Glenn Randall, Ph.D.
Our laboratory investigates the roles of virus-host interactions in replication and pathogenesis. We study viruses of the Flaviviridae, including hepatitis C virus (HCV) and dengue virus (DENV). These are clinically important viruses: over 130 million people are chronically infected with HCV, while an estimated 50-100 million DENV infections occur annually. The most severe pathologies are HCV-associated liver cancer and dengue hemorrhagic fever.
We have applied RNAi interference to identify ~85 host cofactors of viral replication. We now study the importance of these cellular genes in diverse steps of the viral life cycle, including entry, the regulation of viral protein translation and RNA replication, modulation of cellular lipid metabolism, the establishment of viral replication complexes, the secretion of infectious virus, and control of infection by the innate immune system. Additionally, the identification of cellular cofactors of HCV and DENV infection greatly expands the pool of potential targets for drug design. Three major project areas are:
1. HCV trafficking in infected hepatocytes.
We have combined an RNA interference analysis of HCV endocytosis with the development of the first live cell imaging of HCV infection. This approach identified 16 host cofactors of HCV entry, most of which function in sequential stages of clathrin-mediated endocytosis. We observed the trafficking of fluorescent HCV particles with these cellular cofactors and their related pathways, including the actin cytoskeleton, known receptors CD81 and the tight junction protein Claudin-1, clathrin, an E3 ubiquitin ligase, and early endosomes. Surprisingly, given the role of tight junction proteins as HCV entry factors, virion entry generally occurred outside of cell-cell junctions. We are currently characterizing HCV trafficking inside the infected cell and developing polarized primary hepatocyte models to study viral trafficking.
HCV virion (red) enters cell with its receptor, CD81 (green) Coller et al. 2009.
Conceptually similar studies into HCV release and spread have been recently completed. We combined an RNAi analysis of host factors that are required for infectious HCV secretion with live cell imaging of HCV core trafficking. Using this approach, we identified numerous components of the secretory pathway that are both required for infectious HCV release and co-traffic with HCV core. The dynamics of HCV core trafficking, both in terms of frequency of transport, particle velocity, and the corresponding run lengths were quantified. We observe that dynamic core movements in the periphery require NS2, a viral protein required for virion assembly. Core co-traffics with multiple components of the secretory pathway, including the Golgi, recycling endosome, microtubules, VAMP1 secretory vesicles, and ApoE. This study identifies new molecular determinants of HCV secretion and describes the dynamics of their movements with HCV core in real time. We are currently studying a secondary release pathway that directs the virus to spread from cell to cell without extra-cellular release.
2. Viral replication complex formation and cellular remodeling: viral modulation of host lipid metabolism and signaling.
Viruses intimately interact with their host cell to promote infection. This involves two major strategies: (i) mechanisms for circumventing host antiviral surveillance and (ii) and usurping host pathways to redirect their function for the benefit of the virus. Many viruses have evolved a mechanism to remodel intra-cellular membranes to form protected sites of replication. In essence, these are unique virus-induced structures whose sole purpose is to facilitate the production of large amounts of viral genomes in a way that minimizes alerting cellular antiviral responses. Understanding how these structures form is of fundamental importance, with the added potential of uncovering targets for antiviral therapies.
For most viruses, we know very little about how membrane remodeling occurs. We have analyzed how HCV and DENV remodel membrane using a combinatorial approach, involving cellular and viral genetics, drug inhibitors, cell biology, and biochemical approaches. This analysis revealed that both viruses share a common theme: co-opting a cellular enzyme(s) in lipid signaling or synthesis. However, the mechanisms are quite different. While HCV manipulates lipid signaling to redirect cellular vesicle trafficking, DENV usurps a cellular lipid synthesis pathway. HCV NS5A activates the cellular PI4 kinase IIIa at sites of replication, while the DENV protein NS3 binds to FASN, a major cellular enzyme in lipid biosynthesis, recruits it to sites of viral replication and activates it. Both interactions are required for viral replication complex formation. We have also defined a new role for autophagy in viral replication: the regulation of lipid metabolism. DENV induces autophagy to deplete cellular lipid stores and increases their oxidation for energy production.
Of particular interest, approved drugs exist which inhibit fatty acid biosynthesis for obesity therapy. Additionally, this pathway is required for the replication of many other medically relevant viruses, suggesting the possibility of broad-spectrum antivirals. Additionally, we have found that HCV drugs currently in clinical trials target the NS5A-PI4K interaction. Our lab is currently investigating the mechanism by which the viruses hijack these cellular pathways and the outcome on cellular remodeling, in addition to exploring the therapeutic potential of targeting these virus-host interactions as therapeutics.
3-D reconstruction and 2-photon microscopic resolution of FASN-DENV replication complexes. Heaton et al. 2010.
3. Control of HCV infection by the innate immune system
The current HCV antiviral therapy of interferon/ribavirin is successful in approximately half of the treatments. We are investigating the human gene USP18, which is both a predictor of the response of HCV-infected patients to therapy and is a putative drug target to improve the anti-HCV activity of interferon. USP18 is produced in large amounts in patients who fail to respond to anti-HCV interferon therapy. Decreasing USP18 levels in cells greatly improves the anti-HCV activity of interferon. We are investigating how USP18 regulates Type I and III interferon activity. It is part of a larger effort to evaluate the potential of USP18 inhibitors to improve interferon therapy. The hope is that USP18 inhibitors combined with interferon may improve patient response rates to therapy.
Coller, K.E., N.S. Heaton, K.L. Berger, J.D. Cooper, J. Saunders, and G. Randall. In press. Molecular determinants and dynamics of hepatitis C virus secretion. PLoS Pathogens.
Jordan, T.X. and G. Randall. 2011. Manipulation or capitulation: virus interactions with autophagy. Microbes and Infection. Oct. 23 ePub before print.
Berger, K.L., S.M. Kelly, T.X. Jordan, M.A. Tartell and G. Randall. 2011. Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication. J. Virol. 85(17):8870-83.
Heaton, N.S. and G. Randall. 2011. Multifaceted roles for lipids in viral infection. Trends in Microbiology. 19(7):368-375.
Heaton, N.S. and G. Randall. 2011. Dengue virus and autophagy. Viruses. 3:1332-1341.
Vangeloff, A.D., C. Zhang, S. Khadka, P. Siddavatam, N.S. Heaton, R. Perera, R. Sengupta, S. Sahasrabudhe, G. Randall, M. Gribskov, R.J. Kuhn, and D.J. LaCount. 2011. A physical interaction network of dengue virus and human proteins. Mol. Cell. Proteomics. ePub Sept. 12.
Heaton, N.S. and G. Randall. In press. Dengue virus induced autophagy regulates lipid metabolism. Cell Host and Microbe. 8:422-32. PMCID: PMC3026642
Heaton, N.S., K.L. Berger, R. Perera, S. Khadka, D.J. LaCount, R.J. Kuhn and G. Randall. 2010. Dengue virus infection redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis. Proc. Natl. Acad. Sci. USA. 107:17345-50. ePub Sept. 20.
Berger, K.L. and G. Randall. 2010. Possibilities for RNA interference in developing hepatitis C virus therapeutics. Viruses. 2(8): 1647-1665
Coller, K.E., K.L. Berger, N.S. Heaton, J.D. Cooper, R. Yoon, and G. Randall. 2009. RNA interference and single particle tracking analysis of hepatitis C virus endocytosis. PLoS Pathogens. 5(12):1-14. PMCID: PMC2790617.
Berger, K.L. and G. Randall. 2009. Potential roles for cellular cofactors in hepatitis C virus replication complex formation. Commun. Integr. Biol. 2(6): 471-473. PMCID: PMC2829822.
Berger, K.L., J.D. Cooper, N.S. Heaton, R. Yoon, T.E. Oakland, T.X. Jordan, G. Mateu, A. Grakoui, and G. Randall. 2009. Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication. Proc. Natl. Acad. Sci. USA. 106(18):7577-82. ePub April 17, 2009. PMCID: PMC2678598.
Faculty and Research