Timothy L. Tellinghuisen

Cellular cofactors affecting hepatitis C virus infection and replication

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus–host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2′-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the worlds population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56–58 kDa) with an amphipathic α-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-Å resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.

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.

Interaction between hepatitis C virus proteins and host cell factors.

Since the discovery of the hepatitis C virus (HCV) as the causative agent of non-A, non-B hepatitis, significant effort has been devoted to understanding this important pathogen. Despite the difficulty in culturing this virus efficiently, much is known about the organization of the viral genome and the functions of many of the viral proteins. Through the use of surrogate expression systems combined with cellular fractionation, pull-down experiments and yeast two-hybrid screens, numerous interactions between hepatitis C virus proteins and cellular components have been identified. The relevance of many of these interactions to hepatitis C biology remains to be demonstrated. This review discusses recent developments in this area of HCV research.

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

ABSTRACT 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 antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell transmission. 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.

Identification of Residues Required for RNA Replication in Domains II and III of the Hepatitis C Virus NS5A Protein

ABSTRACT The NS5A protein of hepatitis C virus (HCV) plays an important but undefined role in viral RNA replication. NS5A has been proposed to be a three-domain protein, and the crystal structure of the well-conserved amino-terminal domain I has been determined. The remaining two domains of NS5A, designated domains II and III, and their corresponding interdomain regions are poorly understood. We have conducted a detailed mutagenesis analysis of NS5A domains II and III using the genotype 1b HCV replicon system. The majority of the mutants containing 15 small (8- to 15-amino-acid) deletions analyzed were capable of efficient RNA replication. Only five deletion mutations yielded lethal phenotypes, and these were colinear, spanning a 56-amino-acid region within domain II. This region was further analyzed by combining triple and single alanine scanning mutagenesis to identify individual residues required for RNA replication. Based upon this analysis, 23 amino acids were identified that were found to be essential. In addition, two residues were identified that yielded a small colony phenotype while possessing only a moderate defect in RNA replication. These results indicate that the entire domain III region and large portions of domain II of the NS5A protein are not required for the function of NS5A in HCV RNA replication.

Studying Hepatitis C Virus: Making the Best of a Bad Virus

An estimated 3% of the world population is infected with hepatitis C virus (HCV) ([5][1], [232][2]). In most infected individuals, this remarkable RNA virus evades the immune system and establishes a chronic infection that can lead to cirrhosis, liver cancer, and death. While advances have been made

Alphavirus Nucleocapsid Protein Contains a Putative Coiled Coil α-Helix Important for Core Assembly

ABSTRACT The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an α-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with β-branched residues and conserved leucine residues occupying the a andd positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.

Development of novel therapies for hepatitis C.

The current standard of care for the treatment of hepatitis C virus (HCV) infection is a combination of pegylated IFN and ribavirin (Peg-IFN/RBV). Because of the adverse effects associated with both IFN and ribavirin and because Peg-IFN/RBV provides only about a 45-50% sustained virological response (SVR, undetectable HCV RNA for greater than 24 weeks after cessation of therapy) in genotype 1-infected individuals, there is a need for more potent anti-HCV compounds with fewer adverse effects. The twenty-first International Conference on Antiviral Research held in May 2009 in Miami Beach, Florida, featured a special session focused on novel targets for HCV therapy. The session included presentations by world-renowned experts in HCV virology and covered a diverse array of potential targets for the development of new classes of HCV therapies. This review contains concise summaries of discussed topics that included the innate immune response, virus entry, the NS2 protease, the NS3 helicase, NS4B, and NS5A. Each presenter discussed the current knowledge of these targets and provided examples of recent scientific breakthroughs that are enhancing our understanding of these targets. As our understanding of the role of these novel anti-HCV targets increases so will our ability to discover new, more safe and effective anti-HCV therapies.

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

ABSTRACT The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx17CxCx20C 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 Cx22CxCx24C 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.