Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles

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Hepatitis C virus (HCV) core plays a key role in viral particle formation and is involved in viral pathogenesis. Here, constructs for single-domain intrabodies consisting of variable regions derived from mouse mAbs against HCV core were established. Expressed single-domain intrabodies were shown to bind to HCV core, and inhibit the growth of cell culture-produced HCV derived from JFH-1 (genotype 2a) and a TH (genotype 1b)/JFH-1 chimera. Adenovirus vectors expressing intrabodies were also capable of reducing HCV propagation. Intrabody expression did not affect viral entry or genome replication of single-round infectious trans-complemented HCV particles. However, intrabody expression reduced intracellular and extracellular infectious titres in CD81-defective Huh7-25 cells transfected with the HCV genome, suggesting that these intrabodies impair HCV assembly. Furthermore, intrabody expression suppressed HCV core-induced NFk B promoter activity. These intrabodies may therefore serve as tools for elucidating the role of core in HCV pathogenesis.

Section: Resultsmentioning

confidence: 99%

Single-domain intrabodies against hepatitis C virus core inhibit viral propagation and core-induced NFκB activation

Suzuki

Saito

Matsuda

et al. 2016

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“…The NS5A protein of CSFV, BVDV and HCV has been shown to be an essential replicase component (Masaki et al, 2008;Tellinghuisen et al, 2006;Sheng et al, 2010), thus, ANXA2 might participate in the CSFV production process by binding to NS5A protein. In fact, ANXA2 has been found to be involved in the formation of HCV replication complex on the lipid raft (Saxena et al, 2012).…”

Section: W I T H O U T S I R N a C O N T R O L S I R N A A N T I -A Nmentioning

confidence: 99%

“…Previous reports showed that HCV NS5A is necessary for HCV assembly and production and interacts with ANXA2 (Appel et al, 2008;Masaki et al, 2008;Saxena et al, 2012). To investigate whether ANXA2 interacts with CSFV NS5A, several NS5A mutants (Fig.…”

mentioning

confidence: 99%

Annexin A2 is involved in the production of classical swine fever virus infectious particles

Sheng

Liu

Jiang

et al. 2015

Annexin A2 (ANXA2) is an important host factor regulating several key processes in many viruses. To evaluate the potential involvement of ANXA2 in the life cycle of classical swine fever virus (CSFV), an RNA interference (RNAi) approach was utilized. Knockdown of ANXA2 did not impair CSFV RNA replication but significantly reduced CSFV production. A comparable reduction of extracellular and intracellular infectivity levels was detected, indicating that ANXA2 might play a role in CSFV assembly rather than in genome replication and virion release. Furthermore, ANXA2 was found to bind CSFV NS5A, an essential replicase component. Amino acids R338, N359, G378 of NS5A were revealed to be pivotal for the ANXA2-NS5A interaction. Substitutions of these amino acids had no effect on viral RNA replication but substantially reduced CSFV production, which might partly be due to these mutations destroying the ANXA2-NS5A interaction. These results suggested that ANXA2 might participate in CSFV production process by binding NS5A.

“…HCV NS5a is a phosphoprotein involved in replication and virus-particle formation. In both processes, the phosphorylation status of NS5a has been shown to be critical (Appel et al, 2005;Evans et al, 2004;Masaki et al, 2008;Tellinghuisen et al, 2008). The multifunctional protein NS3 might be regulated in a similar way.…”

mentioning

confidence: 99%

Characterization of hepatitis C virus NS3 modifications in the context of replication

Liefhebber

Hensbergen

Deelder

et al. 2009

Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV nonstructural protein 3 (NS3) has many functions. It has protease and helicase activities, interacts with several host-cell proteins and plays a role in translation, replication and virus-particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were digested in gel and analysed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.Post-translational modifications (PTMs) such as phosphorylation, prenylation, methylation and acetylation play an important role in protein activity, localization and protein-protein interactions (Mann & Jensen, 2003;Schweppe et al., 2003). It is not only that eukaryotes use this mechanism to modulate the function(s) of their proteins; viruses in particular require PTMs because their genome is small and events such as translation, replication, virus assembly and release all need to be well-orchestrated (Bartenschlager et al., 2004;Jakubiec & Jupin, 2007). The small 9.6 kb genome of hepatitis C virus (HCV), a positivestranded RNA virus, yields a single polyprotein that, after processing, results in three structural proteins (core, E1 and E2), a peptide (p7) and six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). Together, these 10 proteins coordinate the complete life cycle of HCV (Moradpour et al., 2007). For some of these proteins, it is known that they have a dual function involved in more than one process of the virus life cycle, requiring precise regulation (Kim et al., 2004;Lindenbach et al., 2007;Tellinghuisen et al., 2008;Yu et al., 2006).An apparent example of a multifunctional protein within the HCV genome is NS3, which possesses protease and helicase activities (Bartenschlager, 1999;Kim et al., 1995).The serine protease domain of NS3 is in the N-terminal one-third of the protein. Together with NS4a, it forms a stable complex that cleaves the non-structural proteins downstream of NS3 (Bartenschlager et al., 1995;Failla et al., 1994;Tanji et al., 1995). Besides these cleavages, the NS3/ 4A protease is known to cleave MAVS/IPS-1/VISA/Cardif and TRIF, which affect signalling of the RIG-I and Toll-like receptor 3 pathways, respectively, thereby abrogating the interferon response (Meurs & Breiman, 2007).In addition to its protease activity, the C terminus of NS3 can unwind RNA through ATPase and helicase activities (Kim et al...