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

Date Published: February 7, 2019

Publisher: Public Library of Science

Author(s): Allison E. Roder, Christine Vazquez, Stacy M. Horner, Glenn Randall.


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. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. 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 wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV 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.

Partial Text

Hepatitis C virus (HCV) is a positive-sense RNA virus of the genus Hepacivirus in the Flaviviridae family. Over 70 million people worldwide are chronically infected with HCV and this chronic infection can lead to liver cirrhosis and hepatocellular cancer [1]. In the years spanning 2003–2013, HCV-related deaths numbered more than any other CDC-reported infectious disease [2]. Despite the availability of newly designed, highly effective direct-acting antivirals, disease prevalence remains high, and no vaccine exists for the virus [3–5].

Our results define a new role for NS4A in the late stages of the HCV lifecycle. Specifically, we have found that the acidic domain of NS4A is important for regulating assembly and that mutation of specific amino acids within this domain prevents formation of the viral envelope. Further, we have identified new interactions between NS4A and HCV structural and nonstructural viral proteins. This suggests that NS4A may act to bridge two stages of the HCV lifecycle, linking virion assembly at the lipid droplet to virion envelopment at the ER, similar to the actions of the NS2 protein. Importantly, we found that NS4A binds to E1 and that antagonizing this interaction with one amino acid change in NS4A prevents viral envelopment. We mapped the binding site of NS4A on E1 and found that it interacts with the first hydrophobic region, specifically at D72, an amino acid that is known to be important for viral particle production [49, 50]. Finally, we found that the Y45F mutation in NS4A, which prevents envelopment, also results in secretion of noninfectious, incompletely formed virions that are composed of low-density Core protein oligomers that lack HCV RNA. Together our results reveal a new role for NS4A in coordinating the proper assembly and envelopment of HCV particles to make infectious virus.