Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes

Abstract

Background: Hepatitis C virus (HCV) effectively establishes persistent infection in human livers. The nonstructural (NS) 3/4A complex participates in this process by cleavage of interferon b (IFNb) promoter stimulator-1 (IPS-1; also termed Cardif/MAVS/VISA), which inhibits responses to double stranded (ds) RNA. However, it is not known whether this effect extends beyond innate responses. Aims: To test if HCV NS3/4A affects innate and adaptive immune responses in vivo. Methods: NS3 levels were semi-quantified in human liver biopsies, transfected cells, and in transgenic (Tg) mouse livers by western blot. The effect of NS3/4A on dsRNAmediated signalling and on the integrity of IPS-1 was analysed using in vitro translation, transfected cells and Tg mice. Cytotoxic T cell (CTL)-mediated clearance of transient firefly luciferase (FLuc)-and/or NS3/4A-Tg hepatocytes was determined using in vivo imaging and western blot. Results: NS3 protein levels were in a comparable range (0.1-49 mg/g tissue) in infected human livers and Tg mouse livers. Importantly, these levels of NS3/4A reduced murine innate responses to synthetic dsRNA in vivo, supporting the possibility that this occurs also in infected humans. The likely explanation for this was the NS3/4A-mediated cleavage of mouse IPS-1, albeit less efficiently than human IPS-1. Despite this, FLuc-and/or NS3/4A-expressing murine hepatocytes were effectively eliminated by hepatic CTLs, utilising the classical molecules for virus-infected cell lysis, including CD8, IFNc, perforin and FasL. Conclusions: Although HCV NS3/4A inhibits the innate immunity, this does not prevent CTL-mediated clearance of NS3/4A-expressing hepatocytes in vivo. Thus, other HCV proteins are most likely responsible for interfering with the adaptive immunity.Hepatitis C virus (HCV) is highly efficient in establishing persistent infections in human hepatocytes in vivo, but not in vitro.

Heterologous T Cells Can Help Restore Function in Dysfunctional Hepatitis C Virus Nonstructural 3/4A-Specific T Cells during Therapeutic Vaccination

“…This is consistent with the human infection (2). Additionally, this suggests that the dependence on T cell help for NS3-specific CTL priming is different in a naive host (24,30) and one with a dysfunctional T cell response to NS3.…”

“…2A; p = NS, AUC and ANOVA). Thus, unlike the situation in a naive host (24,30), the priming of NS3-specific CTLs seems to be more dependent on the presence of CD4 + T cells in a host with a dysfunctional T cell response to NS3.…”

“…Cells were transfected using 4 mg plasmid DNA together with 6 ml TurboFect in vitro transfection reagent (Fermentas International, Burlington, ON, Canada) and incubated for 36 h before harvesting for analysis of NS3 and HBcAg protein content by immunoprecipitation and Western blot as described (30,34). In brief, cell lysates were applied to protein A-Sepharose beads conjugated with anti-NS3 or anti-HBcAg Abs for 2 h (+4˚C).…”

“…The RMA-S cell line (provided by Prof. K. Kärre, Karolinska Institutet) was maintained in RPMI 1640 medium (Life Technologies) supplemented with 2 mM L-glutamine, 5% FBS, and 100 U/ml penicillin and 100 mg streptomycin. The cells were incubated in humidified incubators at 37˚C with 5% CO 2 (30).…”

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

“…A total of 60 mg protein was separated on an SDS-PAGE 4-12% Bis-Tris gel. For running and blotting of protein gels, the XCell SureLock Mini-Cell system and iBlot equipment (Invitrogen) were used following the manufacturer's protocol and as described previously (43). Abs targeted to rabbit anti-V5 (diluted 1:1000) and goat anti-rabbit HRP (diluted 1:5000) were purchased from Sigma-Aldrich and DakoCytomation (Glostrup, Denmark), respectively.…”