The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences

Abstract

The role of the hepatitis C virus (HCV) p7 protein in the virus life cycle is not known. Previous in vitro data indicated that this 63-aa polypeptide is located in the endoplasmic reticulum and has two transmembrane domains (TMDs) connected by a cytoplasmic loop; the aminoand carboxyl-terminal tails are oriented toward the endoplasmic reticulum lumen. Furthermore, recent in vitro studies suggested that HCV p7 could function as a virus-encoded ion channel. It might therefore be a relevant target for future drug development. We studied the role of HCV p7 in vivo. Because HCV does not replicate efficiently in cell culture, we mutagenized p7 of an infectious genotype 1a cDNA clone and tested RNA transcripts of each mutant for infectivity in chimpanzees by intrahepatic transfection. Appropriate processing of mutant polypeptides was confirmed by studies in transfected mammalian cells. Mutants with deletions of all or part of p7 and a mutant with substitutions of two conserved residues in the cytoplasmic loop were not viable. Thus, p7 is essential for infectivity of HCV. A chimera in which the p7 of the 1a clone was replaced with p7 from an infectious genotype 2a clone also was not viable. This finding suggests a genotype-specific interaction between p7 and other genomic regions. To define which portions of p7 played the most significant role for this interaction, we tested three chimeras with the 1a backbone in which only specific domains of p7 had the 2a sequence. A p7 chimera with 2a tails and TMDs and the 1a cytoplasmic loop was not viable. A mutant with 2a tails and cytoplasmic loop and 1a TMDs also was not viable. However, a p7 chimera with 2a TMDs and cytoplasmic loop and 1a tails was viable. The transfected chimpanzee became viremic at week 2, and recovered viruses had the chimeric sequence. These data indicate that the amino-and͞or carboxyl-terminal intraluminal tails of p7 contain sequences with genotype-specific function. H epatitis C virus (HCV), a member of the Flaviviridae family, is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. HCV is an enveloped virus with a positive single-stranded RNA genome that encodes a single polyprotein precursor of Ϸ3,000 aa that is cleaved into 10 proteins by host or viral proteases (1, 2). These include three structural proteins [one core (C) and two envelope (E1 and E2) glycoproteins], as well as six nonstructural proteins (NS2-NS5B) involved with polyprotein processing and RNA replication. However, it is not known whether the 10th protein, p7, is a structural or nonstructural protein. Studies of HCV subgenomic replicons showed that p7 is not critical for RNA replication (3), but its actual role in the virus life cycle has not been determined. The corresponding protein of bovine viral diarrhea virus (BVDV), another member of the Flaviviridae family, was critical for cell culture infectivity (4). Transfection with RNA transcripts from an infectious BVDV cDNA clone with in-frame deletions or point mutations of p7 could not produce...

Characterization of GB Virus B Polyprotein Processing Reveals the Existence of a Novel 13-kDa Protein with Partial Homology to Hepatitis C Virus p7 Protein

“…In a reverse genetics study of bovine viral diarrhea virus, it was shown that a large in-frame deletion of p7 does not affect RNA replication but prevents the formation of infectious virions (15). The role of HCV p7 in the virus life cycle is unclear at present, but a genome-length HCV RNA in which the p7 sequence had been deleted was not infectious upon intrahepatic inoculation in a chimpanzee (56). A subsequent analysis of genome-length molecular clones of HCV containing chimeric intertypic p7 sequences revealed that there was a genotype-specific p7 requirement localized to the N-and/or C-terminal regions of this polypeptide, suggesting that it may interact with other proteins encoded by HCV in a genotype-specific manner (56).…”

Viroporins: structure, function and potential as antiviral targets

“…p7 channels displayed both single-channel and burst-like behaviour, consistent with channel-pore dualism. Interest in p7 as a potential ion channel therapeutic target was stimulated by chimpanzee studies that showed it to be essential for HCV propagation in vivo (Sakai et al, 2003).…”

Unravelling hepatitis C virus replication from genome to function

“…As this overlaps with the IRES, there is considerable interest in understanding how this region might modulate translation and replication, which are unlikely to occur simultaneously on the same RNA template. The 3፱ NCR has been found to contain a nonessential variable region, a poly-U/UC tract that must be more than 26 nucleotides long, followed by a highly conserved and essential 3፱X domain 55,57,76 . Recently a conserved stem-loop structure within the NS5B coding region, 5BSL3.2, was found to be required for RNA replication 78 .…”

“…Further studies indicated that 5BSL3.2 forms functionally important long-distance base pairs bled and shown to be infectious by direct intrahepatic injection of RNA transcripts into chimpanzees 52,53 . These infectious clones were used to show that all viral enzyme activities, the p7 gene and the correct genomic 3፱ end are necessary for HCV replication in vivo [54][55][56] . In contrast, the hypervariable N-terminal region of E2 is dispensible 57 .…”