info

  Platelet activation by mechanical means such as shear stress exposure, is a vital driver of thrombotic risk in implantable blood-contacting devices used in the treatment of heart failure. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical shear-mediated platelet activation.
• Wise, L. M., Xi, Y., & Purdy, J. G. (2021). Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis. mBio, 12(2).
  More info

  Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling. Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.
• Xi, Y., Lindenmayer, L., Kline, I., von Einem, J., & Purdy, J. G. (2021). Human Cytomegalovirus Uses a Host Stress Response To Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very-Long-Chain Fatty Acids. mBio, 12(3).
  More info

  Stress and virus infection regulate lipid metabolism. Human cytomegalovirus (HCMV) infection induces fatty acid (FA) elongation and increases the abundance of lipids with very-long-chain FA (VLCFA) tails. While reprogramming of metabolism can be stress related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knock out protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. In HCMV-infected cells, PERK promotes increases in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFA tails. Further, PERK enhances FA elongase 7 (ELOVL7) protein levels, which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection were independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Additionally, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK-and, more broadly, ER stress-may be necessary for the membrane biogenesis needed to generate infectious HCMV virions. HCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator PERK controls FA elongation and the cellular abundance of several types of lipids following HCMV infection. Specifically, PERK promotes FA elongase 7 synthesis and phospholipids with saturated/monounsaturated very-long-chain FA tails. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.
• Sweedo, A., Slepian, M. J., Wise, L. M., Sweedo, A., Slepian, M. J., Sheriff, J., Purdy, J. G., & Bluestein, D. (2020). MCS Hypershear Modulates Platelet Membrane Fluidity, Lipid Species, and is Gender Specific.. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 39(4S), S147. doi:10.1016/j.healun.2020.01.1075
  More info

  Shear-mediated platelet activation (SMPA) is a main driver of MCS thrombosis. A target of MCS shear, driving activation, is the platelet membrane - leading to potential alteration of biophysical properties. Here, we examined the effect of shear stress-accumulation on platelet membrane fluidity and on platelet lipid species profile - both retained (membrane) and free (ejected) post-activation..Human gel-filtered platelets (20,000 plts/uL) were exposed to shear in a hemodynamic shearing device (70 dynes/cm2 x 10'); or to thrombin (1U/mL x 10') as a chemical agonist comparator. Membrane fluidity was determined by ໿fluorescence anisotropy of the probe TMA-DPH (1uM, 5' @ 37C). Platelet lipids - both retained (cellular) and free (ejected) following activation - were extracted and analyzed via high resolution liquid-chromatography tandem mass spectrometry (LC-MS/MS)..Shear stress accumulation increases platelet membrane fluidity - with a 6% increase noted even for the brief shear exposure examined. Shear stress exposure results in significant alteration of overall platelet lipid composition, with increase in supernatant lipid species relative to the non-activated samples such that 72% of identified lipid species have >2-fold increase. Lipid species altered in the supernatant of sheared platelets were unaltered under thrombin treatment. Interestingly, within the limits of our analysis, a gender-based difference in retained lipid species was seen with shear but not with thrombin activation. Male donors showed at least a 2-fold decrease in 53% of lipids identified in the pellet of the sheared platelets using negative mode MS. In contrast, female donors had only 6% of lipids decrease >2-fold relative to non-activated platelets, while 77% of lipids showed a less than 2-fold change in either direction..Shear stress has a specific impact on platelet membranes - leading to an increase in fluidity, and alteration of platelet lipidomic profile, which differs from biochemical activation, with evident gender-based differences. Specifically, shear leads to an increase in the number of lipids ejected across all donors, and a male-preferred decrease in retained lipid species. This sex-based difference in platelet composition resulting from shear exposure raises the issue of altered responsiveness that may impart gender differences in in MCS outcomes.
• Turner, D. L., Korneev, D. V., Purdy, J. G., de Marco, A., & Mathias, R. A. (2020). The host exosome pathway underpins biogenesis of the human cytomegalovirus virion. eLife, 9.
  More info

  Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constituents. To validate this acquisition, we characterized exosomes released from uninfected cells, and demonstrated that over 99% of the protein cargo was subsequently incorporated into HCMV virions during infection. This suggested a common membrane origin, and utilization of host exosome machinery for virion assembly and egress. Thus, we selected a panel of exosome proteins for knock down, and confirmed that loss of 7/9 caused significantly less HCMV production. Saliently, we report that VAMP3 is essential for viral trafficking and release of infectious progeny, in various HCMV strains and cell types. Therefore, we establish that the host exosome pathway is intrinsic for HCMV maturation, and reveal new host regulators involved in viral trafficking, virion envelopment, and release. Our findings underpin future investigation of host exosome proteins as important modulators of HCMV replication with antiviral potential.
• Passalacqua, K. D., Purdy, J. G., & Wobus, C. E. (2019). The inert meets the living: The expanding view of metabolic alterations during viral pathogenesis. PLoS pathogens, 15(7), e1007830.
• Purdy, J. G. (2019). Pathways to Understanding Virus-Host Metabolism Interactions. Current Clinical Microbiology Reports. doi:10.1007/s40588-018-0109-7
• Purdy, J. G., & Luftig, M. A. (2019). Reprogramming of cellular metabolic pathways by human oncogenic viruses. Current opinion in virology, 39, 60-69.
  More info

  Oncogenic viruses, like all viruses, relies on host metabolism to provide the metabolites and energy needed for virus replication. Many DNA tumor viruses and retroviruses will reprogram metabolism during infection. Additionally, some viral oncogenes may alter metabolism independent of virus replication. Virus infection and cancer development share many similarities regarding metabolic reprogramming as both processes demand increased metabolic activity to produce biomass: cell proliferation in the case of cancer and virion production in the case of infection. This review discusses the parallels in metabolic reprogramming between human oncogenic viruses and oncogenesis.
• Purdy, J. G., Wise, L. M., Xi, Y., & Harwood, S. (2019). Human Cytomegalovirus pUL37x1 Is Important for Remodeling of Host Lipid Metabolism. Journal of Virology, 93(21). doi:10.1128/jvi.00843-19
• Sweedo, A., Wise, L. M., Sweedo, A., Slepian, M. J., Sheriff, J., Roka-moiia, Y., Purdy, J. G., & Bluestein, D. (2019). Elevated Shear Associated with MCS Devices Yields Unique Platelet Membrane Lipid Species: Pro-Thrombotic Agonists?. Journal of Heart and Lung Transplantation, 38(4), S233. doi:10.1016/j.healun.2019.01.574
• Xi, Y., Harwood, S., Wise, L. M., & Purdy, J. G. (2019). Human Cytomegalovirus pUL37x1 Is Important for Remodeling of Host Lipid Metabolism. Journal of virology, 93(21).
  More info

  Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential for infection. While multiple host factors, including kinases and transcription factors, important for metabolic changes that occur following HCMV infection have been identified, little is known about the viral factors required to alter metabolism. In this study, we tested the hypothesis that pUL37x1 is important for the metabolic remodeling that is necessary for HCMV replication using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had levels of metabolites similar to those in cells infected with a mutant virus lacking the UL37x1 gene, UL37x1. However, we found that relative to WT-infected cells, UL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infection: fatty acid elongase 7 (ELOVL7) and the endoplasmic reticulum (ER) stress-related kinase PERK. Moreover, we observed that HCMV infection results in an increase in phospholipids with very-long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in UL37x1-infected cells than in WT-infected cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes associated with HCMV infection, it is important for the remodeling of a subset of metabolic changes that occur during infection. Human cytomegalovirus (HCMV) is a common pathogen that asymptomatically infects most people and establishes a lifelong infection. However, HCMV can cause end-organ disease that results in death in the immunosuppressed and is a leading cause of birth defects. HCMV infection depends on host metabolism, including lipid metabolism. However, the viral mechanisms for remodeling of metabolism are poorly understood. In this study, we demonstrate that the viral UL37x1 protein (pUL37x1) is important for infection-associated increases in lipid metabolism, including fatty acid elongation to produce very-long-chain fatty acids (VLCFAs). Furthermore, we found that HCMV infection results in a significant increase in phospholipids, particularly those with VLCFA tails (PL-VLCFAs). We found that pUL37x1 was important for the high levels of fatty acid elongation and PL-VLCFA accumulation that occur in HCMV-infected cells. Our findings identify a viral protein that is important for changes in lipid metabolism that occur following HCMV infection.
• Leng, S. X., Kamil, J., Purdy, J. G., Lemmermann, N. A., Reddehase, M. J., & Goodrum, F. D. (2017). Recent advances in CMV tropism, latency, and diagnosis during aging. GeroScience, 39(3), 251-259.
  More info

  Human cytomegalovirus (CMV) is one of the largest viruses known to cause human diseases. Chronic CMV infection, as defined by anti-CMV IgG serology, increases with age and is highly prevalent in older adults. It has complex biology with significant immunologic and health consequences. This article aims to summarize research findings presented at the 6th International Workshop on CMV and Immunosenescence that relate to advances in the areas of CMV tropism, latency, CMV manipulation of cell metabolism, and T cell memory inflation, as well as novel diagnostic evaluation and translational research of chronic CMV infection in older adults. Information summarized here represents the current state of knowledge in these important fields. Investigators have also identified a number of areas that deserve further and more in-depth investigation, including building more precise parallels between mouse CMV (mCMV) and human CMV (HCMV) research. It is hoped that this article will also stimulate engaging discussion on strategies and direction to advance the science to the next level.
• Purdy, J. G., Shenk, T., & Rabinowitz, J. D. (2015). Fatty acid elongase 7 catalyzes lipidome remodeling essential for human cytomegalovirus replication. Cell reports, 10(8), 1375-85.
  More info

  Human cytomegalovirus (HCMV) infection rewires host-cell metabolism, upregulating flux from glucose into acetyl-CoA to feed fatty acid metabolism, with saturated very-long-chain fatty acids (VLFCAs) required for production of infectious virion progeny. The human genome encodes seven elongase enzymes (ELOVL) that extend long-chain fatty acids into VLCFA. Here, we identify ELOVL7 as pivotal for HCMV infection. HCMV induces ELOVL7 by more than 150-fold. This induction is dependent on mTOR and SREBP-1. ELOVL7 knockdown or mTOR inhibition impairs HCMV-induced fatty acid elongation, HCMV particle release, and infectivity per particle. ELOVL7 overexpression enhances HCMV replication. During HCMV infection, mTOR activity is maintained by the viral protein pUL38. Expression of pUL38 is sufficient to induce ELOVL7, and pUL38-deficient virus is partially defective in ELOVL7 induction and fatty acid elongation. Thus, through its ability to modulate mTOR and SREBP-1, HCMV induces ELOVL7 to synthesize the saturated VLCFA required for efficient virus replication.
• England, M. R., Purdy, J. G., Ropson, I. J., Dalessio, P. M., & Craven, R. C. (2014). Potential role for CA-SP in nucleating retroviral capsid maturation. Journal of virology, 88(13), 7170-7.
  More info

  During virion maturation, the Rous sarcoma virus (RSV) capsid protein is cleaved from the Gag protein as the proteolytic intermediate CA-SP. Further trimming at two C-terminal sites removes the spacer peptide (SP), producing the mature capsid proteins CA and CA-S. Abundant genetic and structural evidence shows that the SP plays a critical role in stabilizing hexameric Gag interactions that form immature particles. Freeing of CA-SP from Gag breaks immature interfaces and initiates the formation of mature capsids. The transient persistence of CA-SP in maturing virions and the identification of second-site mutations in SP that restore infectivity to maturation-defective mutant viruses led us to hypothesize that SP may play an important role in promoting the assembly of mature capsids. This study presents a biophysical and biochemical characterization of CA-SP and its assembly behavior. Our results confirm cryo-electron microscopy (cryo-EM) structures reported previously by Keller et al. (J. Virol. 87:13655-13664, 2013, doi:10.1128/JVI.01408-13) showing that monomeric CA-SP is fully capable of assembling into capsid-like structures identical to those formed by CA. Furthermore, SP confers aggressive assembly kinetics, which is suggestive of higher-affinity CA-SP interactions than observed with either of the mature capsid proteins. This aggressive assembly is largely independent of the SP amino acid sequence, but the formation of well-ordered particles is sensitive to the presence of the N-terminal β-hairpin. Additionally, CA-SP can nucleate the assembly of CA and CA-S. These results suggest a model in which CA-SP, once separated from the Gag lattice, can actively promote the interactions that form mature capsids and provide a nucleation point for mature capsid assembly.
• Hwang, J., Purdy, J. G., Wu, K., Rabinowitz, J. D., & Shenk, T. (2014). Estrogen-related receptor α is required for efficient human cytomegalovirus replication. Proceedings of the National Academy of Sciences of the United States of America, 111(52), E5706-15.
  More info

  An shRNA-mediated screen of the 48 human nuclear receptor genes identified multiple candidates likely to influence the production of human cytomegalovirus in cultured human fibroblasts, including the estrogen-related receptor α (ERRα), an orphan nuclear receptor. The 50-kDa receptor and a 76-kDa variant were induced posttranscriptionally following infection. Genetic and pharmacological suppression of the receptor reduced viral RNA, protein, and DNA accumulation, as well as the yield of infectious progeny. In addition, RNAs encoding multiple metabolic enzymes, including enzymes sponsoring glycolysis (enolase 1, triosephosphate isomerase 1, and hexokinase 2), were reduced when the function of ERRα was inhibited in infected cells. Consistent with the effect on RNAs, a substantial number of metabolites, which are normally induced by infection, were either not increased or were increased to a reduced extent in the absence of normal ERRα activity. We conclude that ERRα is needed for the efficient production of cytomegalovirus progeny, and we propose that the nuclear receptor contributes importantly to the induction of a metabolic environment that supports optimal cytomegalovirus replication.
• Grady, S. L., Purdy, J. G., Rabinowitz, J. D., & Shenk, T. (2013). Argininosuccinate synthetase 1 depletion produces a metabolic state conducive to herpes simplex virus 1 infection. Proceedings of the National Academy of Sciences of the United States of America, 110(51), E5006-15.
  More info

  Herpes simplex virus 1 (HSV-1) infection triggers specific metabolic changes in its host cell. To explore the interactions between cellular metabolism and HSV-1 infection, we performed an siRNA screen of cellular metabolic genes, measuring their effect on viral replication. The screen identified multiple enzymes predicted to influence HSV-1 replication, including argininosuccinate synthetase 1 (AS1), which consumes aspartate as part of de novo arginine synthesis. Knockdown of AS1 robustly enhanced viral genome replication and the production of infectious virus. Using high-resolution liquid chromatography-mass spectrometry, we found that the metabolic phenotype induced by knockdown of AS1 in human fibroblasts mimicked multiple aspects of the metabolic program observed during HSV-1 infection, including an increase in multiple nucleotides and their precursors. Together with the observation that AS1 protein and mRNA levels decrease during wild-type infection, this work suggests that reduced AS1 activity is partially responsible for the metabolic program induced by infection.
• Koyuncu, E., Purdy, J. G., Rabinowitz, J. D., & Shenk, T. (2013). Saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny. PLoS pathogens, 9(5), e1003333.
  More info

  Human cytomegalovirus hijacks host cell metabolism, increasing the flux of carbon from glucose to malonyl-CoA, the committed precursor to fatty acid synthesis and elongation. Inhibition of acetyl-CoA carboxylase blocks the production of progeny virus. To probe further the role of fatty acid metabolism during infection, we performed an siRNA screen to identify host cell metabolic enzymes needed for the production of infectious cytomegalovirus progeny. The screen predicted that multiple long chain acyl-CoA synthetases and fatty acid elongases are needed during infection, and the levels of RNAs encoding several of these enzymes were upregulated by the virus. Roles for acyl-CoA synthetases and elongases during infection were confirmed by using small molecule antagonists. Consistent with a role for these enzymes, mass spectrometry-based fatty acid analysis with ¹³C-labeling revealed that malonyl-CoA is consumed by elongases to produce very long chain fatty acids, generating an approximately 8-fold increase in C26-C34 fatty acid tails in infected cells. The virion envelope was yet further enriched in C26-C34 saturated fatty acids, and elongase inhibitors caused the production of virions with lower levels of these fatty acids and markedly reduced infectivity. These results reveal a dependence of cytomegalovirus on very long chain fatty acid metabolism.
• Rabinowitz, J. D., Purdy, J. G., Vastag, L., Shenk, T., & Koyuncu, E. (2011). Metabolomics in drug target discovery. Cold Spring Harbor symposia on quantitative biology, 76, 235-46.
  More info

  Most diseases result in metabolic changes. In many cases, these changes play a causative role in disease progression. By identifying pathological metabolic changes, metabolomics can point to potential new sites for therapeutic intervention. Particularly promising enzymatic targets are those that carry increased flux in the disease state. Definitive assessment of flux requires the use of isotope tracers. Here we present techniques for finding new drug targets using metabolomics and isotope tracers. The utility of these methods is exemplified in the study of three different viral pathogens. For influenza A and herpes simplex virus, metabolomic analysis of infected versus mock-infected cells revealed dramatic concentration changes around the current antiviral target enzymes. Similar analysis of human-cytomegalovirus-infected cells, however, found the greatest changes in a region of metabolism unrelated to the current antiviral target. Instead, it pointed to the tricarboxylic acid (TCA) cycle and its efflux to feed fatty acid biosynthesis as a potential preferred target. Isotope tracer studies revealed that cytomegalovirus greatly increases flux through the key fatty acid metabolic enzyme acetyl-coenzyme A carboxylase. Inhibition of this enzyme blocks human cytomegalovirus replication. Examples where metabolomics has contributed to identification of anticancer drug targets are also discussed. Eventual proof of the value of metabolomics as a drug target discovery strategy will be successful clinical development of therapeutics hitting these new targets.
• Butan, C., Lokhandwala, P. M., Purdy, J. G., Cardone, G., Craven, R. C., & Steven, A. C. (2010). Suppression of a morphogenic mutant in Rous sarcoma virus capsid protein by a second-site mutation: a cryoelectron tomography study. Journal of virology, 84(13), 6377-86.
  More info

  Retrovirus assembly is driven by polymerization of the Gag polyprotein as nascent virions bud from host cells. Gag is then processed proteolytically, releasing the capsid protein (CA) to assemble de novo inside maturing virions. CA has N-terminal and C-terminal domains (NTDs and CTDs, respectively) whose folds are conserved, although their sequences are divergent except in the 20-residue major homology region (MHR) in the CTD. The MHR is thought to play an important role in assembly, and some mutations affecting it, including the F167Y substitution, are lethal. A temperature-sensitive second-site suppressor mutation in the NTD, A38V, restores infectivity. We have used cryoelectron tomography to investigate the morphotypes of this double mutant. Virions produced at the nonpermissive temperature do not assemble capsids, although Gag is processed normally; moreover, they are more variable in size than the wild type and have fewer glycoprotein spikes. At the permissive temperature, virions are similar in size and spike content as in the wild type and capsid assembly is restored, albeit with altered polymorphisms. The mutation F167Y-A38V (referred to as FY/AV in this paper) produces fewer tubular capsids than wild type and more irregular polyhedra, which tend to be larger than in the wild type, containing approximately 30% more CA subunits. It follows that FY/AV CA assembles more efficiently in situ than in the wild type and has a lower critical concentration, reflecting altered nucleation properties. However, its infectivity is lower than that of the wild type, due to a 4-fold-lower budding efficiency. We conclude that the wild-type CA protein sequence represents an evolutionary compromise between competing requirements for optimization of Gag assembly (of the immature virion) and CA assembly (in the maturing virion).
• Winkler, D. C., Steven, A. C., Purdy, J. G., Heymann, J., Craven, R. C., Cheng, N., Cardone, G., & Butan, C. (2010). Retrovirus Capsid Assembly and Polymorphism Studied by Cryo-EM and Cryo-ET. Microscopy and Microanalysis, 16(S2), 1106-1107. doi:10.1017/s1431927610061210
  More info

  Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.
• Cardone, G., Purdy, J. G., Cheng, N., Craven, R. C., & Steven, A. C. (2009). Visualization of a missing link in retrovirus capsid assembly. Nature, 457(7230), 694-8.
  More info

  For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.
• Purdy, J. G., Flanagan, J. M., Ropson, I. J., & Craven, R. C. (2009). Retroviral capsid assembly: a role for the CA dimer in initiation. Journal of molecular biology, 389(2), 438-51.
  More info

  In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.
• Purdy, J. G., Flanagan, J. M., Ropson, I. J., Rennoll-Bankert, K. E., & Craven, R. C. (2008). Critical role of conserved hydrophobic residues within the major homology region in mature retroviral capsid assembly. Journal of virology, 82(12), 5951-61.
  More info

  During retroviral maturation, the CA protein undergoes dramatic structural changes and establishes unique intermolecular interfaces in the mature capsid shell that are different from those that existed in the immature precursor. The most conserved region of CA, the major homology region (MHR), has been implicated in both immature and mature assembly, although the precise contribution of the MHR residues to each event has been largely undefined. To test the roles of specific MHR residues in mature capsid assembly, an in vitro system was developed that allowed for the first-time formation of Rous sarcoma virus CA into structures resembling authentic capsids. The ability of CA to assemble organized structures was destroyed by substitutions of two conserved hydrophobic MHR residues and restored by second-site suppressors, demonstrating that these MHR residues are required for the proper assembly of mature capsids in addition to any role that these amino acids may play in immature particle assembly. The defect caused by the MHR mutations was identified as an early step in the capsid assembly process. The results provide strong evidence for a model in which the hydrophobic residues of the MHR control a conformational reorganization of CA that is needed to initiate capsid assembly and suggest that the formation of an interdomain interaction occurs early during maturation.

Presentations

• Purdy, J. G. (2022, Summer). Viral Remodeling of the Metabolic Program. American Society for Virolgoy Annual Conference. Madison, WI: American Socity for Virology (ASV).
• Purdy, J. G. (2021). Metabolic Perspective of Virus-Host Interactions. Division of Infectious Disease Seminar Series. Virtual: Division of Infectious Disease, University of Arizona COM-T.
• Purdy, J. G. (2021). Metabolites in Metabolism and Beyond in Human Cytomegalovirus Infection. American Society for Virology 40th Annual Conference. Virtual: American Society for Virology.
• Purdy, J. G. (2020, September). Investigating how Host Metabolism makes Viruses become Alive. University of Maryland, College Park Host-Pathogen Interactions Research Stream. College Park, MD: University of Maryland, College Park.
• Purdy, J. G. (2019, Apr). Host Metabolism: Feeding and Starving Viral Infections. UAHS Infectious Disease Seminar Series. UAHS: UAHS Infectious Disease.
• Purdy, J. G. (2019, Apr). Host Metabolism: Feeding and Starving Viral Infections. ASU Virology. Arizona State University, BIO Design.
• Purdy, J. G. (2019, Dec). Defining Human Cytomegalovirus Remodeling of Lipid Synthesis using Untargeted Lipidomics. Oncogenic Viruses & Metabolism Workshop. NIH NCI, Washington DC: NIH NCI.
• Purdy, J. G. (2019, January). HIF1a Suppression of IDO-1 Kynurenine Synthesis is Antiviral during Human Cytomegalovirus Infection. Endorcinology Works in Progress. Tucson, Arizona: University of Arizona College of Medicine.
• Purdy, J. G. (2019, Jul). Hypoxia Inducible Factor 1α (HIF1α) Inhibits Human Cytomegalovirus by Suppressing IDO-1 Kynurenine Synthesis. International Herpesvirus Workshop. Knoxville, TN: International Herpesvirus Workshop.
• Purdy, J. G. (2019, Jun). Metabolic Limits to Human Cytomegalovirus Infection. AZ Wellbeing Commons: Virology, Immunology, Microbiome and Infectious Disease (VIMID). UA COM-Phoenix: AZ Wellbeing Commons.
• Purdy, J. G. (2019, Jun). Targeting Host Metabolism during Cytomegalovirus Infection. UAHS K Club. UAHS.
• Purdy, J. G. (2019, May). Hypoxia Inducible Factor 1α (HIF1α) Limits Human Cytomegalovirus by Suppressing IDO-1 Kynurenine Synthesis. ABRC Research Conference. UA COM-Phoenix: UAHS and ABRC.
• Purdy, J. G. (2019, Oct). Modulation of metabolism by cytomegalovirus: What can a virus teach us about human metabolism?. UACC. UACC: CBIO, UACC.
• Purdy, J. G. (2018, February). Integrating Metabolomics and Lipidomics to Define Viral Hijacking of Host Metabolism. Core Fair. Tucson, Arizona: University of Arizona RDI.
• Purdy, J. G. (2018, October). Targeting Human Cytomegalovirus Infection using Untargeted Lipidomics and Metabolomics. University of Michigan Medical School Dept of Microbiology and Immunology. Ann Arbor, Michigan: University of Michigan Medical School.
• Purdy, J. G., Xi, Y., & Wise, L. (2018, July). Untargeted Lipidomics reveals that Human Cytomegalovirus Induces PC Lipid Synthesis. 43rd Annual International Herpesvirus Workshop. Vancouver, British Columbia Canada: International Herpesvirus Workshop.
• Purdy, J. G. (2017, April). Targeting Metabolism to Block Virus Replication. Invited Speaker, Department of Basic Medical Sciences Seminar Series, UA COM-Phoenix. Phoenix, AZ: Department of Basic Medical Sciences, UA COM-Phoenix.
• Purdy, J. G. (2017, November). Cytomegalovirus remodeling of host metabolism. ASU/UA Joint Meeting of Virologists. Phoenix, AZ: BioDesign Institute.
• Purdy, J. G., Mustacich, D., & Wise, L. (2017, May). Human Cytomegalovirus Infection uses Host Metabolism to Control Cell Stress. Viruses and Cells Gordon Research Conference. II Ciocco Lucca (Barga), Italy: Gordon Research Conference.
• Purdy, J. G., Sohrab, N., & Wise, L. (2017, Summer). Human Cytomegalovirus Remodeling of Host Metabolism. 42nd International Herpesvirus Workshop. Ghent, Belgium: International Herpesvirus Workshop.
• Purdy, J. G., Wise, L., Mustacich, D., & Sohrab, N. (2017, June). Human Cytomegalovirus Remodeling of Host Metabolism. American Society for Virology Annual Meeting. Madison, WI: American Society for Virology.
• Purdy, J. G. (2016, 03/30/2016). Viral Hijacking of Host Cell Metabolism. Analytical Chemistry Seminar Series. University of Arizona: Chemsitry and Biochemistry Deparment,.
• Purdy, J. G. (2016, Fall). Human Cytomegalovirus Remodeling of Host Glucose Metabolism. Division of Endocrinology/Center for Disparities in Diabetes, Obesity, and Metabolism (CDDOM) seminar series. Tucson, AZ: Division of Endocrinology, College of Medicine, University of Arizona.
• Purdy, J. G. (2016, Fall). Human Cytomegalovirus Remodeling of Host Metabolism to Favor Replication. 6th International Workshop on CMV and Immunosenescence. Tucson, AZ: International Workshop on CMV and Immunosenescence.
• Purdy, J. G. (2016, Fall). Human Cytomegalovirus Remodeling of Host Metabolism. Arizona Biology Retreat. Tucson, AZ: University of Arizona.
• Purdy, J. G. (2016, Fall). Lipidomic and Metabolomic Approaches for Treating Viral Infections. Biological Chemistry Program Seminar Series. Tucson, AZ: Biological Chemistry Program, University of Arizona.
• Purdy, J. G. (2016, Fall). Targeting Host Metabolism for Treatment of Viral Infections. Drug Discovery and Development. Tucson, AZ: College of Pharmacy, University of Arizona.
• Purdy, J. G. (2016, March). Fattening up the cell: remodeling of metabolism by HCMV. Frontiers in Immunobiology and Immunopathogenesis Symposium. University of Arizona: Immunobiology Department.
• Purdy, J. G. (2016, Winter). Understanding Human Metabolomics and Lipidomics using Viruses as the Teachers. BIO5 Fellow Seminar Series. Tucson, AZ: BIO5 Institute.
• Purdy, J. G., & Wise, L. (2016, May). Fatty Acid Elongation is Required for Human Cytomegalovirus Infection. LIPID MAPS. San Diego, CA: LIPID Metabolites And Pathways Strategy (LIPID MAPS).

Poster Presentations

• Xi, Y., Kline, I., Wise, L., & Purdy, J. G. (2021). Remodeling of Host Lipidome following Infection with Enveloped Viruses. ABRC-Flinn 6th Annual Research Conference. Virtual: ABRC, Flinn Foundation, COM-P.
• Purdy, J. G. (2019, May). HIF1α Suppression of IDO-1 / Kynurenine Synthesis Limits Human Cytomegalovirus. Gordon Research Conference Virsuses and Cells. Tuscany Il Ciocco in Lucca (Barga) Italy: Gordon Research Conferences.
• Purdy, J. G., & Xi, Y. (2019, Jul). Human Cytomegalovirus Remodeling of Host Phospholipid Metabolism Requires PERK, a member of the ER Stress Response. International Herpesvirus Workshop. Knoxville, TN: International Herpesvirus Workshop.
• Purdy, J. G., Wise, L., & Sohrab, N. (2018, May). Lipidomic Analysis of Viral Infection: Identification of New Lipids with Very Long Chain Fatty Acid Tails. ASMS Conference on Mass Spectrometry and Allied Topics. San Diego, CA: American Society for Mass Spectrometry.
• Purdy, J. G. (2016, Winter). Human Cytomegalovirus Infection uses Host Metabolism to Control Cell Stress. Cellular Stress Responses and Infectious Agents. Santa Fe, NM: Keystone Symposia on Molecular and Cellular Biology.