Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line

Abstract

SUMMARY To investigate the functions of signal transducers and activators of transcription 1 (STAT1)-induced anti-hepatitis C viral (HCV) effects, a stable Huh7.5 cell line (Huh7.5-STAT1ER) was established that constitutively expresses a fusion protein (STAT1ER) of STAT1 and the mouse oestrogen receptor (ER), which forms STAT1ER homodimers after 4-hydroxytamoxifen (4-HT) treatment. This inducible and cytokine/receptor-independent STAT1 activation system allowed us to investigate the anti-HCV effects of STAT1ER activation after inducing IFN-stimulated gene (ISG) expression. The anti-HCV effects of dimerized STAT1ER fusion protein were determined by real-time PCR in a time-dependent fashion post-HCV (JFH-1) infection. HCV (JFH-1) RNA decreased 48% at 72 h after 4-HT treatment. To distinguish the inhibitory effects of STAT1ER activation on HCV RNA replication or HCV internal ribosomal entry site (IRES)–mediated translation, a dicistronic pRL-HL construct was used in the studies. Both cellular (Cap-dependent) and HCV IRES–mediated (Cap-independent) translation were decreased by 63% and 57% at 72 h post-STAT1ER activation in the STAT1ER cell line. In our previous studies, interferon-induced transmembrane protein 3 [(IFITM3) (1-8U)] was found to inhibit HCV RNA replication. Subsequently, elevated expression of the 1-8U gene was confirmed by Western blotting in the Huh7.5-STAT1ER cell line. To further investigate the 1-8U function with both in vivo and in vitro studies, the 1-8U gene was found to suppress cellular and HCV IRES–mediated translation.

Innate immune responses in hepatitis C virus infection

“…These authors showed that IFITM3 was induced by IL1 as well as by IFN-α and that over-expression of IFITM3 reduced HCV RNA abundance in replicon-bearing cells. How IFITM3 exerts its anti-HCV effect remains elusive, but it was recently reported that IFITM3 suppresses both cellular and HCV IRES-dependent translation (182). It should be noted, however, IFITM3 was reported not to inhibit HCV-N replicon in HEK293 cells (71).…”

Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells

“…In Figure 5A, the 1-8U protein was detected by Western blotting and was up-regulated in Huh7.5-IRF3ER cells after 4-HT treatment. Due to auto-dimerization of IRF-3ER fusion protein in Huh7.5-IRF3ER cells, the fold-induction of 1-8U protein (Figure 5A, lane 1, 2, and 3) is not as robust as described in our previous report in which the STAT1 gene was activated [27]. Real-time quantitative reverse-transcription PCR was used to detect and measure hnRNP M mRNA expression in Huh7.5-IRF3ER cells.…”

Hepatitis C Virus: Strategies to Evade Antiviral Responses

“…Interestingly, the HCV NS5A protein has been proposed to impair OAS1 function, thus counteracting the antiviral effect of this pathway [120]. At least two members of the IFN-inducible transmembrane (IFITM) family, IFITM1 and IFITM3, are antiviral towards HCV in vitro [105, 121–123]. IFITM1 blocks HCV entry by binding to and preventing the interaction between the HCV co-receptors CD81 and occludin at tight junctions in hepatocytes [122].…”

“…IFITM1 blocks HCV entry by binding to and preventing the interaction between the HCV co-receptors CD81 and occludin at tight junctions in hepatocytes [122]. IFITM3 has been proposed to function as an anti-HCV ISG by impairing HCV IRES-mediated translation [123]. However, it could also affect the HCV life cycle by perturbing cholesterol- and lipid-related biology within the cell.…”