HCV NS A inhibits induction of the type I
HCV NS3/4A inhibits induction of the type I interferon response by cleaving MAVS from the mitochondrial surface (Li et al., 2005b). MAVS is cleaved at cysteine 508, and allopurinol zyloprim australia of cysteine 508 to arginine (C508R) abrogates cleavage (Li et al., 2005b). GBV-B and the related canine hepatitis virus also cleave MAVS (Chen et al., 2007, Parera et al., 2012).
Toll-like receptor 3 (TLR3) is another dsRNA sensor that detects dsRNA inside endosomes after uptake by endocytosis. TLR3 signals through TIR-domain-containing adapter-inducing interferon-β (TRIF), which also leads to activation of IRF3 and induces the type I interferon response. HCV NS3/4A has been reported to cleave TRIF based on studies using an in vitro translation system (Li et al., 2005a). However Dansako et al. showed that HCV NS3/4A does not cleave TRIF in cell culture and that extracellular dsRNA interferon-mediated signaling remains intact (Dansako et al., 2007).
HPgV and HCV coinfection occur in approximately 30–40% of HIV patients, and studies have shown a beneficial effect of HPgV in HIV/HCV coinfection (Berzsenyi et al., 2007, Berzsenyi et al., 2011). HCV NS3-specific protease inhibitors have recently been developed for treatment of chronic HCV infection. Telaprevir and Boceprevir, which are covalent, reversible inhibitors of HCV NS3/4A protease are used in concert with pegylated interferon-α (PEG-IFN) and ribavirin (RBV) to treat chronic HCV infection. Second generation inhibitors such as Simeprevir, which has recently been approved for clinical use (Jacobson et al., 2013, Lawitz et al., 2013, Manns et al., 2013), and Danoprevir, are also being developed.
We hypothesized that HPgV NS3/4A cleaves MAVS and inhibits induction of the type I interferon response similar to HCV and GBV-B NS3/4A. We also sought to better characterize HPgV NS3 protease cleavage kinetics and determine whether or not cleavage activity is affected by HCV protease inhibitors. Given the similarities between HCV and HPgV NS3/4A, HCV protease inhibitors could affect HPgV NS3/4A activity and inhibit HPgV replication, potentially modulating HPgV׳s beneficial effects in HIV and HIV/HCV coinfection. To study HPgV NS3/4A cleavage activity, we expressed the complete NS3/4AB polyprotein to be certain all of the domains required for maximal activity were present (George et al., 2012).
Results r> Discussion We have previously shown that HPgV NS3/4A protease activity inhibits HIV growth in Jurkat cells (George et al., 2012). Therefore we wanted to better characterize its cleavage activity and kinetics and identify cellular targets cleaved by HPgV. We also sought to determine if HCV NS3 protease inhibitors affect HPgV NS3 activity and whether HPgV NS3 affected the type I interferon system strongly enough to affect viral growth. We found that HPgV NS3 cleavage activity in Jurkat cells is not affected by the anti-HCV NS3/4A protease inhibitors Telaprevir, Boceprevir, or Danoprevir at concentrations up to 100µM (Fig. 3). Thus there was a wide difference in sensitivity to HCV protease inhibitors between HCV and HPgV NS3 proteases. We also found that HPgV cleaves MAVS near the C508 position (Fig. 4, Fig. 5) similar to HCV and GBV-B (Chen et al., 2007, Li et al., 2005b), but does not cleave TRIF in Jurkat, Huh7 or 293T cells (Fig. 4). MAVS was cleaved by HPgV NS3/4AB-HA with similar efficiency and at a site similar to the HCV NS3/4A cleavage site (Li et al., 2005b) in three different cell lines. This cleavage inhibited the type I interferon response as suggested by a reporter assay (Fig. 6A) and confirmed by measuring IFN-β transcript levels and the ability of supernatants from NS3-expressing cells to suppress CPE in response to a viral infection (Fig. 6B and C). The mutations made in the catalytic triad were expected to disable protease activity. Similar results have been seen in HCV for H57 and S139 (Grakoui et al., 1993). In yellow fever virus mutations to alanine in the H53, D77, and S138 positions all resulted in loss of cleavage activity (Chambers et al., 1990). However, the retention of partial activity by HPgV NS3/4AB(D79) may be explained by the relatively less important role of the aspartic acid in cleavage activity compared with the histidine and serine. The catalytic serine acts as a nucleophile while the histidine accepts the hydrogen from the serine facilitating the attack, and the aspartic acid hydrogen bonds with the histidine to make it more negative (Polgar, 2005).