Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

Stapleford, Kenneth A.; Lindenbach, Brett D.

doi:10.1128/jvi.02268-10

Cited by 122 publications

(147 citation statements)

References 75 publications

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“…One well-characterized ion channel-independent p7 function is its interaction with NS2, targeting the latter to defined loci within infected cells where it is thought to act as a 'particle assembly scaffold' (Boson et al, 2011;Jirasko et al, 2008Jirasko et al, , 2010Ma et al, 2011;Popescu et al, 2011;Stapleford & Lindenbach, 2011;Tedbury et al, 2011). p7 and NS2 in concert control sub/genotypedependent compartmentalization of HCV core protein between the ER and lipid droplets, with more efficient particle production resulting from ER-associated core (Boson et al, 2011).…”

Section: Hcv P7mentioning

confidence: 99%

Viroporins: structure, function and potential as antiviral targets

Scott

Griffin

2015

Journal of General Virology

124

146

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The channel-forming activity of a family of small, hydrophobic integral membrane proteins termed 'viroporins' is essential to the life cycles of an increasingly diverse range of RNA and DNA viruses, generating significant interest in targeting these proteins for antiviral development. Viroporins vary greatly in terms of their atomic structure and can perform multiple functions during the virus life cycle, including those distinct from their role as oligomeric membrane channels. Recent progress has seen an explosion in both the identification and understanding of many such proteins encoded by highly significant pathogens, yet the prototypic M2 proton channel of influenza A virus remains the only example of a viroporin with provenance as an antiviral drug target. This review attempts to summarize our current understanding of the channel-forming functions for key members of this growing family, including recent progress in structural studies and drug discovery research, as well as novel insights into the life cycles of many viruses revealed by a requirement for viroporin activity. Ultimately, given the successes of drugs targeting ion channels in other areas of medicine, unlocking the therapeutic potential of viroporins represents a valuable goal for many of the most significant viral challenges to human and animal health.

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Section: Hcv P7mentioning

confidence: 99%

Viroporins: structure, function and potential as antiviral targets

Scott

Griffin

2015

Journal of General Virology

124

146

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“…It was suggested that NS5A is responsible for recruiting a replication complex to the vicinity of the LD since the localization of other nonstructural proteins in close proximity of LD depended on the localization of NS5A to LD (19). Several independent studies, including ours, showed that NS2 is involved in virus particle assembly by interacting with both the structural proteins E1 and E2 and also the nonstructural proteins NS3 and NS5A (25)(26)(27)(28). Interestingly, these NS2-mediated interactions are associated with colocalization of E1, E2, NS2, NS3, and NS5A at dot-like structures near LD, and the relative percentages of HCV replicating cells exhibiting these dot-like structures positively correlated with the infectious virus titer (25,26,28).…”

mentioning

confidence: 99%

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

Shanmugam

2013

J Virol

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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...

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“…The E1 and E2 envelope proteins are incorporated into ER membranes with ectodomains facing the ER lumen (9, 10). Later, they are recruited to assembly sites via the NS2 complex (11,12). Upon recruitment of all required viral components, HCV assembly is thought to occur at the surface of LDs (6-8, 13).…”

mentioning

confidence: 99%

Hepatitis C Virus Is Released via a Noncanonical Secretory Route

Bayer

Banning

Bruss

et al. 2016

J Virol

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We analyzed hepatitis C virus (HCV) morphogenesis using viral genomes encoding a mCherry-tagged E1 glycoprotein. HCV-E1-mCherry polyprotein expression, intracellular localization, and replication kinetics were comparable to those of untagged HCV, and E1-mCherry-tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and nonstructural NS5A followed a temporospatial pattern with a succinct decrease in the number of replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core, and E2 increased at E1-mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating an absence of viral glycoprotein processing by the Golgi apparatus. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggest that assembled HCV particles are released via a noncanonical secretory route involving the endosomal compartment. IMPORTANCEThe goal of this study was to shed light on the poorly understood trafficking and release routes of hepatitis C virus (HCV). For this, we generated novel HCV genomes which resulted in the production of fluorescently labeled viral particles. We used live-cell microscopy and other imaging techniques to follow up on the temporal dynamics of virus particle formation and trafficking in HCV-expressing liver cells. While viral particles and viral structural protein were found in endosomal compartments, no overlap of Golgi structures could be observed. Furthermore, biochemical and inhibitor-based experiments support a HCV release route which is distinguishable from canonical Golgi-mediated secretion. Since viruses hijack cellular pathways to generate viral progeny, our results point toward the possible existence of a not-yet-described cellular secretion route. Hepatitis C virus (HCV) belongs to the Flavivirus genus and has a positive-strand RNA genome. This encodes a polyprotein which is posttranslationally cleaved into six nonstructural (NS) proteins, the ion channel p7 protein, and the structural proteins Core, E1, and E2 (1). The NS proteins reside at the outer leaflet of the endoplasmic reticulum (ER) membrane where NS4B and NS5A in particular induce membrane alterations resulting in the formation of the membranous web, which is the major site for HCV replication (2-5). Core is targeted to adjacent lipid droplets (LDs) (6, 7), which represent intracellular lipid deposits and are considered important for production of infectious particles (1,7,8). The E1 and E2 envelope proteins are incorporated into ER membranes with ectodomains facing the ER lumen (9, 10). Later, they are recruited to assembly sites via the NS2 complex (11,12). Upon recruitment of all requi...

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Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

Cited by 122 publications

References 75 publications

Viroporins: structure, function and potential as antiviral targets

Viroporins: structure, function and potential as antiviral targets

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

Hepatitis C Virus Is Released via a Noncanonical Secretory Route

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