Neutralizing Antibody-Resistant Hepatitis C Virus Cell-to-Cell Transmission

Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal anti- bodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins rep- resenting the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell trans- mission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver

Complete replication of hepatitis C virus in cell culture.

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals

Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein

Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important

A Major Determinant of Cyclophilin Dependence and Cyclosporine Susceptibility of Hepatitis C Virus Identified by a Genetic Approach

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials

The NS5A Protein of Bovine Viral Diarrhea Virus Contains an Essential Zinc-Binding Site Similar to That of the Hepatitis C Virus NS5A Protein

The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx(17)CxCx(20)C and the presence of a similar sequence element in the NS5A proteins of members of the Pestivirus genus has led to the hypothesis that the NS5A protein of the pestivirus bovine viral diarrhea virus (BVDV) is a zinc-binding protein. A method for the expression and partial purification of BVDV NS5A was developed, and the partially purified protein was analyzed for zinc content by atomic absorption spectroscopy. BVDV NS5A was found to coordinate a single zinc atom per protein molecule. Mutation of any of the four cysteines of the predicted zinc-binding motif eliminated zinc coordination. Furthermore, analysis of mutations at these cysteine residues in the context of a BVDV replicon system indicated that these residues were absolutely essential for RNA replication. The recently determined crystal structure of the N-terminal zinc-binding domain of the HCV NS5A protein, combined with secondary structure predictions of the region surrounding the mapped BVDV zinc-binding region, indicates that the BVDV zinc-binding motif fits the general template Cx(22)CxCx(24)C and likely comprises a three-stranded antiparallel β-sheet fold. These data highlight the similarities between the Hepacivirus and Pestivirus NS5A proteins and suggest that both proteins perform a not-yet-defined function in RNA replication that requires coordination of a single zinc atom

Single amino acid mutations that mimic phosphorylation of serine 457 alter particle assembly.

<p>A). IHC analysis of cells electroporated with indicated point mutation bearing viral RNAs. AEED indicates mutation of serine 457 to alanine, DEED is the same residue changed to aspartic acid, SEEDcc is a change of the codon of serine 457 while retaining serine at this position. Rows of images are as described in 1B, 2B and 2D. B). RNA replication 48 hour time course of J6/JFH-1 (red line), J6/JFH-1 deletion B (blue line), J6/JFH-1 pol- (black line), AEED RNA (green line), DEED RNA (purple line), and SEEDcc RNA (light blue line) as determined by real time PCR analysis. C). Infectivity release 48 hours post electroporation from indicated viral RNA electroporated cells in units of tissue culture infectious dose 50% value per milliliter. D). Western blot analysis of NS5A from cells electroporated with replication competent full-length viral genomes as shown in panel A. Nomenclature used is as described in A.</p

Core protein release.

a<p>Values reported as average of three independent experiments in the format “average value (+/−standard deviation)” in units of femptomoles of HCV core protein per liter 24 hours post electroporation.</p

A Deletion in NS5A Domain III Disrupts the Production of Infectious Virus.

<p>A). Schematic representation of the domain organization of NS5A indicating the locations of the three domains and low complexity domain connectors (LCS). The location of the membrane anchoring helix is also shown. Numbers indicate amino acid positions on the Con1 genotype 1b NS5A sequence. Sequences of the C terminus of NS5A indicated by the black box on the schematic are shown below for representative sequences of major HCV genotypes. The sequences shown are 1a H77 (AF011753), 1b Con1 (AJ238799), 2a JFH-1 (AB047639), 2b HC-J8 (D10988), 3a HCVCEN (X76918), 4a ED43 (Y11604), 5a EUH1480 (Y13184), and 6a 6a33 (AY859526). B). IHC analysis of cells electroporated with indicated viral RNAs (top of image rows) for NS5A 48 hours post electroporation (top row of panels) or 48 hours post infection (bottom row of panels). C). Western blot of wild type (WT) and deletion B (del B) NS5A from cells electroporated with replication competent full-length genomes shown in B and D. D). RNA replication 48 hour time course of J6/JFH-1 (blue line), J6/JFH-1 deletion B (red line), or J6/JFH-1 pol- (black line) as determined by real time PCR analysis. E). RNA replication 48 hour time course of indicated subgenomic replicon RNAs. pSGR-JFH-1 (red line), pSGR-JFH-1 deletion B (blue line), and pSGR-JFH-1 pol- (black line). F). Infectivity release 48 hours (gray bars) and 6 days post (black bars) electroporation from indicated viral RNA electroporated cells in units of tissue culture infectious dose 50% value per milliliter. G). Infectivity released from cells via freeze-thaw treatment 48 hours post electroporation from indicated viral RNA electroporated cells. H). Relative HCV RNA release from cells electroporated with the indicated HCV constructs. Values reported are arbitrary units normalized to the RNA release by a non-replicating J6/JFH-1 pol-.</p