Abstract
BackgroundThe antiviral therapy of chronic hepatitis B virus (HBV) infection pursues the dual goals, virological response (undetectable serum HBV DNA) and hepatitis B e antigen (HBeAg) serological response (serum HBeAg loss/seroconversion). It is relatively difficult, however, to realize the serological response, especially for nucleotide/nucleoside analogs. Furin, a proprotein convertase, is involved in HBeAg maturation. The suppression of furin using inhibitors accordingly reduces HBeAg secretion, but possibly enhances HBV replication. For these reasons, the strategy based on the combination of nucleoside analog entecavir (ETV) and furin inhibitors to inhibit HBV replication and HBeAg secretion simultaneously were studied here.MethodsThe suppression of furin was performed using inhibitors decanoyl-RVKR-chloromethylketone (CMK) and hexa-D-arginine (D6R) or the expression of furin inhibitory prosegment. The influence of furin suppression on HBV replication and the effect of CMK combined with nucleoside analog entecavir (ETV) on HBV replication and HBeAg secretion was investigated in HepG2.2.15 cells. HBeAg level in media was detected using enzyme-linked immunosorbent assay. Intracellular viral antigens and HBV DNA were detected using Western and Southern blotting analyses, respectively.ResultsCMK, D6R and the expression of inhibitory prosegment all significantly reduced HBeAg secretion, but only CMK enhance HBV replication. Concordantly, only CMK post-transcriptionally accumulated cytosolic HBV replication-essential hepatitis B core antigen (HBcAg). The HBcAg-accumulating effect of CMK was further found to be resulted from its redundant inhibitory effect on the trypsin-like activity of cellular proteasomes that are responsible for HBcAg degradation. Moreover, the viral replication-enhancing effect of CMK was abrogated by ETV and ETV combined with CMK reduced HBV replication and HBeAg secretion simultaneously.ConclusionThe suppression of furin itself does not enhance HBV replication. Nucleotide/nucleoside analogs combined with furin inhibitors may be a potential easy way to realize the dual goals of the antiviral therapy for chronic hepatitis B in the future.
Highlights
The antiviral therapy of chronic hepatitis B virus (HBV) infection pursues the dual goals, virological response and hepatitis B e antigen (HBeAg) serological response
Furin suppressed by measures other than CMK does not enhance HBV replication A strain of HBeAg-defective variant (G1896A) harboring additional mutations, T147A and V149I, adjacent to a putative furin recognition site in the C-terminus of hepatitis B core antigen (HBcAg), has higher replication efficiency than the conventional variant
CMK reduces Hepatitis B surface antigen (HBsAg) secretion at high concentrations (15), perhaps by interfering with furin proteolyzation of HBV large surface antigen pre-S1 that simultaneously affect HBV virion release, implying that CMK at high concentrations may interfere with HBV virion release; the enhancements based on the increased level of intracellular core-associated HBV DNA were not resulted from the limitation to virion release since the virion level in media did not significantly decrease (Figure 1B), and were neither related to the influence of CMK on cell viability
Summary
The antiviral therapy of chronic hepatitis B virus (HBV) infection pursues the dual goals, virological response (undetectable serum HBV DNA) and hepatitis B e antigen (HBeAg) serological response (serum HBeAg loss/seroconversion). Compared with virological response only, virological response plus HBeAg serological response have a low relapse relate while off treatment of current antiviral therapy [7] Though those patients with HBeAg seroconversion (HBeAg-negative chronic hepatitis B) due to infection of HBeAg-defective variants have poor prognoses, the significance of HBeAg to HBV infection is not minimized since these variants rarely cause a de novo chronic infection [8], implying that HBeAg loss may be helpful for termination of chronic HBV infection. Nucleoside analog entecavir (ETV) blocks HBV replication rapidly, but induce HBeAg seroconversion unpredictably For these reasons, ETV combined with some direct HBeAg secretion-inhibitory measures seems a strategy to improve the current antiviral therapy of chronic hepatitis B
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