Modulation of Hepatitis C Virus Genome Encapsidation by Nonstructural Protein 4B

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Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIII␣ enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC. IMPORTANCELike most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex and is required for HCV genome replication and replication complex formation. More importantly, this study demonstrates, for the first time, the crucial role of glycosphingolipids in the HCV life cycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.

mentioning

confidence: 81%

Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

Khan

Katikaneni

Han

et al. 2014

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“…In terms of replication, the NS3-5B region is sufficient to sustain viral RNA synthesis (12), and each of the proteins derived from this polyprotein is also known to contribute to virus production (30,(35)(36)(37)(38)(39)(40)(41)(42)(44)(45)(46)(47)(48). Our previous studies have shown that the kinetics of cleavage at boundaries between the NS proteins plays a critical role in reg- , and G1911A refer to the NS3-5B polyprotein expressed in the second cistron for each construct; wt denotes wt versions of NS5A and NS4B, S2208I indicates the mutation in NS5A, and G1911A denotes the mutation in NS4B.…”

Section: Discussionmentioning

confidence: 99%

“…Cell imaging shows that the RC lies proximal to sites where virus assembly probably occurs, since double-stranded RNA and MAF colocalize with envelope proteins and core-coated lipid droplets (30)(31)(32); the latter structures are thought to represent both a repository and a transitionary step in the maturation of core from a monomeric protein to a capsid shell (33,34). In addition, it has been conclusively demonstrated that mutations in the NS proteins can affect the efficiency of virus particle production independently of any effect that these changes may have on RNA replication (30,(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). Such data illustrate the tight link between viral RNA replication and virion assembly.…”

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confidence: 99%

Genetic Complementation of Hepatitis C Virus Nonstructural Protein Functions Associated with Replication Exhibits Requirements That Differ from Those for Virion Assembly

Herod

Schregel

Hinds

et al. 2014

Within the polyprotein encoded by hepatitis C virus (HCV), the minimum components required for viral RNA replication lie in the NS3-5B region, while virion assembly requires expression of all virus components. Here, we have employed complementation systems to examine the role that HCV polyprotein precursors play in RNA replication and virion assembly. In a trans-complementation assay, an HCV NS3-5A polyprotein precursor was required to facilitate efficient complementation of a replicationdefective mutation in NS5A. However, this requirement for precursor expression was partially alleviated when a second functional copy of NS5A was expressed from an additional upstream cistron within the RNA to be rescued. In contrast, rescue of a virion assembly mutation in NS5A was more limited but exhibited little or no requirement for expression of functional NS5A as a precursor, even when produced in the context of a second replicating helper RNA. Furthermore, expression of NS5A alone from an additional cistron within a replicon construct gave greater rescue of virion assembly in cis than in trans. Combined with the findings of confocal microscope analysis examining the extent to which the two copies of NS5A from the various expression systems colocalize, the results point to NS3-5A playing a role in facilitating the integration of nonstructural (NS) proteins into viral membrane-associated foci, with this representing an early stage in the steps leading to replication complex formation. The data further imply that HCV employs a minor virion assembly pathway that is independent of replication. IMPORTANCEIn hepatitis C virus-infected cells, replication is generally considered an absolute prerequisite for virus particle formation. Here we investigated the role that the viral protein NS5A has in both replication and particle assembly using complementation assays and microscopy. We found that efficient rescue of replication required NS5A to be expressed as part of a larger polyprotein, and this correlated with detection of NS5A at sites where replication occurred. In contrast, rescue of particle assembly did not require expression of NS5A within the context of a polyprotein. Interestingly, although only partial restoration of particle assembly was possible by complementation, that proportion that could be rescued benefitted from expressing NS5A from the same RNA being packaged. Collectively, these findings provide new insight into aspects of polyprotein function. They also support the existence of a minor virion assembly pathway that bypasses replication.

“…For pestiviruses, almost all nonstructural proteins have been shown to be essential for the production of infectious viral particles (15)(16)(17)(18)(19). Pestiviral NS4B has not yet been characterized with regard to its role in virion morphogenesis but has been shown to be critical in HCV virion morphogenesis, suggesting a similar function in pestiviruses (20).…”

mentioning

confidence: 99%

Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses

Klemens

Dubrau

Tautz

2015

A peculiarity of the Flaviviridae is the critical function of nonstructural (NS) proteins for virus particle formation. For pestiviruses, like bovine viral diarrhea virus (BVDV), uncleaved NS2-3 represents an essential factor for virion morphogenesis, while NS3 is an essential component of the viral replicase. Accordingly, in natural pestivirus isolates, processing at the NS2-3 cleavage site is not complete, to allow for virion morphogenesis. Virion morphogenesis of the related hepatitis C virus (HCV) shows a major deviation from that of pestiviruses: while RNA replication also requires free NS3, virion formation does not depend on uncleaved NS2-NS3. Recently, we described a BVDV-1 chimera based on strain NCP7 encompassing the NS2-4B*-coding region of strain Osloss (E. Lattwein, O. Klemens, S. Schwindt, P. Becher, and N. Tautz, J Virol 86:427-437, 2012, doi:10.1128/JVI.06133-11). This chimera allowed for the production of infectious virus particles in the absence of uncleaved NS2-3. The Osloss sequence deviates in the NS2-4B* part from NCP7 in 48 amino acids and also has a ubiquitin insertion between NS2 and NS3. The present study demonstrates that in the NCP7 backbone, only two amino acid exchanges in NS2 (E1576V) and NS3 (V1721A) are sufficient and necessary to allow for efficient NS2-3-independent virion morphogenesis. The adaptation of a bicistronic virus encompassing an internal ribosomal entry site element between the NS2 and NS3 coding sequences to efficient virion morphogenesis led to the identification of additional amino acids in E2, NS2, and NS5B that are critically involved in this process. The surprisingly small requirements for approximating the packaging schemes of pestiviruses and HCV with respect to the NS2-3 region is in favor of a common mechanism in an ancestral virus. IMPORTANCEFor positive-strand RNA viruses, the processing products of the viral polyprotein serve in RNA replication as well as virion morphogenesis. For bovine viral diarrhea virus, nonstructural protein NS2-3 is of critical importance to switch between these processes. While free NS3 is essential for RNA replication, uncleaved NS2-3, which accumulates over time in the infected cell, is required for virion morphogenesis. In contrast, the virion morphogenesis of the related hepatitis C virus is independent from uncleaved NS2-NS3. Here, we demonstrate that pestiviruses can adapt to virion morphogenesis in the absence of uncleaved NS2-3 by just two amino acid exchanges. While the mechanism behind this gain of function remains elusive, the fact that it can be achieved by such minor changes is in line with the assumption that an ancestral virus already used this mechanism but lost it in the course of adapting to a new host/infection strategy. T he family Flaviviridae comprises the genera Flavivirus, Hepacivirus, Pestivirus, and Pegivirus (1-3). Pestiviruses, like bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV), are important pathogens causing significant economic damage in livestock industries (3)....