Nicholas S. Eyre

The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A

The interferon‐stimulated gene, viperin, has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here, we demonstrate that viperin plays an integral part in the ability of interferon to limit the replication of cell‐culture–derived HCV (JFH‐1) that accurately reflects the complete viral life cycle. Using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis, we demonstrate that viperin localizes and interacts with HCV nonstructural protein 5A (NS5A) at the lipid‐droplet (LD) interface. In addition, viperin also associates with NS5A and the proviral cellular factor, human vesicle‐associated membrane protein‐associated protein subtype A (VAP‐A), at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C‐terminus, as well as an N‐terminal amphipathic helix. Removal of the amphipathic helix‐redirected viperin from the cytosolic face of the endoplasmic reticulum and the LD to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C‐terminal viperin mutants still localized to the LD interface and replication complexes, but did not interact with NS5A proteins, as determined by FRET analysis. Conclusion: In conclusion, we propose that viperin interacts with NS5A and the host factor, VAP‐A, to limit HCV replication at the replication complex. This highlights the complexity of the host control of viral replication by interferon‐stimulated gene expression. (HEPATOLOGY 2011;)

Viperin Is Induced following Dengue Virus Type-2 (DENV-2) Infection and Has Anti-viral Actions Requiring the C-terminal End of Viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.

Hepatitis B virus and hepatitis C virus interaction in Huh-7 cells

BACKGROUND/AIMS Co-infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) increases the risk of development and the severity of chronic liver disease. Although dominant and suppressive effects of each virus over the other have been reported in vivo, in vitro studies of HBV/HCV co-infection have been limited to analysis of the effects of over-expression of HCV proteins on HBV replication. METHODS We have re-examined HBV/HCV interactions in Huh-7 cells following co-infection with cell culture-propagated HCV (HCVcc; genotype 2a) and a recombinant adenovirus vector capable of delivering a replication-competent HBV genome (AdHBV; genotype A). RESULTS While intracellular HCV RNA levels were significantly increased when cells were pre-infected with AdHBV, HCV replication and virion secretion were not altered by simultaneous infection with AdHBV or AdHBV superinfection of HCV-infected cells. Likewise intracellular and secreted HBV DNA levels and HBV promoter activities were either unchanged or modestly increased by HCVcc infection. Despite this, HCV E2 and HBsAg proteins colocalized extensively in co-infected cells suggesting shared stages in viral egress. CONCLUSIONS These studies indicate that there is little direct interaction of HBV and HCV in co-infected hepatocytes and imply that indirect effects of host-viral interactions dictate viral dominance in HBV/HCV co-infected individuals.

The SR-BI Partner PDZK1 Facilitates Hepatitis C Virus Entry

Entry of hepatitis C virus (HCV) into hepatocytes is a multi-step process that involves a number of different host cell factors. Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes. Physiologically, SR-BI is the major receptor for high-density lipoproteins (HDL) in the liver, where its expression is primarily controlled at the post-transcriptional level by its interaction with the scaffold protein PDZK1. However, the importance of interaction with PDZK1 to the involvement of SR-BI in HCV entry is unclear. Here we demonstrate that stable shRNA-knockdown of PDZK1 expression in human hepatoma cells significantly reduces their susceptibility to HCV infection, and that this effect can be reversed by overexpression of full length PDZK1 but not the first PDZ domain of PDZK1 alone. Furthermore, we found that overexpression of a green fluorescent protein chimera of the cytoplasmic carboxy-terminus of SR-BI (amino acids 479–509) in Huh-7 cells resulted in its interaction with PDZK1 and a reduced susceptibility to HCV infection. In contrast a similar chimera lacking the final amino acid of SR-BI (amino acids 479–508) failed to interact with PDZK1 and did not inhibit HCV infection. Taken together these results indicate an indirect involvement of PDZK1 in HCV entry via its ability to interact with SR-BI and enhance its activity as an HCV entry factor.

Signal transducer and activator of transcription 3 is a proviral host factor for hepatitis C virus

Host factors play an important role in all facets of the hepatitis C virus (HCV) life cycle and one such host factor is signal transducer and activator of transcription 3 (STAT3). The HCV core protein has been shown to directly interact with and activate STAT3, while oxidative stress generated during HCV replication in a replicon‐based model also induced STAT3 activation. However, despite these findings the precise role of STAT3 in the HCV life cycle remains unknown. We have established that STAT3 is actively phosphorylated in the presence of replicating HCV. Furthermore, expression of a constitutively active form of STAT3 leads to marked increases in HCV replication, whereas, conversely, chemical inhibition and small interfering RNA (siRNA) knockdown of STAT3 leads to significant decreases in HCV RNA levels. This strongly implicates STAT3 as a proviral host factor. As STAT3 is a transcription factor, up‐regulation of a distinct set of STAT3‐dependent genes may create an environment that is favorable for HCV replication. However, STAT3 has recently been demonstrated to positively regulate microtubule (MT) dynamics, by way of a direct sequestration of the MT depolymerizing protein Stathmin 1 (STMN1), and we provide evidence that STAT3 may exert its effect on the HCV life cycle by way of positive regulation of MT dynamics. Conclusion: We have demonstrated that STAT3 plays a role in the life cycle of HCV and have clarified the role of STAT3 as a proviral host factor. (HEPATOLOGY 2013;58:1558–1568)

Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake

This study investigates the role of the cytoplasmic C terminus of fatty acid translocase (FAT/CD36) in localization of the molecule to the plasma membrane, its insertion into lipid rafts, and its ability to enhance long-chain fatty acid uptake in transfected H4IIE rat hepatoma cells. In these cells, wild-type FAT/CD36 is localized to both lipid raft and nonraft domains of the plasma membrane. Interestingly, a FAT/CD36 truncation mutant lacking the final 10 amino acids of the cytoplasmic C terminus was retained within the cell in detergent-resistant membranes, and unlike wild-type FAT/CD36, it did not enhance oleate uptake. Furthermore, expression of FAT/CD36 in these cells increased the incorporation of oleate into diacylglycerol, a property that was not shared by truncated FAT/CD36. To examine whether the C terminus itself has an intrinsic ability to dictate the plasma membrane localization of FAT/CD36, this region was fused in-frame to enhanced green fluorescent protein (EGFP). This domain was sufficient to attach EGFP to cellular membranes, suggesting an involvement in the intracellular traffic of the molecule. We conclude that the C terminus of FAT/CD36 is required for localization of the receptor to the cell surface and its ability to enhance cellular oleate uptake.

Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection

ABSTRACT Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways. IMPORTANCE Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.

The interferon-induced transmembrane proteins - IFITM1, IFITM2 and IFITM3 inhibit hepatitis C virus entry

Background: Interferon-induced transmembrane (IFITM) proteins limit a broad range of RNA viruses. Results: Tyrosine phosphorylation of IFITM2 and IFITM3, and S-palmitoylation of the IFITM proteins, are crucial for anti-hepatitis C virus (HCV) activity. Conclusion: IFITM2 and IFITM3 are able to limit HCV infection by targeting the late entry stages of the virus. Significance: IFITM proteins inhibit HCV at early and late stages of entry. The interferon-induced transmembrane (IFITM) family of proteins have recently been identified as important host effector molecules of the type I interferon response against viruses. IFITM1 has been identified as a potent antiviral effector against hepatitis C virus (HCV), whereas the related family members IFITM2 and IFITM3 have been described to have antiviral effects against a broad range of RNA viruses. Here, we demonstrate that IFITM2 and IFITM3 play an integral role in the interferon response against HCV and act at the level of late entry stages of HCV infection. We have established that in hepatocytes, IFITM2 and IFITM3 localize to the late and early endosomes, respectively, as well as the lysosome. Furthermore, we have demonstrated that S-palmitoylation of all three IFITM proteins is essential for anti-HCV activity, whereas the conserved tyrosine residue in the N-terminal domain of IFITM2 and IFITM3 plays a significant role in protein localization. However, this tyrosine was found to be dispensable for anti-HCV activity, with mutation of the tyrosine resulting in an IFITM1-like phenotype with the retention of anti-HCV activity and co-localization of IFITM2 and IFITM3 with CD81. In conclusion, we propose that the IFITM proteins act in a coordinated manner to restrict HCV infection by targeting the endocytosed HCV virion for lysosomal degradation and demonstrate that the actions of the IFITM proteins are indeed virus and cell-type specific.

Viperin is an important host restriction factor in control of Zika virus infection

Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-β and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin−/− mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate.

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.