Interactions with phospholipids and membrane remodeling by nonstructural protein 5A of hepatitis C virus

Question Hepatitis C virus (HCV) is a global health threat with approximately 71 million individuals chronically infected worldwide. The nonstructural protein 5A (NS5A) of HCV plays a key role in the virus life cycle by (i) inducing formation of an RNA replication compartment, known as membranous web, and by (ii) controlling the switch from genomic RNA replication to viral particle assembly. NS5A consists of an N-terminal amphipathic helix (AH) intercalating into the cytosolic leaflet of cellular membranes, a folded domain 1 and domains 2 and 3 that are intrinsically unfolded in solution. The aim of the work was to gain insight into the molecular mechanisms underlying membrane sensing and remodeling by NS5A.

Methods We (i) expressed the full length wt protein (fl-NS5A), comprising its membrane attachment region, truncated protein constructs and proteins containing amino acid substitutions; (ii) analyzed their interactions with lipids in a protein lipid overlay assay (PLOA) and (iii) reconstituted fl-NS5A into liposomes and examined the liposomes by negative staining and transmission electron microscopy.

Results We show that AH is not the only membrane interacting domain of NS5A. We characterize a pattern of specific interactions of NS5A with phospholipids and show amino acids determining specificity of the interaction with phospholipids to localize in domain 1 of NS5A. We find NS5A-lipid interactions enhanced by domain 2 and 3, whereas mutagenesis of positively charged amino acid residues (K20A and K26A) in AH or deletion of AH and/or deletion of domains 2 and 3 from NS5A do not affect the specific interaction pattern of NS5A with phospholipids in PLOA. By using an in vitro liposome remodeling assay, we demonstrate that fl-NS5A senses lipid membranes followed by membrane curvature induction when reconstituted into liposomes.

Conclusions The data obtained elucidate novel aspects of NS5A function and unravel a distinct role of the protein domain architecture in the induction of the membranous web.

Publication History

Article published online:
04 January 2021