Karla J. Helbig

Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector

Interferon (IFN) α inhibits hepatitis C virus (HCV) replication both clinically and in vitro; however, the complete spectrum of interferon‐stimulated genes (ISGs) expressed in the HCV‐infected liver or the genes responsible for control of HCV replication have not been defined. To better define ISG expression in the chronically infected HCV liver, DNA microarray analysis was performed on 9 individuals with chronic hepatitis C (CHC). A total of 232 messenger RNAs were differentially regulated in CHC compared with nondiseased liver controls. A significant proportion of these were potential ISGs that were transcriptionally elevated, suggesting an ongoing response to endogenous IFN and/or double‐stranded RNA. One ISG significantly elevated in all patients was viperin, an evolutionary conserved ISG that has antiviral activity against human cytomegalovirus. Stimulation of Huh‐7 and HepG2 cells with IFN‐α or ‐γ revealed viperin is predominantly a type I ISG. Furthermore, viperin expression could also be induced following transfection of Huh‐7 cells with either poly(I:C) or HCV RNA. Transient expression of viperin in cells harboring the HCV genomic replicon resulted in a significant decrease in HCV replication, suggesting that viperin has anti‐HCV activity. In conclusion, even in the face of a persistent HCV infection, there is an active ISG antiviral cellular response, highlighting the complexity of the host viral relationship. Furthermore, ISG viperin has anti‐HCV activity in vitro; we postulate that viperin, along with other ISGs, acts to limit HCV replication. (HEPATOLOGY 2005.)

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;)

HIV-1 infection of human macrophages directly induces viperin which inhibits viral production.

Macrophages are key target cells for HIV-1. HIV-1(BaL) induced a subset of interferon-stimulated genes in monocyte-derived macrophages (MDMs), which differed from that in monocyte-derived dendritic cells and CD4 T cells, without inducing any interferons. Inhibition of type I interferon induction was mediated by HIV-1 inhibition of interferon-regulated factor (IRF3) nuclear translocation. In MDMs, viperin was the most up-regulated interferon-stimulated genes, and it significantly inhibited HIV-1 production. HIV-1 infection disrupted lipid rafts via viperin induction and redistributed viperin to CD81 compartments, the site of HIV-1 egress by budding in MDMs. Exogenous farnesol, which enhances membrane protein prenylation, reversed viperin-mediated inhibition of HIV-1 production. Mutagenesis analysis in transfected cell lines showed that the internal S-adenosyl methionine domains of viperin were essential for its antiviral activity. Thus viperin may contribute to persistent noncytopathic HIV-1 infection of macrophages and possibly to biologic differences with HIV-1-infected T cells.

Expression of the CXCR3 ligand I‐TAC by hepatocytes in chronic hepatitis C and its correlation with hepatic inflammation

The factors that regulate lymphocyte traffic in chronic hepatitis C (CHC) are not completely defined. Interferon (IFN)‐inducible T cell α chemoattractant (I‐TAC) is a relatively new member of the CXCR3 chemokine ligand family that selectively recruits activated T cells to sites of inflammation. To determine if I‐TAC plays a role in CHC, we investigated I‐TAC expression in hepatitis C virus (HCV)‐infected liver biopsy material. I‐TAC messenger RNA (mRNA) levels were significantly increased in HCV‐infected liver compared with normal liver, which correlated with both portal and lobular inflammation. I‐TAC expression was localized to hepatocytes throughout the liver lobule, with those in close proximity to active areas of inflammation expressing the highest concentration of I‐TAC. In vitro, I‐TAC mRNA and protein expression was inducible in Huh‐7 cells following either IFN‐α or ‐γ stimulation and synergistically with tumor necrosis factor (TNF)‐α. Furthermore, transfection of Huh‐7 cells with either poly(I:C) or HCV RNA representing the HCV subgenomic replicon induced I‐TAC mRNA expression. HCV replication was also found to modulate I‐TAC expression, with stimulation of Huh‐7 cells harboring either the HCV subgenomic or genomic replicon showing significantly increased synergistic effects compared with those previously seen in Huh‐7 cells alone with IFN‐γ and TNF‐α. In conclusion, these results suggest I‐TAC, one of the most potent chemoattractants for activated T cells, is produced by hepatocytes in the HCV‐infected liver and plays an important role in T cell recruitment and ultimately the pathogenesis of CHC. (HEPATOLOGY 2004;39:1220–1229.)

The Role of Viperin in the Innate Antiviral Response

Viral infection of the cell is able to initiate a signaling cascade of events that ultimately attempts to limit viral replication and prevent escalating infection through expression of host antiviral proteins. Recent work has highlighted the importance of the host antiviral protein viperin in this process, with its ability to limit a large variety of viral infections as well as play a role in the production of type I interferon and the modulation of a number of transcription factor binding sites. Viperin appears to have the ability to modulate varying conditions within the cell and to interfere with proviral host proteins in its attempts to create an unfavorable environment for viral replication. The study of the mechanistic actions of viperin has come a long way in recent years, describing important functional domains of the protein for its antiviral and immune modulator actions as well as demonstrating its role as a member of the radical SAM enzyme family. However, despite the rapid expansion of knowledge regarding the functions of this highly conserved and ancient antiviral protein, there still remains large gaps in our understanding of the precise mechanisms at play for viperin to exert such a wide variety of roles within the cell.

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.

CD4+ T-Cell Deficiency in HIV Patients Responding to Antiretroviral Therapy Is Associated With Increased Expression of Interferon-Stimulated Genes in CD4+ T Cells

Most patients with human immunodeficiency virus (HIV) who remain CD4(+) T-cell deficient on antiretroviral therapy (ART) exhibit marked immune activation. As CD4(+) T-cell activation may be mediated by microbial translocation or interferon-alpha (IFN-α), we examined these factors in HIV patients with good or poor CD4(+) T-cell recovery on long-term ART. Messenger RNA levels for 3 interferon-stimulated genes were increased in CD4(+) T cells of patients with poor CD4(+) T-cell recovery, whereas levels in patients with good recovery did not differ from those in healthy controls. Poor CD4(+) T-cell recovery was also associated with CD4(+) T-cell expression of markers of activation, senescence, and apoptosis, and with increased serum levels of the lipopolysaccharide receptor and soluble CD14, but these were not significantly correlated with expression of the interferon-stimulated genes. Therefore, CD4(+) T-cell recovery may be adversely affected by the effects of IFN-α, which may be amenable to therapeutic intervention.

Differential expression of the CXCR3 ligands in chronic hepatitis C virus (HCV) infection and their modulation by HCV in vitro

ABSTRACT To investigate chemokine expression networks in chronic hepatitis C virus (HCV) infection, we used microarray analysis to determine chemokine expression in human infection and in chimpanzees experimentally infected with HCV. The CXCR3 chemokine family was highly expressed in both human and chimpanzee infection. CXCL10 was the only CXCR3 chemokine elevated in the serum, suggesting that it may neutralize any CXCR3 chemokine gradient established between the periphery and liver by CXCL11 and CXCL9. Thus, CXCR3 chemokines may not be responsible for recruitment of T lymphocytes but may play a role in positioning these cells within the liver. The importance of the CXCR3 chemokines, in particular CXCL11, was highlighted by replicating HCV (JFH-1) to selectively upregulate its expression in response to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). This selective upregulation was confirmed at the transcriptional level by using the CXCL11 promoter driving the luciferase reporter gene. This synergistic increase in expression was not a result of HCV protein expression but the nonspecific innate response to double-stranded RNA (dsRNA), as both in vitro-transcribed HCV RNA and the dsRNA analogue poly(I:C) increased CXCL11 expression and promoter activity. Furthermore, we show that CXCL11 is an IRF3 (interferon regulatory factor 3) response gene whose expression is selectively enhanced by IFN-γ and TNF-α. In conclusion, the CXCR3 chemokines are the most significantly expressed chemokines in chronic hepatitis C and most likely play a role in positioning T cells in the liver. Furthermore, HCV can selectively increase CXCL11 expression in response to IFN-γ and TNF-α stimulation that may play a role in the pathogenesis of HCV-related liver disease.

Alcohol Metabolism Increases the Replication of Hepatitis C Virus and Attenuates the Antiviral Action of Interferon

The interactions between hepatitis C virus (HCV) and alcohol metabolism are not well understood. To determine the effect that alcohol metabolism has on HCV replication and the antiviral action of interferon (IFN), Huh-7 cells that harbor HCV replication and metabolize ethanol via the introduced expression of cytochrome P450 2E1 (Cyp2e1) were treated with ethanol and IFN-alpha. Treatment of these cells with ethanol (0-100 mmol/L) significantly increased HCV replication. This effect was dependent on Cyp2e1 expression and alcohol-metabolized oxidative stress (OS), because the antioxidant N-acetylcysteine blocked this effect. Furthermore, the anti-HCV action of IFN-alpha was attenuated in the presence of ethanol metabolism, most likely via attenuation of Stat1 tyrosine-701 phosphorylation. These in vitro results mimic what is often noted clinically, and further dissection of this model system will aid in our understanding of interactions between HCV and alcohol metabolism.

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)