NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly

Abstract

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

“…NS2 and p7) (24 -26) do not localize to LDs in the absence of core or NS5A and do not interact with DGAT1 in our assays. These factors seem to be required at later stages in infectious particle production and regulate relocation of core from LDs to the ER as well as association of viral envelope protein E2 with viral factors, such as core and NS5A (24,25).…”

Viroporins: structure, function and potential as antiviral targets

“…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).…”

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

“…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).…”