Completion of Hepatitis C Virus Replication Cycle in Heterokaryons Excludes Dominant Restrictions in Human Non-liver and Mouse Liver Cell Lines

Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model

Susceptibility of Chikungunya Virus to Inactivation by Heat and Commercially and World Health Organization-Recommended Biocides.

Despite increasing clinical relevance of Chikungunya virus (CHIKV) infection, caused by a rapidly emerging pathogen, recommended guidelines for its inactivation do not exist. In this study, we investigated the susceptibility of CHIKV to inactivation by heat and commercially available hand, surface, and World Health Organization-recommended disinfectants to define CHIKV prevention protocols for healthcare systems

Subcellular localization and function of an epitope-tagged p7 viroporin in hepatitis C virus-producing cells.

The hepatitis C virus (HCV) viroporin p7 is crucial for production of infectious viral progeny. However, its role in the viral replication cycle remains incompletely understood, in part due to the poor availability of p7-specific antibodies. To circumvent this obstacle, we inserted two consecutive hemagglutinin (HA) epitope tags at its N terminus. HA-tagged p7 reduced peak virus titers ca. 10-fold and decreased kinetics of virus production compared to the wild-type virus. However, HA-tagged p7 rescued virus production of a mutant virus lacking p7, thus providing formal proof that the tag does not disrupt p7 function. In HCV-producing cells, p7 displayed a reticular staining pattern which colocalized with the HCV envelope glycoprotein 2 (E2) but also partially with viral nonstructural proteins 2, 3, and 5A. Using coimmunoprecipitation, we confirmed a specific interaction between p7 and NS2, whereas we did not detect a stable interaction with core, E2, or NS5A. Moreover, we did not observe p7 incorporation into affinity-purified virus particles. Consistently, there was no evidence supporting a role of p7 in viral entry, as an anti-HA antibody was not able to neutralize Jc1 virus produced from an HA-p7-tagged genome. Collectively, these findings highlight a stable interaction between p7 and NS2 which is likely crucial for production of infectious HCV particles. Use of this functional epitope-tagged p7 variant should facilitate the analysis of the final steps of the HCV replication cycle

Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation▿

Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing

How stable is the hepatitis C virus (HCV)? Environmental stability of HCV and its susceptibility to chemical biocides.

In the absence of a cell culture system for propagation of the hepatitis C virus (HCV), the antiviral activity of disinfectants against HCV was extrapolated from studies with the bovine viral diarrhea virus. The recent development of an HCV infection system allowed the direct assessment of environmental stability and susceptibility to chemical disinfectants

Inactivation and survival of hepatitis C virus on inanimate surfaces.

Hepatitis C virus (HCV) cross-contamination from inanimate surfaces or objects has been implicated in transmission of HCV in health-care settings and among injection drug users. We established HCV-based carrier and drug transmission assays that simulate practical conditions to study inactivation and survival of HCV on inanimate surfaces

Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment.

Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7

Landscape of protein-protein interactions during hepatitis C virus assembly and release

Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites