Abstract
So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.
Highlights
Hepatitis C virus (HCV), a leading cause of chronic liver diseases, is an enveloped, single-stranded and positive-sense RNA virus which belongs to Hepacivirus genus within the family Flaviviridae [1]
To understand how HCV interacts with cells under a more defined cell-virus context, we investigated the importance of viral replication status to subsequent viral propagation and how HCV establishes persistent infection
We first established a cell model capable of naturally or actively expressing HCV by genetically engineering a full-length JFH1 RNA genome containing a blasticidin-resistant cassette (Bla) gene inserted at the a.a. position 420 of the NS5A domain III, i.e., 420Bla genome (Figure 1B, scheme 3)
Summary
Hepatitis C virus (HCV), a leading cause of chronic liver diseases, is an enveloped, single-stranded and positive-sense RNA virus which belongs to Hepacivirus genus within the family Flaviviridae [1]. The genome of HCV is an uncapped linear single stranded RNA molecule with a size of about 9.6 kb, which is flanked by untranslated regions (UTRs) at its 59and 39 ends [2]. The structural proteins, core, E1 and E2, are the components of the viral particle [1,2], whereas the NS gene products participate in the genome replication and the assembly of viral particle [2]. Among these NS proteins, NS5A is a zincbinding protein composed of three domains separated by low complex (LC) regions LCI and LCII (Figure 1A) [3]. Domain II contains the interferon sensitivity determining region (ISDR) and participates in viral RNA replication [3]
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