Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction

Abstract

Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets).In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCEHepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.A pproximately 180 million persons are estimated to be chronically infected by hepatitis C virus (HCV) worldwide, the majority of whom are ignorant of their carrier status until chronic infection progresses toward serious symptom...

Differential regulation of the Wnt/β-catenin pathway by hepatitis C virus recombinants expressing core from various genotypes

“…Therefore, it was essential to assess the sequence-specific effect of HCV core variants on the Wnt/β-catenin signaling pathway in the context of a relevant HCV infection model. Most existing HCV infection systems rely on Huh-7.5 cell line and Japanese fulminant hepatitis 1 strain (JFH1) of HCV genotype 2a 23 or cell culture-adapted intergenotypic recombinants encoding proteins associated with viral morphogenesis (core C, envelope glycoproteins E1/E2, ion channel protein p7, and nonstructural protein NS2) from a non-2a genotype connected to JFH1 nonstructural proteins NS3 to NS5B 24 , 25 . Here, we engineered core intergenotypic recombinant cDNAs within the backbone of a robust cell culture-adapted derivative of HCV JFH1 (Jad) 15 , 26 by replacing only the core coding sequence of JFH1 by that of 1aH77, 1aC, 1aCvar, 4aR, and 4fC (Fig.…”

“…Plasmid pJad 26 was derived from pJFH1 (genome-length HCV cDNA of subtype 2a) 23 and contains cDNA substitutions resulting in three amino acid changes that confer high-titer cell culture adaptation 15 . Plasmids pJad/C(1aH77), /C(1aC), /C(1aCvar), /C(4aR), and /C(4fC) were generated by an overlapping-PCR strategy 24 designed to substitute Jad 2a core coding sequence (nucleotides 341–913 of the genome length cDNA) by core sequences of the indicated HCV strains. The resulting PCR fragments contained unique Age I and Bsi WI restriction sites at their 5′ and 3′ extremities, respectively, which were used for cloning into pJad at the corresponding sites.…”

“…For normalization purposes, geometric means of mRNA levels of SFRS4, GAPDH, HMBS164, and BC002942 housekeeping genes were quantified in Huh-7.5 cell extracts 50 , 51 , while GAPDH gene was used in HEK293 cell extracts. Viral RNA was quantified by one step reverse transcription-quantitative PCR using 20 ng of total intracellular RNA and TaqMan RNA-to-CT™ 1-Step Kit (Applied Biosystems) with primers and probe targeting the HCV 5′ nontranslated region as described previously 24 . Viral RNA levels were normalized with respect to 18S RNA levels quantified in parallel using TaqMan ribosomal RNA control reagents (Applied Biosystems).…”

“…For sequencing purposes, viral RNA extracted from RNA-transfected or virus-infected cells was converted to cDNA using SuperScript II reverse transcriptase and d(pN)6, then amplified by PCR using One Taq 2 Master Mix with standard buffer (New England BioLabs) and a series of primer pairs spanning genomic segments of about 1500 bp. Following purification, the amplified DNAs were then subjected to direct sequencing as described previously 24 .…”

Distantly related hepaciviruses share common entry factor, Claudin-1

“…In fact, GBV-B has yet to be isolated from any animals other than those experimentally infected [14,16]. Nonetheless, due to HCV's restricted host tropism, infection of small New World monkeys (tamarins, marmosets, owl monkeys) with GBV-B has previously been employed as a surrogate to study HCV disease and correlates of protections [17][18][19][20][21]. GBV-B, like HCV, is associated with hepatitis and is primarily found in the liver of the infected host [22,23].…”