Heat Stress Cognate 70 Host Protein as a Potential Drug Target against Drug Resistance in Hepatitis B Virus

Abstract

Heat stress cognate 70 (Hsc70) is a host protein associated with hepatitis B virus (HBV) replication. The goal of this study was to investigate whether Hsc70 could be an anti-HBV drug target. Our results showed that introducing Hsc70 increased HBV replication in HBV ؉ human hepatocytes (HepG2.2.15 cells). The coiled-coil region on Hsc70 (nucleotides 1533 to 1608; amino acids 511 to 536) was the key sequence for HBV replication. Knockdown of Hsc70 expression by RNA interference (RNAi) largely inhibited HBV replication with no cytotoxicity to the host. Using an Hsc70 mRNA screening assay, the natural compound oxymatrine (OMTR) was found to be a selective inhibitor for Hsc70 expression. Then, OMTR was used to investigate the potential of Hsc70 as an anti-HBV drug target. OMTR inhibited Hsc70 mRNA expression by 80% and HBV DNA replication by over 60% without causing cytotoxicity. The anti-HBV effect of OMTR appeared to be mediated by destabilizing Hsc70 mRNA. The half-life (T 1/2 ) of Hsc70 mRNA decreased by 50% in OMTR-treated hepatocytes. The Hsc70 mRNA 3-untranslated-region (UTR) sequence was the element responsible for OMTR's destabilization activity. OMTR suppressed HBV de novo synthesis at the reverse transcription stage from pregenomic RNA (pgRNA) to DNA and was active against either wild-type HBV or strains resistant to lamivudine, adefovir, and entecavir. Therefore, host Hsc70 could be a novel drug target against HBV, and OMTR appears to inhibit HBV replication by destabilizing Hsc70 mRNA. As the target is not a viral protein, OMTR is active for either wild-type HBV or strains resistant to reverse transcriptase (RT) inhibitors. Antiviral chemotherapy can select for drug-resistant viral mutants (21). For chronic infections that need long-term chemotherapy, such as infection with hepatitis B virus (HBV), the challenge to clinical therapy is substantial (27, 31). Reverse transcriptase (RT) inhibitors, such as lamivudine, adefovir, entecavir, telbivudine, and tenofovir, are potent drugs for HBV infections, but their use in the clinical setting often selects for drug resistance (13,14,27,31). The incidence of lamivudine resistance rises from 15 to 32% in the first year to 67 to 69% by the fifth year of treatment (7, 9, 28). Many drug-induced mutations in the HBV polymerase gene have been characterized. For instance, rtM204I/V/S ("rt" means "resistant"), rtL180M, rtL80V/I, and rtV173L are signature mutations for lamivudine; rtN236T and rtA181T/V are signature mutations for adefovir; rtS202G/I, rtI169T, rtS184G, and rtM250V are signature mutations for entecavir; rtM204I is a signature mutation for telbivudine; and rtA194T is a signature mutation for tenofovir (9,27,30,31). The mutations in RT result from the intrinsic high variability due to the lack of an editing function of the enzyme (18,20), and they alter the three-dimensional (3D) interaction between HBV polymerase and the drugs (27). This challenges the current anti-HBV strategy, which is directed at viral enzymes.However, HBV strains rely heavily on host c...

Host factors involved in hepatitis B virus maturation, assembly, and egress

“…Hsc70 binds to the CAD sequence of i-preS in a productive manner, as the modulation of its in vivo activity interferes with L topogenesis. Hyper-active Hsc70 suppresses preS posttranslocation, while hypoactive Hsc70 has opposite effects, with the consequences that the improper topological reorientation of L leads to defects in virus formation [12,30]. RNA interference studies yielded similar results, since the knock-down of Hsc70 inhibits HBV replication [30].…”

“…Hyper-active Hsc70 suppresses preS posttranslocation, while hypoactive Hsc70 has opposite effects, with the consequences that the improper topological reorientation of L leads to defects in virus formation [12,30]. RNA interference studies yielded similar results, since the knock-down of Hsc70 inhibits HBV replication [30]. Of note, however, Hsc70/Hsp70 not only regulates the proper folding of L, but also activates the hepadnaviral polymerase [31].…”

Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators

“…Infectious organisms such as bacteria and viruses also require Hsp70s for survival, replication, and infectivity. Therefore targeting these specialized proteins is a viable approach to antiproliferative and antiinfective drug discovery (22)(23)(24). Nevertheless, until recently, relatively few small-molecule modulators of Hsp70 were known (20,(22)(23)(24)(25)(26).…”

A Review of Novel Non-Nucleoside Anti-HBV Agents and Their Mechanism of Action

“…The antiviral mechanisms may be interfering with the reverse transcription process from pgRNA to DNA by destabilizing Hsc70 mRNA, and then inhibiting the replication of HBV [28]. Cepharanthine hydrochloride (CH) (Figure 7), a natural alkaloid, was reported to suppress HBeAg production and HBV DNA replication by downregulatng significantly the Hsc70 mRNA levels, indicating that its activity could be associated with its inhibitory effect on host Hsc70 [29].…”