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The arginine clusters of the carboxy-terminal domain of the core protein of hepatitis B virus make pleiotropic contributions to genome replication.

ABSTRACT: The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual arginine clusters to genome replication are unknown. We analyzed core protein variants in which the individual arginine clusters were substituted with either alanine or lysine residues. We developed assays to analyze these variants at specific steps throughout genome replication. We used a replication template that was not spliced in order to study the replication of only pgRNA. We found that alanine substitutions caused defects at both early and late steps in genome replication. Lysine substitutions also caused defects, but primarily during later steps. These findings demonstrate that the CTD contributes to DNA synthesis pleiotropically and that preserving the charge within the CTD is not sufficient to preserve function.

SUBMITTER: Lewellyn EB

PROVIDER: S-EPMC3020508 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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The arginine clusters of the carboxy-terminal domain of the core protein of hepatitis B virus make pleiotropic contributions to genome replication.

Lewellyn Eric B EB Loeb Daniel D DD

Journal of virology 20101117 3

The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual ...[more]

PMID: 21084467

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Project description:UnlabelledPhosphorylation of serines 157, 164, and 172 within the carboxyl-terminal SPRRR motif of the hepatitis B virus (HBV) core (C) protein modulates HBV replication at multiple stages. Threonine 162 and serines 170 and 178, located within the carboxyl-terminal conserved RRRS/T motif of HBV C protein, have been proposed to be protein kinase A phosphorylation sites. However, in vivo phosphorylation of these residues has never been observed, and their contribution to HBV replication remains unknown. In this study, [(32)P]orthophosphate labeling of cells expressing C proteins followed by immunoprecipitation with anti-HBc antibody revealed that threonine 162 and serines 170 and 178 are phosphoacceptor residues. A triple-alanine-substituted mutant, mimicking dephosphorylation of all three residues, drastically decreased pregenomic RNA (pgRNA) encapsidation, thereby decreasing HBV DNA synthesis. In contrast, a triple-glutamate-substituted mutant, mimicking phosphorylation of these residues, decreased DNA synthesis without significantly decreasing encapsidation. Neither triple mutant affected C protein expression or core particle assembly. Individual alanine substitution of threonine 162 significantly decreased minus-strand, plus-strand, and relaxed-circular DNA synthesis, demonstrating that this residue plays multiple roles in HBV DNA synthesis. Double-alanine substitution of serines 170 and 178 reduced HBV replication at multiple stages, indicating that these residues also contribute to HBV replication. Thus, in addition to serines 157, 164, and 172, threonine 162 and serines 170 and 178 of HBV C protein are also phosphorylated in cells, and phosphorylation and dephosphorylation of these residues play multiple roles in modulation of HBV replication.ImportanceThreonine 162, within the carboxyl-terminal end of the hepatitis B virus (HBV adw) core (C) protein, has long been ignored as a phosphoacceptor, even though it is highly conserved among mammalian hepadnaviruses and in the overlapping consensus RxxS/T, RRxS/T, and TP motifs. Here we show, for the first time, that in addition to the well-known phosphoacceptor serines 157, 164, and 172 in SPRRR motifs, threonine 162 and serines 170 and 178 in the RRRS/T motif are phosphorylated in cells. We also show that, like serines 157, 164, and 172, phosphorylated and dephosphorylated threonine 162 and serines 170 and 178 contribute to multiple steps of HBV replication, including pgRNA encapsidation, minus-strand and plus-strand DNA synthesis, and relaxed-circular DNA synthesis. Of these residues, threonine 162 is the most important. Furthermore, we show that phosphorylation of C protein is required for efficient completion of HBV replication.

Project description:Hepatitis B virus (HBV) core protein (HBc) contains an N-terminal domain (NTD, assembly domain) and a C-terminal domain (CTD), which are linked by a flexible linker region. HBc plays multiple essential roles in viral replication, including capsid assembly, packaging of the viral pregenomic RNA (pgRNA) into nucleocapsids, viral reverse transcription that converts pgRNA to the genomic DNA, and secretion of DNA-containing (complete) virions or genome-free (empty) virions. The HBc linker is generally assumed to act merely as a spacer between NTD and CTD but some results suggest that the linker may affect NTD assembly. To determine its role in viral replication, we have made a number of deletion and substitution mutants in the linker region, in either the presence or absence of CTD, and tested their abilities to support capsid assembly and viral replication in human cells. Our results indicate that the linker could indeed impede NTD assembly in the absence of CTD, which could be partially relieved by partial linker deletion. In contrast, when CTD was present, the linker deletions or substitutions did not affect capsid assembly. Deletion of the entire linker or its C-terminal part resulted in a partial defect in pgRNA packaging and severely impaired viral DNA synthesis. In contrast, deletion of the N-terminal part of the linker, or substitutions of the linker sequence, had little to no effect on RNA packaging or first-strand DNA synthesis. However, the N-terminal linker deletion and two linker substitution mutants were defective in the production of mature double-stranded viral DNA. Secretion of empty virions was blocked by all the linker deletions and substitutions tested. In particular, a conservative linker substitution that allowed mature viral DNA synthesis and secretion of complete virions severely impaired the secretion of empty virions, thus increasing the ratio of complete to empty virions that were secreted. Together, these results demonstrate that the HBc linker region plays critical and complex roles at multiple stages of HBV replication.

Project description:BackgroundHepatitis B core-related antigen (HBcrAg) is a promising disease-monitoring marker for chronic hepatitis B (CHB). We investigated correlations between HBcrAg with antiviral efficacy and virological and histological variables.MethodsOne hundred and forty-five CHB patients from the mainland of China between August 2013 and September 2016 who underwent liver biopsy received entecavir therapy and had paired liver biopsy at 78 weeks. We analyzed correlations between HBcrAg and virological and histological variables in hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients. We also explored the predictors of HBeAg loss after 78 weeks of antiviral therapy. Pearson correlation analysis and logistic forward stepwise regression were the main statistic methods.ResultsHBeAg-positive patients (n = 93) had higher baseline HBcrAg (median 7.4 vs. 5.3 log10 U/mL P < 0.001) and greater HBcrAg declines (median 1.6 vs. 0.9 log10 U/mL P = 0.007) than HBeAg-negative patients after 78 weeks of therapy. At baseline, HBcrAg correlated with hepatitis B virus (HBV) DNA in both HBeAg-positive (r = 0.641, P < 0.001) and -negative patients (r = 0.616, P < 0.001), with hepatitis B surface antigen (HBsAg) in HBeAg-positive patients (r = 0.495, P < 0.001), but not with anti-hepatitis B virus core antibody (anti-HBc). Weak correlations existed between HBcrAg, histology activity index (HAI; r = 0.232, P = 0.025), and Ishak fibrosis score (r = -0.292, P = 0.005) in HBeAg-positive patients. At 78 weeks, significant correlations existed only between HBcrAg and anti-HBc in HBeAg-positive (r = -0.263, P = 0.014) and HBeAg-negative patients (r = -0.291, P = 0.045). Decreased HBcrAg significantly correlated with reduced HBV DNA (r = 0.366, P = 0.001; r = 0.626, P < 0.001) and HBsAg (r = 0.526, P = 0.001; r = 0.289, P = 0.044) in HBeAg-positive and -negative patients, respectively, and with reduced HAI in HBeAg-positive patients (r = 0.329, P = 0.001). Patients with HBeAg loss (n = 29) showed a larger reduction in HBcrAg than those without (median 2.3 vs. 1.3 log10 U/mL, P = 0.001). In multivariate analysis, decreased HBcrAg was an independent predictor of HBeAg loss (P = 0.005).ConclusionsHBcrAg reflects viral replication and protein production. Decreased HBcrAg could predict HBeAg loss after antiviral therapy.Trial registrationClinical Trials.gov: NCT01962155; https://www.clinicaltrials.gov/ct2/show/NCT01962155?term=NCT01962155&draw=2&rank=1.

Dataset Information

The arginine clusters of the carboxy-terminal domain of the core protein of hepatitis B virus make pleiotropic contributions to genome replication.

Publications

The arginine clusters of the carboxy-terminal domain of the core protein of hepatitis B virus make pleiotropic contributions to genome replication.

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