Immunohistochemistry for hepatitis E virus capsid protein cross-reacts with cytomegalovirus-infected cells: a potential diagnostic pitfall.

Immunohistochemistry for hepatitis E virus (HEV) ORF2 (capsid) protein is a powerful tool for tissue-based diagnosis of hepatitis E, particularly useful in evaluating abnormal liver values in immunocompromised patients. We report here a previously unobserved reactivity of the HEV ORF2 antibody to human cytomegalovirus (CMV) proteins and contrast the staining patterns encountered in HEV and CMV infection, respectively. As part of a routine diagnostic work-up, the liver biopsy of an immunocompromised patient with elevated liver values was examined histologically for infection with viruses including CMV and HEV. Cytopathic changes were found, suggestive of CMV infection, which was confirmed by immunohistochemistry. Surprisingly, reactivity of a portion of CMV-infected cells with a mouse monoclonal antibody (clone 1E6) against HEV ORF2 protein was also detected. This observation prompted a screening of 22 further specimens (including liver, gastrointestinal, lung, brain and placental biopsies) with confirmed CMV infection/reactivation. Immunoreactivity of CMV-infected cells with HEV ORF2 antibody was observed in 18 of 23 specimens. While the HEV ORF2 antibody showed cytoplasmic, nuclear and canalicular positivity in hepatitis E cases, positivity in CMV-infected cells was limited to the nucleus. In conclusion, the HEV ORF2 antibody (clone 1E6) shows unexpected immunoreactivity against CMV proteins. In contrast to the hepatitis E staining pattern with cytoplasmic, nuclear and occasional canalicular positivity, reactivity in CMV-infected cells is restricted to the nucleus. Awareness of this cross-reactivity and knowledge of the differences in staining patterns will prevent pathologists from misinterpreting positive HEV ORF2 immunohistochemistry in liver specimens.

Hepatitis E virus RNA-dependent RNA polymerase is involved in RNA replication and infectious particle production.

BACKGROUND AND AIMS: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and is known as the viral replicase. The RNA-dependent RNA polymerase (RDRP) domain is responsible for the synthesis of viral RNA. APPROACH AND RESULTS: Here, we identified a highly conserved α-helix located in the RDRP thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RDRP palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release. CONCLUSIONS: Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RDRP with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RDRP thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production.

OCIAD1 is a host mitochondrial substrate of the hepatitis C virus NS3-4A protease.

The hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) protease is a key component of the viral replication complex and the target of protease inhibitors used in current clinical practice. By cleaving and thereby inactivating selected host factors it also plays a role in the persistence and pathogenesis of hepatitis C. Here, we describe ovarian cancer immunoreactive antigen domain containing protein 1 (OCIAD1) as a novel cellular substrate of the HCV NS3-4A protease. OCIAD1 was identified by quantitative proteomics involving stable isotopic labeling using amino acids in cell culture coupled with mass spectrometry. It is a poorly characterized membrane protein believed to be involved in cancer development. OCIAD1 is cleaved by the NS3-4A protease at Cys 38, close to a predicted transmembrane segment. Cleavage was observed in heterologous expression systems, the replicon and cell culture-derived HCV systems, as well as in liver biopsies from patients with chronic hepatitis C. NS3-4A proteases from diverse hepacivirus species efficiently cleaved OCIAD1. The subcellular localization of OCIAD1 on mitochondria was not altered by NS3-4A-mediated cleavage. Interestingly, OCIAD2, a homolog of OCIAD1 with a cysteine residue in a similar position and identical subcellular localization, was not cleaved by NS3-4A. Domain swapping experiments revealed that the sequence surrounding the cleavage site as well as the predicted transmembrane segment contribute to substrate selectivity. Overexpression as well as knock down and rescue experiments did not affect the HCV life cycle in vitro, raising the possibility that OCIAD1 may be involved in the pathogenesis of hepatitis C in vivo.

Recombinant Hepatitis E Viruses Harboring Tags in the ORF1 Protein.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.

Glycogen Synthase Kinase 3ß Enhances Hepatitis C Virus Replication by Supporting miR-122.

Hepatitis C virus (HCV) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the HCV life cycle on key molecules in these metabolic pathways. Yet, little is known on the role in the HCV life cycle of glycogen synthase kinase 3 (GSK3), one of the most important kinases in cellular metabolism. Therefore, the impact of GSK3 on the HCV life cycle was assessed in human hepatoma cell lines harboring subgenomic genotype 1b and 2a replicons or producing cell culture-derived HCV genotype 2a by exposure to synthetic GSK3 inhibitors, GSK3 gene silencing, overexpression of GSK3 constructs and immunofluorescence analyses. In addition, the role of GSK3 in hepatitis E virus (HEV) replication was investigated to assess virus specificity of the observed findings. We found that both inhibition of GSK3 function by synthetic inhibitors as well as silencing of GSK3ß gene expression resulted in a decrease of HCV replication and infectious particle production, whereas silencing of the GSK3α isoform had no relevant effect on the HCV life cycle. Conversely, overexpression of GSK3ß resulted in enhanced HCV replication. In contrast, GSK3ß had no effect on replication of subgenomic HEV replicon. The pro-viral effect of GSK3ß on HCV replication was mediated by supporting expression of microRNA-122 (miR-122), a micro-RNA which is mandatory for wild-type HCV replication, as GSK3 inhibitors suppressed miR-122 levels and as inhibitors of GSK3 had no antiviral effect on a miR-122-independent HCV mutant. In conclusion, we have identified GSK3ß is a novel host factor supporting HCV replication by maintaining high levels of hepatic miR-122 expression.

Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion.

Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle.

Differential modulation of hepatitis C virus replication and innate immune pathways by synthetic calcitriol-analogs.

BACKGROUND AND AIMS: Vitamin D signaling is involved in infectious and non-infectious liver diseases, yet the natural vitamin D metabolites are suboptimal therapeutic agents. In the present study, we therefore aimed to explore the potential and mechanism of selected calcitriol analogs to regulate the hepatocellular transcriptome and to inhibit hepatitis C virus (HCV) in comparison with calcitriol. METHODS: Human hepatoma cell lines and primary human macrophages were stimulated with calcitriol and selected calcitriol analogs. The effect of calcitriol and its derivatives on hepatocellular gene expression and vitamin D receptor (VDR) signaling as well as on replication of HCV were assessed by quantitative PCR, microarray analyses and in silico analyses of ligand-VDR complexes. RESULTS: The structurally related vitamin D analogs calcipotriol and tacalcitiol, but not calcitriol itself, suppressed HCV replication in a VDR-dependent manner. Using a residue-interaction network approach we outline structural and functional differences between VDR-ligand complexes. In particular we find characteristics in the VDR structure bound to calcipotriol with distinct local residue interaction patterns that affect key functional residues that pertain to the VDR charge clamp, H397 and F422, a VDR regulatory element for interaction with co-activators and -repressors. As a consequence, we show calcipotriol in comparison to calcitriol to induce stronger regulatory actions on the transcriptome of hepatocytes and macrophages including key antimicrobial peptides. CONCLUSION: Calcipotriol induces local structure rearrangements in VDR that could possibly translate into a superior clinical potential to execute important non-classical vitamin D effects such as inhibition of HCV replication.

Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular Systems.

BACKGROUND & AIMS: The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. METHODS: Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. RESULTS: HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. CONCLUSIONS: Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin.

Identification of GBF1 as a cellular factor required for hepatitis E virus RNA replication.

The hepatitis E virus (HEV) genome is a single-stranded, positive-sense RNA that encodes three proteins including the ORF1 replicase. Mechanisms of HEV replication in host cells are unclear, and only a few cellular factors involved in this step have been identified so far. Here, we used brefeldin A (BFA) that blocks the activity of the cellular Arf guanine nucleotide exchange factors GBF1, BIG1, and BIG2, which play a major role in reshuffling of cellular membranes. We showed that BFA inhibits HEV replication in a dose-dependent manner. The use of siRNA and Golgicide A identified GBF1 as a host factor critically involved in HEV replication. Experiments using cells expressing a mutation in the catalytic domain of GBF1 and overexpression of wild type GBF1 or a BFA-resistant GBF1 mutant rescuing HEV replication in BFA-treated cells, confirmed that GBF1 is the only BFA-sensitive factor required for HEV replication. We demonstrated that GBF1 is likely required for the activity of HEV replication complexes. However, GBF1 does not colocalise with the ORF1 protein, and its subcellular distribution is unmodified upon infection or overexpression of viral proteins, indicating that GBF1 is likely not recruited to replication sites. Together, our results suggest that HEV replication involves GBF1-regulated mechanisms.

Visualization of hepatitis E virus RNA and proteins in the human liver.

BACKGROUND & AIMS: Although hepatitis E constitutes a substantial disease burden worldwide, surprisingly little is known about the localization of hepatitis E virus (HEV) in the human liver. We therefore aimed to visualize HEV RNA and proteins in situ. METHODS: A panel of 12 different antibodies against HEV open reading frame (ORF) 1-3 proteins was evaluated for immunohistochemistry (IHC) and two probes for in situ hybridization (ISH) in formalin-fixed, paraffin-embedded (FFPE) HuH7 cells transfected with HEV ORF1-3 expression vectors. IHC (and partly ISH) were then applied to Hep293TT cells replicating infectious HEV and liver specimens from patients with hepatitis E (n=20) and controls (n=134). RESULTS: Whereas ORF1-3 proteins were all detectable in transfected, HEV protein-expressing cells, only ORF2 and 3 proteins were traceable in cells replicating infectious HEV. Only the ORF2-encoded capsid protein was also unequivocally detectable in liver specimens from patients with hepatitis E. IHC for ORF2 protein revealed a patchy expression in individual or grouped hepatocytes, generally stronger in chronic compared to acute hepatitis. Besides cytoplasmic and canalicular, ORF2 protein also displayed a hitherto unknown nuclear localization. Positivity for ORF2 protein in defined areas correlated with HEV RNA detection by ISH. IHC was specific and comparably sensitive as PCR for HEV RNA. CONCLUSIONS: ORF2 protein can be reliably visualized in the liver of patients with hepatitis E, allowing for sensitive and specific detection of HEV in FFPE samples. Its variable subcellular distribution in individual hepatocytes of the same liver suggests a redistribution of ORF2 protein during infection and interaction with nuclear components. LAY SUMMARY: The open reading frame (ORF) 2 protein can be used to visualize the hepatitis E virus (HEV) in the human liver. This enabled us to discover a hitherto unknown localization of the HEV ORF2 protein in the nucleus of hepatocytes and to develop a test for rapid histopathologic diagnosis of hepatitis E, the most common cause of acute hepatitis worldwide.

Update on hepatitis E virology: Implications for clinical practice.

Hepatitis E virus (HEV) is a positive-strand RNA virus transmitted by the fecal-oral route. The 7.2kb genome encodes three open reading frames (ORF) which are translated into (i) the ORF1 polyprotein, representing the viral replicase, (ii) the ORF2 protein, corresponding to the viral capsid, and (iii) the ORF3 protein, a small protein involved in particle secretion. Although HEV is a non-enveloped virus in bile and feces, it circulates in the bloodstream wrapped in cellular membranes. HEV genotypes 1 and 2 infect only humans and cause mainly waterborne outbreaks. HEV genotypes 3 and 4 are widely represented in the animal kingdom and are transmitted as a zoonosis mainly via contaminated meat. HEV infection is usually self-limited but may persist and cause chronic hepatitis in immunocompromised patients. Reduction of immunosuppressive treatment or antiviral therapy with ribavirin have proven effective in most patients with chronic hepatitis E but therapy failures have been reported. Alternative treatment options are needed, therefore. Infection with HEV may also cause a number of extrahepatic manifestations, especially neurologic complications. Progress in the understanding of the biology of HEV should contribute to improved control and treatment of HEV infection.

Hepatitis C virus variants resistant to macrocyclic NS3-4A inhibitors subvert IFN-ß induction by efficient MAVS cleavage.

BACKGROUND & AIMS: The hepatitis C virus (HCV) NS3-4A protease is essential for the HCV life cycle and a prime target of antiviral treatment strategies. Protease inhibitors, however, are limited by emergence of resistance-associated amino acid variants (RAVs). The capacity to cleave and inactivate mitochondrial antiviral-signaling protein (MAVS) in the RIG-I-signaling pathway is a cardinal feature of NS3-4A, by which HCV blocks induction of interferon-(IFN)-ß, thereby promoting viral persistence. Here, we aimed to investigate the impact of NS3-4A RAVs on MAVS cleavage. METHODS: The impact of NS3-4A RAVs on MAVS cleavage was assessed using immunoblot analyses, luciferase reporter assays and molecular dynamics simulations to study the underlying molecular principles. IFN-ß was quantified in serum from patients with different NS3-4A RAVs. RESULTS: We show that macrocyclic NS3-4A RAVS with substitutions at residue D168 of the protease result in an increased capacity of NS3-4A to cleave MAVS and suppress IFN-ß induction compared with a comprehensive panel of RAVs and wild type HCV. Mechanistically, we show the reconstitution of a tight network of electrostatic interactions between protease and the peptide substrate that allows much stronger binding of MAVS to D168 RAVs than to the wild-type protease. Accordingly, we could show IFN-ß serum levels to be lower in patients with treatment failure due to the selection of D168 variants compared to R155 RAVs. CONCLUSIONS: Our data constitutes a proof of concept that the selection of RAVs against specific classes of direct antivirals can lead to the predominance of viral variants with possibly adverse pathogenic characteristics.

Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B.

GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer.

BACKGROUND: Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have undergone an EMT. RESULTS: Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3 expression correlates with EMT markers and is prognostic of poor overall survival. CONCLUSIONS: Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy for the treatment of patients with poorly differentiated tumors.

NS2 proteins of GB virus B and hepatitis C virus share common protease activities and membrane topologies.

UNLABELLED: GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Determinants for membrane association of the hepatitis C virus NS2 protease domain.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is required for HCV polyprotein processing and particle assembly. It comprises an N-terminal membrane domain and a C-terminal, cytosolically oriented protease domain. Here, we demonstrate that the NS2 protease domain itself associates with cellular membranes. A single charged residue in the second α-helix of the NS2 protease domain is required for proper membrane association, NS2 protein stability, and efficient HCV polyprotein processing.

Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease.

UNLABELLED: The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. CONCLUSION: We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease.

Genetic analyses reveal a role for vitamin D insufficiency in HCV-associated hepatocellular carcinoma development.

BACKGROUND: Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99-1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12-2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13-1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis. CONCLUSIONS/SIGNIFICANCE: Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.

Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic α-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.

BMP-2 and TGF-beta1 differentially control expression of type II procollagen and alpha 10 and alpha 11 integrins in mouse chondrocytes.

Bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 are multifunctional cytokines both proposed as stimulants for cartilage repair. Thus it is crucial to closely examine and compare their effects on the expression of key markers of the chondrocyte phenotype, at the gene and protein level. In this study, the expression of alpha 10 and alpha 11 integrin subunits and the IIA/IIB spliced forms of type II procollagen have been monitored for the first time in parallel in the same in vitro model of mouse chondrocyte dedifferentiation/redifferentiation. We demonstrated that TGF-beta1 stimulates the expression of the non-chondrogenic form of type II procollagen, IIA isoform, and of a marker of mesenchymal tissues, i.e. the alpha 11 integrin subunit. On the contrary, BMP-2 stimulates the cartilage-specific form of type II procollagen, IIB isoform, and a specific marker of chondrocytes, i.e. the alpha 10 integrin subunit. Collectively, our results demonstrate that BMP-2 has a better capability than TGF-beta1 to stimulate chondrocyte redifferentiation and reveal that the relative expressions of type IIB to type IIA procollagens and alpha 10 to alpha 11 integrin subunits are good markers to define the differentiation state of chondrocytes. In addition, adenoviral expression of Smad6, an inhibitor of BMP canonical Smad signaling, did not affect expression of total type II procollagen or the ratio of type IIA and type IIB isoforms in mouse chondrocytes exposed to BMP-2. This result strongly suggests that signaling pathways other than Smad proteins are involved in the effect of BMP-2 on type II procollagen expression.