Abstract
Hepatitis B virus (HBV) capsid or core protein (HBc) contains an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. HBc plays a critical role in virtually every step of viral replication, which is further modulated by dynamic phosphorylation and dephosphorylation of its CTD. While several cellular kinases have been identified that mediate HBc CTD phosphorylation, there is little information on the cellular phosphatases that mediate CTD dephosphorylation. Herein, a consensus binding motif for the protein phosphatase 2A (PP2A) regulatory subunit B56 was recognized within the HBc linker peptide. Mutations within this motif designed to block or enhance B56 binding showed pleiotropic effects on CTD phosphorylation state as well as on viral RNA packaging, reverse transcription, and virion secretion. Furthermore, linker mutations affected the HBV nuclear episome (the covalently closed circular or CCC DNA) differentially during intracellular amplification vs. infection. The effects of linker mutations on CTD phosphorylation state varied with different phosphorylation sites and were only partially consistent with the linker motif serving to recruit PP2A-B56, specifically, to dephosphorylate CTD, suggesting that multiple phosphatases and/or kinases may be recruited to modulate CTD (de)phosphorylation. Furthermore, pharmacological inhibition of PP2A could decrease HBc CTD dephosphorylation and increase the nuclear HBV episome. These results thus strongly implicate the HBc linker in recruiting PP2A and other host factors to regulate multiple stages of HBV replication.
Highlights
Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and the cause of acute and chronic hepatitis B, liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) [1,2,3]
The dynamic phosphorylation and dephosphorylation of the viral capsid protein (HBc), which are controlled by host cell protein kinases and phosphatases, play a critical role in regulating multiple stages of HBV replication
The HBV replicon plasmid pCIΔA-HBV-HBV core protein (HBc)-WT was constructed by subcloning the entire HBV insert together with the linked human cytomegalovirus (HCMV) promoter sequences from pCMVHBV [57,58] to the pCIΔA vector and has been described recently [27,28]. pCIΔA-HBV-HBc-C, was constructed by substitution of core ORF 4th codon (GAC) to a stop codon (TAG), which is capable of supporting viral replication upon complementation with HBc. pCIΔA-HBV-HBc-L, was constructed by substitution of the L ORF start codon (ATG) to ACG. pCIΔA-HBV-HBc-TT146/ 147AA was constructed by introducing the TT146/147AA linker double substitutions into pCIΔA-HBV-HBc-WT and expresses the mutated HBV pregenomic RNA (pgRNA) encoding the same HBc linker
Summary
Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and the cause of acute and chronic hepatitis B, liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) [1,2,3]. HBV genome is a small (3.2 kb), partially double-stranded (DS), relaxed circular (RC) DNA, in which neither strand is covalently closed [4]. In complete HBV virions, RC DNA is enclosed in an icosahedral capsid formed by the HBV core protein (HBc), which is in turn enclosed by the viral envelope containing three surface proteins (large, middle, small or L, M, S). RC DNA-containing (mature) NCs can be enveloped by the viral surface proteins to secrete extracellularly as complete virions [10]. In addition to complete virions, empty HBV capsids, without any RNA or DNA packaged, are formed inside cell, enveloped by the surface proteins, and secreted as empty virions at high levels [10,16,17]
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