Mechanism insights into the role of Smc5/6 in HBV inhibition

Smc5/6 is a protein complex with a ring structure that suppresses HBV transcription and proliferation. HBV counters this restriction by encoding the regulatory protein HBx, which targets Smc5/6 for proteasomal degradation. However, the molecular mechanism of HBV inhibition by Smc5/6 remains elusive. Here, we take advantage of a luciferase reporter gene in a cell-free expression system and measured the transcriptional activity using the purified Smc5/6 holo-complex, subcomplexes or individual subunits. Besides Smc5/6 holo-complex, NSMCE1/3 subcomplex is sufficient to inhibit transcription in vitro. NSMCE1/3 represses HBx expression at both the mRNA level and the protein level as revealed by the RT-PCR and a cycloheximide chase experiment, respectively. Overexpression of NSMCE1/3 causes degradation of HBx and this effect is blocked by a proteasome inhibitor but not by an inhibitor of the ubiquitin-activating enzyme E1. NSMCE1/3 interacts with the 20S proteasome but does not stimulate the ubiquitination of HBx, indicating that NSMCE1/3 leads to HBx degradation via a ubiquitin-independent proteasomal mechanism. Overexpression of NSMCE1/3 results in inhibition of HBV proliferation in hepatoma cell lines while knockdown of NSMCE3 leads to proliferation promotion. These new findings provide insights into the molecular mechanism of HBV inhibition by Smc5/6.