Post-translational regulation of hepatitis B virus large envelope protein

点击翻译按钮获取中文摘要

Project Summary/Abstract The large (L) envelope protein of hepatitis B virus (HBV) is essential for virion production and infectivity, and its high intracellular level can cause liver cancer. It has extra preS1 + preS2 domains than small (S) envelope protein. Majority of the S protein is secreted as noninfectious subviral particles (SVPs), which have been implicated in the induction of immune tolerance for establishment of chronic HBV infection. L protein is not secreted when expressed alone. It interacts with core particle to initiate virion morphogenesis, and recruits the S protein for virion secretion. In doing so it also suppresses SVP secretion according to L/S protein ratio. L protein is also essential for initiation of HBV infection by interacting with the high-affinity receptor. Despite its critical importance in HBV biology and pathogenesis, little is known about regulation of its intracellular level at the post-translational step. We previously found that preventing S protein expression by mutating the initiating ATG into GCG abolished M protein, suggesting its reliance on S protein for stability. The intracellular level of L protein was also markedly reduced despite blocked secretion. Very recently, we discovered this particular mutant could produce a shortened and non-secreted S protein through translation initiation from a downstream ATG. Further mutating that ATG into GCG diminished intracellular L protein to extremely low level. Nevertheless, mutating the first and second in-frame ATG codons in the S gene into GCG introduces amino acid substitutions in the S domain of L protein. To verify that reduced L protein level is attributed to lost S protein expression rather than mutations in L protein, in Aim 1 we will test the impact of mutating the S gene ATG codon into other codons such as ATA, AAG, and TTG. The preS1 region harbors the promoter for 2.1-kb RNA for M/S proteins, and we recently found many naturally occurring in-frame preS1 deletions abolished S protein expression. Whether lost S protein expression at the transcriptional level also diminishes intracellular level of shortened L protein will be determined. Aim 2 will establish the role of core protein in sustaining L protein level and check for its synergy with S protein. Our pilot study suggested that core protein expression was required to sustain both intracellular and extracellular levels of L protein. In Aim 3, we will verify whether much reduced intracellular level of L protein is attributed to accelerated degradation, and examine if providing S or core protein in trans can rescue the L protein level. We hypothesize that during HBV virion morphogenesis, L protein is stabilized by its molecular interaction with core particles or with S protein. The proposed studies will reveal a novel mechanism to control L protein level at the post-translational level, which should have therapeutic implications.