HCV Core Residues Critical for Infectivity Are Also Involved in Core-NS5A Complex Formation

Abstract

Hepatitis C virus (HCV) infection is a major cause of liver disease. The molecular machinery of HCV assembly and particle release remains obscure. A better understanding of the assembly events might reveal new potential antiviral strategies. It was suggested that the nonstructural protein 5A (NS5A), an attractive recent drug target, participates in the production of infectious particles as a result of its interaction with the HCV core protein. However, prior to the present study, the NS5A-binding site in the viral core remained unknown. We found that the D1 domain of core contains the NS5A-binding site with the strongest interacting capacity in the basic P38-K74 cluster. We also demonstrated that the N-terminal basic residues of core at positions 50, 51, 59 and 62 were required for NS5A binding. Analysis of all substitution combinations of R50A, K51A, R59A, and R62A, in the context of the HCVcc system, showed that single, double, triple, and quadruple mutants were fully competent for viral RNA replication, but deficient in secretion of viral particles. Furthermore, we found that the extracellular and intracellular infectivity of all the mutants was abolished, suggesting a defect in the formation of infectious particles. Importantly, we showed that the interaction between the single and quadruple core mutants and NS5A was impaired in cells expressing full-length HCV genome. Interestingly, mutations of the four basic residues of core did not alter the association of core or NS5A with lipid droplets. This study showed for the first time that basic residues in the D1 domain of core that are critical for the formation of infectious extracellular and intracellular particles also play a role in core-NS5A interactions.

Dynamic Interaction of Stress Granules, DDX3X, and IKK-α Mediates Multiple Functions in Hepatitis C Virus Infection

“…The core protein promotes the accumulation of LDs to facilitate virus assembly. It has been proposed that HCV core protein recruits nonstructural proteins, mainly NS5A, viral RNA, and the RC-to LD-associated membranes (6), and that NS5A plays a dual role in HCV replication and assembly and meanwhile acts as a switch between these two processes (53,54). In addition to NS5A, other nonstructural proteins, such as NS2, NS3, NS4A, and NS4B, were shown to be critical for HCV assembly (44,(55)(56)(57).…”

Naturally occurring resistance mutations within the core and NS5B regions in hepatitis C genotypes, particularly genotype 5a, in South Africa

“…There is evidence that mutations in the BD2 region (contains the major binding site for NS5A) Fig.1. Hepatitis C core region 1-177 depicting the domains, functions, mutations identified in the study and known epitope regions can suppress binding to lipid droplets and reduce viral particle production, infectivity and release (Murray et al, 2007;Gawlik et al, 2014). The core: R70Q and L91M mutations have been associated with poor treatment response in genotype 1b infection to IFN (Akuta et al, 2010, Alhamlan et al, 2014, resistance to telaprevir combination therapy (Akuta et al, 2010), rate of progression to HCC (Araujo et al, 2014, Nakamoto et al, 2010, Khan et al, 2010, increased steatosis and hepatic oxidative stress (Tachi et al, 2010).…”

Immune modulation by the hepatitis C virus core protein

“…In mammalian cell lines, HCV core protein can be targeted away from the ER due to lack of specific cellular factors or the presence of inhibitory factors. Alternative localizations of core could be correlated with failure to assemble HCV capsids or virions in cultured mammalian cell lines . Related to these findings, naked viral core/nucleocapsids without a lipid envelope have been observed in the circulation of infected individuals …”

“…Alternative localizations of core could be correlated with failure to assemble HCV capsids or virions in cultured mammalian cell lines. 22,[26][27][28] Related to these findings, naked viral core/nucleocapsids without a lipid envelope have been observed in the circulation of infected individuals. 29 Furthermore, by means of a baculovirus expression system, uptake of naked HCV core particles generated in the absence of other HCV proteins has been shown.…”