The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein

Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from WT cells. These results suggest that this Y45F mutation in NS4A causes secretion of low density Core particles devoid of genomic HCV RNA. These results corroborate previous findings showing that mutation of the first hydrophobic region of E1 also causes secretion of Core complexes lacking RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.Author SummaryRNA viruses, which encompass both established and emerging pathogens, pose significant public health challenges. Viruses in the familyFlavivirdae, including Dengue virus, Zika virus and hepatitis C virus (HCV), continue to cause morbidity and mortality worldwide. One HCV protein, NS4A, has known functions in several steps of the viral lifecycle, however, how it contributes to viral particle production is not understood. Here, we investigated the role of one region of NS4A, the C-terminal acidic domain, in regulating the viral lifecycle. We found that some of the amino acids within this domain are important for viral envelopment to make infectious particles, specifically through interaction with the E1 glycoprotein. NS4A interacts with the first hydrophobic domain of E1. Disruption of this interaction prevents the production of infectious virus particles and instead results in release of low density Core protein complexes that lack HCV RNA into the cellular supernatant. Overall, our results reveal that NS4A is important for late stages of the HCV lifecycle and suggest that the interaction between NS4A and E1 may regulate the incorporation of viral RNA into the virion for the formation of infectious HCV particles.