Structural and Functional Studies of Nonstructural Protein 2 of the Hepatitis C Virus Reveal Its Key Role as Organizer of Virion Assembly

Abstract

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.

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

“…Our result that DGAT1 is a key determinant for the localization of NS5A to LDs in the absence of other viral components is in agreement with recent reports showing that the presence of other viral components (e.g. NS2 or core) modulates, but is not critical to the intracellular trafficking of NS5A (22,23). Other non-structural viral proteins implicated in viral assembly (e.g.…”

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

“…Along these lines, HCV NS2 has been shown to interact either directly or indirectly with E1, E2, p7, NS3, NS4A, NS5A, and NS5B (44)(45)(46)(47)(48). Another study revealed interactions of NS2 with E1, E2, and p7, which are mediated by the transmembrane segments of the respective proteins (49). Similar studies for pestiviruses will significantly benefit from our identified gain-of-function mutants in NS2 and NS3.…”

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).…”

“…However, the 16 Å resolution of this structure was not sufficient to discern the precise arrangement of protomers within the channel complex, making further atomic structural information highly desirable. Early solution NMR studies yielded the structure of the genotype 1b p7 C terminus (PDB ID: 2K8J) (Saint et al, 2009), as well as an NMRguided molecular dynamics model of the complete monomer in a hairpin conformation (Montserret et al, 2010). Subsequent solid-state NMR investigations also supported a monomeric hairpin, albeit with altered helical positioning (Cook & Opella, 2010.…”