Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites

Multiple novel members of the genus IMPORTANCEVirus infection is recognized by cellular sensor proteins triggering innate immune signaling and antiviral defenses. While viruses have evolved strategies to thwart these antiviral programs in their cognate host species, these evasion mechanisms are often ineffective in a novel host, thus limiting viral transmission across species. HCV, the best-characterized member of the genus Hepacivirus within the family Flaviviridae, uses its NS3/4A protease to disrupt innate immune signaling by cleaving the cellular adaptor protein MAVS. Recently, a large number of HCV-related viruses have been discovered in various animal species, including wild, livestock, and companion animals. We show that the NS3/4A proteases of these hepaciviruses from different animals and representing various clades of the genus cleave their cognate host MAVS proteins in addition to human MAVS. Therefore, cleavage of MAVS is a common strategy of hepaciviruses, and human MAVS is likely unable to limit replication of these nonhuman viruses upon zoonotic exposure.

Section: Methodsmentioning

confidence: 99%

Hepacivirus NS3/4A Proteases Interfere with MAVS Signaling in both Their Cognate Animal Hosts and Humans: Implications for Zoonotic Transmission

Anggakusuma

Brown

Banda

et al. 2016

“…It was based on the sequence of NS4B genotype 1a (pCV/H77C), which was analyzed with the programs TMHMM (24), HMMTOP (51), and MEMSAT (18). Since the NS3 protease, which is responsible for processing the polyprotein in the nonstructural region, is located on the cytoplasmic side of the ER membrane (1,12,14,53,54), we further assumed that both the N terminal and the C terminal of the protein were cytoplasmic.…”

Section: Prediction Of the Topology Of Ns4b In The Membranementioning

confidence: 99%

Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

Lundin

Monné

Widell

et al. 2003

183

220

Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in Flaviviridae.

“…It has been estimated that more than 3% of the world population is infected with HCV. The plus-strand HCV RNA genome is approximately 9,600 nucleotides in length and encodes a polyprotein precursor of about 3,010 amino acids, which is cleaved co-and posttranslationally by cellular and viral proteases to produce structural and nonstructural (NS) proteins (8,13,15). One of the NS proteins, NS5B, is an RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of HCV (5,30).…”

mentioning

confidence: 99%

Exploiting cis -Acting Replication Elements To Direct Hepatitis C Virus-Dependent Transgene Expression

Zhang

Sakamoto

Yoshida

et al. 2005

We describe here a novel targeting gene therapy strategy to direct gene expression responsive to hepatitis C virus (HCV). The goal was approached by engineering a construct containing the antisense sequence of the transgene and internal ribosome entry site of encephalomyocarditis virus flanked by 5-and 3-end sequences of HCV cDNA that contain cis-acting replication elements. Thus, expression of the transgene is only promoted when the minus-strand RNA has been synthesized by the functional replication machinery present in infected cells. Reporter assay and strand-specific reverse transcription-PCR showed selective transgene expression in Huh-7 cells harboring an autonomously replicating HCV subgenome but remaining silent in uninfected cells. Furthermore, using the cytosine deaminase suicide gene as a transgene coupled with recombinant adenovirus delivery, we demonstrated that cytosine deaminase was specifically expressed in replicon cells, resulting in marked chemosensitization of replicon cells to the cytotoxic effects of flucytosine. This new targeting strategy could be extended to other single-stranded RNA viruses encoding the unique RNA-dependent RNA polymerase that has no parallel in mammalian cells.Chronic hepatitis C virus (HCV) infection, which frequently leads to liver cirrhosis and hepatocellular carcinoma (3, 34), remains a major public health problem worldwide. It has been estimated that more than 3% of the world population is infected with HCV. The plus-strand HCV RNA genome is approximately 9,600 nucleotides in length and encodes a polyprotein precursor of about 3,010 amino acids, which is cleaved co-and posttranslationally by cellular and viral proteases to produce structural and nonstructural (NS) proteins (8,13,15). One of the NS proteins, NS5B, is an RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of HCV (5, 30).Current treatment modalities available for HCV infection, including alpha interferon, has limited effectiveness. Only 20 to 30% of alpha interferon-treated patients develop a sustained remission, and increased or prolonged systemic administration is often associated with severe side effects or viral resistance. The combination of ribavirin and interferon is known to be significantly more effective than interferon monotherapy in naïve and relapser patients, but it induces a sustained response only in 41% of patients and in less than 30% of patients infected with genotype 1 (18). The therapeutic potential of small inhibitory molecules that target serine protease, helicase, or RdRp has proved to be promising (4, 6, 10), however, one unavoidable problem of this approach is selection of resistant mutants conferred by single or multiple mutations due to the error-prone nature of RdRp (24,26,29). Thus, an alternative approach for treating HCV patients that results in the death of infected cells, thereby limiting or eliminating virus production, while leaving uninfected cells unharmed would have an advantage over the HCV therapies now available.Here we describe a targeting gen...