Efficiency of E2-p7 Processing Modulates Production of Infectious Hepatitis C Virus

Abstract

Previous studies indicate that the processing of hepatitis C virus (HCV) E2-p7-NS2 precursor mediated by host signal peptidase is relatively inefficient, resulting in the accumulation of E2-p7-NS2 and E2-p7 precursors in addition to E2 in mammalian cells. In this study, we discovered that a significant inhibition of the processing at an E2-p7 junction site is detrimental for HCV production, whether it was caused by the mutations in p7 or by the strategic introduction of a mutation at a terminal residue of E2 to block the signal peptidase-mediated cleavage of this junction site. However, complete separation of E2 and p7 by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between these two proteins also moderately inhibited virus production. These results indicate that optimal processing of the E2-p7 junction site is critical for efficient HCV production. We further demonstrated that disrupting E2-p7 processing inhibits both NS2 localization to the putative virus assembly sites near lipid droplets (LD) and NS2 interaction with NS3 and E2. However, the impact, if any, of the p7-NS2 processing efficiency on HCV production seems relatively minor. In conclusion, these results imply that effective release of E2 and p7 from the precursor E2-p7 promotes HCV production by enhancing NS2-associated virus assembly complex formation near LD. Hepatitis C virus (HCV) is one of the major agents responsible for causing severe liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 2). It is an enveloped, positive-stranded RNA virus belonging to the Hepacivirus genus in the Flaviviridae family of viruses (3, 4). HCV encodes a single, large open reading frame encoding an ϳ3,000-amino-acid polyprotein flanked by 5= and 3= noncoding sequences. The internal ribosome entry site (IRES) located at the 5= noncoding region mediates translation of a polyprotein that is subsequently processed by both host-and virus-encoded proteases to yield 10 different proteins. The N-terminal one-third of the polyprotein encoding the structural proteins, including core and envelope proteins (E1 and E2), followed by p7 and NS2, is processed by host signal peptidase (5-7). Core protein is further processed by signal peptide peptidase into a mature form (8). The junction site between NS2 and NS3 is cleaved by an NS2-NS3 autoprotease (9, 10), and the remaining nonstructural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) are processed by NS3 protease with its cofactor NS4A (11-13). Proteins NS3 to NS5B, along with the 5=-and 3=-terminal noncoding regions, were shown to be sufficient for the autonomous replication of HCV RNA (14).Recent advances in the infectious HCV cell culture model allowed the investigation of HCV virus particle assembly processes (15-18). We have learned that the association of HCV core protein with lipid droplets (LD) is critical for virus assembly since disrupting the core protein localization to LD inhibited infectious virus production (19,20). We also learned that the...

Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication

“…The results of our recent study suggested that p7 regulates NS2 localization to punctate sites near lipid droplets (punctate LD [see arrows in Fig. 1C]), which are considered putative virus assembly sites (10,12,13,16,20), and modulate virus production (10). However, the biochemical characteristics of these virus assembly sites are currently unknown.…”

“…Construction of HJ3-5 and its derivatives, including p7(KRAA), E2(AR), E2(AR)/IRES, HA p7, and HA p7/IRES, were described previously (8,10,16). To generate ⌬p7 mutants, the p7 sequence from HJ3-5/p7/IRES (10) was deleted by using the QuikChange II XL sitedirected mutagenesis kit (Stratagene, La Jolla, CA) with primer pairs encoding the C-terminal sequence of E2 and the N-terminal sequence of encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES): 5=-CATATCCCAAGCGGAGGCGTGAGTTTAAACAGACCAC AACGG-3= and 5=-CCGTTGTGGTCTGTTTAAACTCACGCCTCCGCT TGGGATATG-3=, respectively.…”

“…p7 is also involved in the virus particle assembly process by modulating capsid assembly and envelopment of viral particles (25). Although detailed mechanisms of how p7 affects viral assembly have not been characterized, one of the mechanisms may involve p7's regulation of NS2's subcellular localization (10,26). As we have shown recently, E2-p7 processing modulates infectious virus production by regulating NS2 localization to virus assembly sites, probably by controlling the release of p7 involved in this process (10).…”

“…The NS3-NS5B proteins are sufficient to form active replicase complexes involved in viral RNA replication (6). Interestingly, in addition to viral structural proteins, most of the nonstructural proteins were shown to affect HCV particle assembly (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). However, detailed mechanisms of HCV assembly are still unclear.…”

“…Although detailed mechanisms of how p7 affects viral assembly have not been characterized, one of the mechanisms may involve p7's regulation of NS2's subcellular localization (10,26). As we have shown recently, E2-p7 processing modulates infectious virus production by regulating NS2 localization to virus assembly sites, probably by controlling the release of p7 involved in this process (10). Interestingly, Tedbury and colleagues showed that the inframe expression of p7 in the context of NS2-5B subgenomic replicon induced the NS2 subcellular localization to the punctate sites near replication complexes and detergent-insoluble fractions (DIF) (26).…”