SPCS1-Dependent E2-p7 processing determines HCV Assembly efficiency.

Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1's roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.

Delayed by Design: Role of Suboptimal Signal Peptidase Processing of Viral Structural Protein Precursors in Flaviviridae Virus Assembly.

The Flaviviridae virus family is classified into four different genera, including flavivirus, hepacivirus, pegivirus, and pestivirus, which cause significant morbidity and mortality in humans and other mammals, including ruminants and pigs. These are enveloped, single-stranded RNA viruses sharing a similar genome organization and replication scheme with certain unique features that differentiate them. All viruses in this family express a single polyprotein that encodes structural and nonstructural proteins at the N- and C-terminal regions, respectively. In general, the host signal peptidase cleaves the structural protein junction sites, while virus-encoded proteases process the nonstructural polyprotein region. It is known that signal peptidase processing is a rapid, co-translational event. Interestingly, certain signal peptidase processing site(s) in different Flaviviridae viral structural protein precursors display suboptimal cleavage kinetics. This review focuses on the recent progress regarding the Flaviviridae virus genus-specific mechanisms to downregulate signal peptidase-mediated processing at particular viral polyprotein junction sites and the role of delayed processing at these sites in infectious virus particle assembly.

Extended interaction networks with HCV protease NS3-4A substrates explain the lack of adaptive capability against protease inhibitors.

Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168 To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.

Multispectral Imaging Enables Characterization of Intrahepatic Macrophages in Patients With Chronic Liver Disease.

Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared with stellate cells, the role of macrophages in the development of fibrosis remains unclear. Multispectral imaging allows detection of multiple markers in situ in human formalin-fixed, paraffin-embedded tissue. This cutting-edge technology is ideal for analyzing human liver tissues, as it allows spectral unmixing of fluorophore signals, subtraction of auto-fluorescence, and preservation of hepatic architecture. We analyzed five different antibodies commonly observed on macrophage populations (CD68, MAC387, CD163, CD14, and CD16). After optimization of the monoplex stains and development of a Spectral Library, we combined all of the antibodies into a multiplex protocol and used them to stain biopsies collected from representative patients with chronic liver diseases, including chronic hepatitis C, nonalcoholic steatohepatitis, and autoimmune hepatitis. Various imaging modalities were tested, including cell phenotyping, tissue segmentation, t-distributed stochastic neighbor embedding plots, and phenotype matrices that facilitated comparison and visualization of the identified macrophage and other cellular profiles. We then tested the feasibility of this platform to analyze numerous regions of interest from liver biopsies with multiple patients per group, using batch analysis algorithms. Five populations showed significant differences between patients positive for hepatitis C virus with advanced fibrosis when compared with controls. Three of these were significantly increased in patients with advanced fibrosis when compared to controls, and these included CD163+CD16+, CD68+, and CD68+MAC387+. Conclusion: Spectral imaging microscopy is a powerful tool that enables in situ analysis of macrophages and other cells in human liver biopsies and may lead to more personalized therapeutic approaches in the future.

Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly.IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

Fibrogenic Gene Expression in Hepatic Stellate Cells Induced by HCV and HIV Replication in a Three Cell Co-Culture Model System.

Retrospective studies indicate that co-infection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) accelerates hepatic fibrosis progression. We have developed a co-culture system (MLH) comprising primary macrophages, hepatic stellate cells (HSC, LX-2), and hepatocytes (Huh-7), permissive for active replication of HCV and HIV, and assessed the effect of these viral infections on the phenotypic changes and fibrogenic gene expression in LX-2 cells. We detected distinct morphological changes in LX-2 cells within 24 hr post-infection with HCV, HIV or HCV/HIV in MLH co-cultures, with migration enhancement phenotypes. Human fibrosis microarrays conducted using LX-2 cell RNA derived from MLH co-culture conditions, with or without HCV and HIV infection, revealed novel insights regarding the roles of these viral infections on fibrogenic gene expression in LX-2 cells. We found that HIV mono-infection in MLH co-culture had no impact on fibrogenic gene expression in LX-2 cells. HCV infection of MLH co-culture resulted in upregulation (>1.9x) of five fibrogenic genes including CCL2, IL1A, IL1B, IL13RA2 and MMP1. These genes were upregulated by HCV/HIV co-infection but in a greater magnitude. Conclusion: Our results indicate that HIV-infected macrophages accelerate hepatic fibrosis during HCV/HIV co-infection by amplifying the expression of HCV-dependent fibrogenic genes in HSC.

HCV NS5A dimer interface residues regulate HCV replication by controlling its self-interaction, hyperphosphorylation, subcellular localization and interaction with cyclophilin A.

The HCV NS5A protein plays multiple roles during viral replication, including viral genome replication and virus particle assembly. The crystal structures of the NS5A N-terminal domain indicated the potential existence of the NS5A dimers formed via at least two or more distinct dimeric interfaces. However, it is unknown whether these different forms of NS5A dimers are involved in its numerous functions. To address this question, we mutated the residues lining the two different NS5A dimer interfaces and determined their effects on NS5A self-interaction, NS5A-cyclophilin A (CypA) interaction, HCV RNA replication and infectious virus production. We found that the mutations targeting either of two dimeric interfaces disrupted the NS5A self-interaction in cells. The NS5A dimer-interrupting mutations also inhibited both viral RNA replication and infectious virus production with some genotypic differences. We also determined that reduced NS5A self-interaction was associated with altered NS5A-CypA interaction, NS5A hyperphosphorylation and NS5A subcellular localization, providing the mechanistic bases for the role of NS5A self-interaction in multiple steps of HCV replication. The NS5A oligomers formed via different interfaces are likely its functional form, since the residues at two different dimeric interfaces played similar roles in different aspects of NS5A functions and, consequently, HCV replication. In conclusion, this study provides novel insight into the functional significance of NS5A self-interaction in different steps of the HCV replication, potentially, in the form of oligomers formed via multiple dimeric interfaces.

Efficient Suppression of Hepatitis C Virus Replication by Combination Treatment with miR-122 Antagonism and Direct-acting Antivirals in Cell Culture Systems.

Direct-acting antivirals (DAAs) against Hepatitis C virus (HCV) show effective antiviral activity with few side effects. However, the selection of DAA-resistance mutants is a growing problem that needs to be resolved. In contrast, miR-122 antagonism shows extensive antiviral effects among all HCV genotypes and a high barrier to drug resistance. In the present study, we evaluated three DAAs (simeprevir, daclatasvir, and sofosbuvir) in combination with anti-miR-122 treatment against HCV genotype 1a in cell cultures. We found that combination treatments with anti-miR-122 and a DAA had additive or synergistic antiviral effects. The EC50 values of simeprevir in simeprevir-resistant mutants were significantly decreased by combining simeprevir with anti-miR-122. A similar reduction in EC50 in daclatasvir-resistant mutants was achieved by combining daclatasvir with anti-miR-122. Combination treatment in HCV-replicating cells with DAA and anti-miR-122 sharply reduced HCV RNA amounts. Conversely, DAA single treatment with simeprevir or daclatasvir reduced HCV RNA levels initially, but the levels later rebounded. DAA-resistant mutants were less frequently observed in combination treatments than in DAA single treatments. In summary, the addition of miR-122 antagonism to DAA single treatments had additive or synergistic antiviral effects and helped to efficiently suppress HCV replication and the emergence of DAA-resistant mutants.

Femoral Trochlear Groove Morphometry Assessed on Oblique Coronal MR Images.

OBJECTIVE: The objective of our study as to assess several indexes relevant to patellofemoral instability (PFI) associated with femoral trochlear dysplasia as measured on oblique coronal MR images at three standardized reference levels. MATERIALS AND METHODS: A total of 30 knee MRI examinations were selected as the study group of PFI patients. Sixty knee MRI examinations were included as a control group. MRI protocols included sagittal T2-weighted, axial proton density-weighted, and oblique coronal T2-weighted imaging. On a midline sagittal image, the following three levels of the femoral trochlear groove cartilage were determined: level 1 (one-fourth level of the trochlear groove in the midsagittal plane), level 2 (one-half level of the trochlear groove in the midsagittal plane), and level 3 (three-fourths level of the trochlear groove in the midsagittal plane). Three-level axial and oblique coronal images were selected using the sagittal image as a scout. Femoral trochlear indexes including the sulcus angle, sulcus depth, facet length, and trochlear groove area were measured on the axial and oblique coronal images. RESULTS: Most indexes showed significant differences between the PFI and control groups in the axial and oblique coronal planes at all three levels (p < 0.05). Almost all indexes measured on the oblique coronal plane images were significantly different from those measured on the axial plane images (p < 0.05). Oblique coronal images showed little variability in the sulcus angle among the three levels in contrast to a marked decrease in the angle from the proximal to distal level on axial images. CONCLUSION: Femoral trochlear indexes measured on oblique coronal knee MR images can be used to assess femoral trochlear dysplasia. Oblique coronal images showed less morphologic distortion of the distal femoral trochlear groove than axial images.

Unilateral Sacroiliitis: Differential Diagnosis Between Infectious Sacroiliitis and Spondyloarthritis Based on MRI Findings.

OBJECTIVE: The purpose of this study was to identify the MRI features that aid in the differentiation between infectious sacroiliitis and unilateral sacroiliitis associated with spondyloarthritis. MATERIALS AND METHODS: The MR images of 54 patients who received a diagnosis unilateral sacroiliitis between August 2001 and August 2013 were reviewed. MR images were evaluated for bone lesions (extent and distribution of bone marrow edema and presence and size of bone erosions), soft-tissue lesions (capsulitis, extracapsular fluid collections, and periarticular muscle edema), and joint space enhancement. The Fisher exact test was used for comparison of categoric data, and multivariate stepwise logistic regression analysis was performed. RESULTS: Thick capsulitis, extracapsular fluid collection, and periarticular muscle edema were all more frequently observed in infectious sacroiliitis (p < 0.001). Iliac-dominant bone marrow edema and joint space enhancement were statistically significantly more common in spondyloarthritis (p < 0.001 and p = 0.014, respectively). The presence of periarticular muscle edema was the only independently differentiating variable on multivariate stepwise logistic regression analysis. When periarticular muscle edema was the sole predictor, unilateral sacroiliitis in spondyloarthritis was correctly identified in 77.3% of cases, and infectious sacroiliitis was correctly identified in 90.6% of cases. The overall accuracy was 85.2%. CONCLUSION: MRI features of the bone lesions, soft-tissue lesions, and joint space enhancement in unilateral sacroiliitis aid in the differential diagnosis between infection and spondyloarthritis. Among various findings, periarticular muscle edema was the single most important predictor of infectious sacroiliitis.

Immune and non-immune responses to hepatitis C virus infection.

The host innate and adaptive immune systems are involved in nearly every step of hepatitis C virus (HCV) infection. In patients, the outcome is determined by a series of complex host-virus interactions, whether it is a natural infection or results from clinical intervention. Strong and persistent CD8(+) and CD4(+) T-cell responses are critical in HCV clearance, as well as cytokine-induced factors that can directly inhibit virus replication. Newly available direct-acting antivirals (DAAs) are very effective in viral clearance in patients. DAA treatment may further result in the down-regulation of programmed death-1, leading to rapid restoration of HCV-specific CD8(+) T cell functions. In this review, we focus on recent studies that address the host responses critical for viral clearance and disease resolution. Additional discussion is devoted to the prophylactic vaccine development as well as to current efforts aimed at understanding the host innate responses against HCV infection. Current theories on how the ubiquitin system and interferon-stimulated genes may affect HCV replication are also discussed.

Coronary CT angiography findings based on smoking status: Do ex-smokers and never-smokers share a low probability of developing coronary atherosclerosis?

The relationship of coronary artery disease (CAD) in ex-smokers has not been elucidated, although smoking is considered to be one of the major risk factors of CAD. We investigate subclinical coronary atherosclerosis (SCA) in asymptomatic subjects with coronary computed tomography angiography (CCTA), according to smoking status, and determine whether ex-smokers share a low probability of developing CAD with never-smokers. We retrospectively enrolled 6930 self-referred asymptomatic adults who underwent both coronary artery calcium score (CACS) and CCTA. The prevalence and characteristics of SCA were assessed according to smoking status (never-, ex- and current smokers). After adjusting for variable risk factors, we used multivariate logistic regression for adjusted odds ratios (AOR) of high CACS (>100), SCA (any plaque), significant stenosis (>50 % in luminal stenosis) and each plaque type (non-calcified, mixed and calcified plaque) among the three groups. The prevalence of SCA was highest in the ex-smokers (35.4 %) and the prevalence of significant stenosis in ex-smokers (6.9 %) was as high as in current smokers (6.4 %). However, after adjusting for variable risk factors, SCA was significantly correlated with both ex-smokers (AOR; 1.21) and current smokers (AOR; 1.25), whereas significant stenosis was correlated only with current smokers (AOR; 1.91). The association between SCA and ex-smokers is as strong as with current smokers, although significant stenosis is only correlated with current smokers; thus, not only quitting smoking but also never initiating smoking would be helpful to reduce the progression of the SCA.

Detergent-resistant membrane association of NS2 and E2 during hepatitis C virus replication.

UNLABELLED: Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-ß-cyclodextrin (MßCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MßCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. IMPORTANCE: The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-ß-cyclodextrin (MßCD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process.

Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.

Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

C2 nerve root on magnetic resonance imaging of occipital neuralgia.

STUDY DESIGN: Review and grade the morphology of the C1-C2 neural foramina, from the MR images of patients who underwent C1-C2 spinal surgery, and determine the relationship with ON. OBJECTIVE: To evaluate the feasibility of MRI for C1-C2 neural foramen evaluation with a new grading system and to correlate the C1-C2 neural foramen grade with ON. SUMMARY OF BACKGROUND DATA: There have been no MRI studies of patients with and without ON in relation to C2 nerve root ganglion findings. METHODS: Among the registry of 124 patients who underwent C1-C2 spinal surgery between July 2004 and May 2012 in Seoul National University Bundang Hospital, we enrolled 101 patients who had information about ON and a relevant preoperative cervical spine MR image. A total of 202 neural foramina were evaluated with our new C1-C2 neural foramen grading system (grade, 0-3) using consensus reading by 2 experienced radiologists who were blinded to the clinical information. The relationship between the C1-C2 grading system and ON was assessed using a χ test and Fisher exact test. Inter- and intraobserver reliability agreement was assessed using the κ statistic. RESULTS: All C1-C2 neural foramina were delineated on T2 parasagittal images. Among 202 C1-C2 neural foramina, grade zero was found in 49 foramina (24.3%), grade 1 in 95 (47.0%), grade 2 in 30 (14.9%), and grade 3 in 28 (13.9%). Grade 1 stenosis was most frequently noted. The grade 2 group had the most frequent prevalence of ON (43.3%), followed by grade 3 (35.7%), grade zero (30.6%), and grade 1 (29.5%). However, the relationship between the grade and ON was not statistically significant. Inter- and intraobserver agreements were substantially high. CONCLUSION: C1-C2 neural foramina can be depicted on MR image. However, the relationship between the new grading system for C1-C2 neural foramina and ON was not statistically significant. LEVEL OF EVIDENCE: 4.

Evolution of a cell culture-derived genotype 1a hepatitis C virus (H77S.2) during persistent infection with chronic hepatitis in a chimpanzee.

UNLABELLED: Persistent infection is a key feature of hepatitis C virus (HCV). However, chimpanzee infections with cell culture-derived viruses (JFH1 or related chimeric viruses that replicate efficiently in cell culture) have been limited to acute-transient infections with no pathogenicity. Here, we report persistent infection with chronic hepatitis in a chimpanzee challenged with cell culture-derived genotype 1a virus (H77S.2) containing 6 cell culture-adaptive mutations. Following acute-transient infection with a chimeric H77/JFH1 virus (HJ3-5), intravenous (i.v.) challenge with 10(6) FFU H77S.2 virus resulted in immediate seroconversion and, following an unusual 4- to 6-week delay, persistent viremia accompanied by alanine aminotransferase (ALT) elevation, intrahepatic innate immune responses, and diffuse hepatopathy. This first persistent infection with cell culture-produced HCV provided a unique opportunity to assess evolution of cell culture-adapted virus in vivo. Synonymous and nonsynonymous nucleotide substitution rates were greatest during the first 8 weeks of infection. Of 6 cell culture-adaptive mutations in H77S.2, Q1067R (NS3) had reverted to Q1067 and S2204I (NS5A) was replaced by T2204 within 8 weeks of infection. By 62 weeks, 4 of 6 mutations had reverted to the wild-type sequence, and all reverted to the wild-type sequence by 194 weeks. The data suggest H77S.2 virus has greater potential for persistence and pathogenicity than JFH1 and demonstrate both the capacity of a nonfit virus to persist for weeks in the liver in the absence of detectable viremia as well as strong selective pressure against cell culture-adaptive mutations in vivo. IMPORTANCE: This study shows that mutations promoting the production of infectious genotype 1a HCV in cell culture have the opposite effect and attenuate replication in the liver of the only fully permissive animal species other than humans. It provides the only example to date of persistent infection in a chimpanzee challenged with cell culture-produced virus and provides novel insight into the forces shaping molecular evolution of that virus during 5 years of persistent infection. It demonstrates that a poorly fit virus can replicate for weeks within the liver in the absence of detectable viremia, an observation that expands current concepts of HCV pathogenesis and that is relevant to relapses observed with direct-acting antiviral therapies.

Efficiency of E2-p7 processing modulates production of infectious hepatitis C virus.

Previous studies indicate that the processing of hepatitis C virus (HCV) E2-p7-NS2 precursor mediated by host signal peptidase is relatively inefficient, resulting in the accumulation of E2-p7-NS2 and E2-p7 precursors in addition to E2 in mammalian cells. In this study, we discovered that a significant inhibition of the processing at an E2-p7 junction site is detrimental for HCV production, whether it was caused by the mutations in p7 or by the strategic introduction of a mutation at a terminal residue of E2 to block the signal peptidase-mediated cleavage of this junction site. However, complete separation of E2 and p7 by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between these two proteins also moderately inhibited virus production. These results indicate that optimal processing of the E2-p7 junction site is critical for efficient HCV production. We further demonstrated that disrupting E2-p7 processing inhibits both NS2 localization to the putative virus assembly sites near lipid droplets (LD) and NS2 interaction with NS3 and E2. However, the impact, if any, of the p7-NS2 processing efficiency on HCV production seems relatively minor. In conclusion, these results imply that effective release of E2 and p7 from the precursor E2-p7 promotes HCV production by enhancing NS2-associated virus assembly complex formation near LD.

MicroRNA-27a regulates lipid metabolism and inhibits hepatitis C virus replication in human hepatoma cells.

The replication and infectivity of the lipotropic hepatitis C virus (HCV) are regulated by cellular lipid status. Among differentially expressed microRNAs (miRNAs), we found that miR-27a was preferentially expressed in HCV-infected liver over hepatitis B virus (HBV)-infected liver. Gene expression profiling of Huh-7.5 cells showed that miR-27a regulates lipid metabolism by targeting the lipid synthetic transcription factor RXRα and the lipid transporter ATP-binding cassette subfamily A member 1. In addition, miR-27a repressed the expression of many lipid metabolism-related genes, including FASN, SREBP1, SREBP2, PPARα, and PPARγ, as well as ApoA1, ApoB100, and ApoE3, which are essential for the production of infectious viral particles. miR-27a repression increased the cellular lipid content, decreased the buoyant density of HCV particles from 1.13 to 1.08 g/cm(3), and increased viral replication and infectivity. miR-27a overexpression substantially decreased viral infectivity. Furthermore, miR-27a enhanced in vitro interferon (IFN) signaling, and patients who expressed high levels of miR-27a in the liver showed a more favorable response to pegylated IFN and ribavirin combination therapy. Interestingly, the expression of miR-27a was upregulated by HCV infection and lipid overload through the adipocyte differentiation transcription factor C/EBPα. In turn, upregulated miR-27a repressed HCV infection and lipid storage in cells. Thus, this negative feedback mechanism might contribute to the maintenance of a low viral load and would be beneficial to the virus by allowing it to escape host immune surveillance and establish a persistent chronic HCV infection.

Contrasting roles of mitogen-activated protein kinases in cellular entry and replication of hepatitis C virus: MKNK1 facilitates cell entry.

The human kinome comprises over 800 individual kinases. These contribute in multiple ways to regulation of cellular metabolism and may have direct and indirect effects on virus replication. Kinases are tempting therapeutic targets for drug development, but achieving sufficient specificity is often a challenge for chemical inhibitors. While using inhibitors to assess whether c-Jun N-terminal (JNK) kinases regulate hepatitis C virus (HCV) replication, we encountered unexpected off-target effects that led us to discover a role for a mitogen-activated protein kinase (MAPK)-related kinase, MAPK interacting serine/threonine kinase 1 (MKNK1), in viral entry. Two JNK inhibitors, AS601245 and SP600125, as well as RNA interference (RNAi)-mediated knockdown of JNK1 and JNK2, enhanced replication of HCV replicon RNAs as well as infectious genome-length RNA transfected into Huh-7 cells. JNK knockdown also enhanced replication following infection with cell-free virus, suggesting that JNK actively restricts HCV replication. Despite this, AS601245 and SP600125 both inhibited viral entry. Screening of a panel of inhibitors targeting kinases that may be modulated by off-target effects of AS601245 and SP600125 led us to identify MKNK1 as a host factor involved in HCV entry. Chemical inhibition or siRNA knockdown of MKNK1 significantly impaired entry of genotype 1a HCV and HCV-pseudotyped lentiviral particles (HCVpp) in Huh-7 cells but had only minimal impact on viral RNA replication or cell proliferation and viability. We propose a model by which MKNK1 acts to facilitate viral entry downstream of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), both of which have been implicated in the entry process.

Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step.

The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited infection at an early postattachment step. Receptor binding studies demonstrated that H77.39 inhibited binding of soluble E2 protein to both CD81 and SR-B1, J6.36 blocked attachment to SR-B1 and modestly reduced binding to CD81, and H77.16 blocked attachment to SR-B1 only. Using yeast surface display, we localized epitopes for the neutralizing MAbs on the E2 protein. Two of the strongly inhibitory MAbs, H77.16 and J6.36, showed markedly reduced binding when amino acids within hypervariable region 1 (HVR1) and at sites ∼100 to 200 residues away were changed, suggesting binding to a discontinuous epitope. Collectively, these studies help to define the structural and functional complexity of antibodies against HCV E2 protein with neutralizing potential.