Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation

Wittkop, Linda; Schwarz, Alexandra; Cassany, Aurélia; Grün-Bernhard, Stefanie; Delaleau, Mildred; Rabe, Birgit; Cazenave, Christian; Gerlich, Wolfram H.; Glebe, Dieter; Kann, Michael

doi:10.1111/j.1462-5822.2010.01444.x

Cited by 42 publications

(47 citation statements)

References 61 publications

(124 reference statements)

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“…Based on prior observations that phosphoacceptor serines 157, 164, and 172 (adw subtype) modulate HBV replication at multiple steps (4,7,11,(21)(22)(23)(24)(25), we hypothesized that phosphorylation and dephosphorylation of threonine 162 and serines 170 and 178 may also contribute to HBV replication.…”

Section: Resultsmentioning

confidence: 99%

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Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication

Jung

Hwang

Chwae

et al. 2014

J Virol

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Phosphorylation 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. Hepatitis B virus (HBV), a prototype hepadnavirus, has a partially double-stranded relaxed-circular (RC) DNA genome that contains four open reading frames (ORFs), encoding the core (C; also called HBc), viral polymerase (P), X (HBx), and surface (S; also called HBs) proteins. HBV replicates by reverse transcription of a pregenomic RNA (...

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Section: Resultsmentioning

confidence: 99%

“…The conserved phosphoacceptor serines 157, 164, and 172 in the SPRRR motif are important for core particle formation, RNA packaging (7,11,13,21), DNA synthesis (4,7,11,22,23), and subcellular localization (24,25). Threonine 162 and serines 170 and 178 in the RRRS/T motif are also conserved among mammalian hepadnaviruses (Fig.…”

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confidence: 98%

Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication

Jung

Hwang

Chwae

et al. 2014

J Virol

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“…In vivo, the core proteins become phosphorylated at different serine residues (Gerlich et al, 1982) by cellular protein kinases. Amongst these is PKC alpha, which was shown to be essential for genome maturation (Wittkop et al, 2010). Core proteins expose a nuclear localization signal (NLS), which causes nuclear import by the nuclear transport receptors of the importin family (Rabe et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Deroubaix

Osseman

Cassany

et al. 2015

Journal of General Virology

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Biopsies from patients show that hepadnaviral core proteins and capsids -collectively called core -are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that e, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

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“…Several kinases have been reported to phosphorylate the core protein in vitro, including protein kinase C (PKC) (23)(24)(25)(26)64), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (12), a 46-kDa serine kinase (26), and serine-arginine protein kinases 1 and 2 (SRPK1 and -2) (10). In all these cases, the site(s) of core phosphorylation was never defined, except that SRPK1 and -2 were shown to phosphorylate the HBc CTD in vitro (10,45) and in Escherichia coli (8).…”

mentioning

confidence: 99%

“…Since HBV encodes no proteins with kinase capability, it has long been presumed that the virus encapsidates a kinase of cellular origin. PKC has been reported to be incorporated into HBV capsids (23,25,48,64). However, other reports have argued that neither PKC, PKA, nor casein kinase II (CKII) is the endogenous kinase (12,25,26).…”

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confidence: 99%

Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

Ludgate

Ning

Nguyen

et al. 2012

J Virol

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Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.T he human hepatitis B virus (HBV) continues to pose a significant health risk worldwide, causing more than one million deaths annually (52). Chronic HBV infection, estimated to affect 350 million people globally, dramatically elevates the risk for developing serious liver diseases, including cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family, which includes hepatotropic DNA viruses that consist of an enveloped icosahedral capsid enclosing an approximately 3-kb DNA genome in a partially double-stranded, relaxed circular (RC) form. These DNA viruses are also retroid viruses and encode a reverse transcriptase (RT) enzyme that converts a so-called pregenomic RNA (pgRNA) template to the RC DNA through reverse transcription within cytoplasmic capsids. Capsids are composed of multiple copies (180 or 240) of one virally encoded protein, the core or capsid protein (9,63,65,71).Phosphorylation of the hepadnavirus core protein is important for RNA packaging, DNA synthesis, and subcellular localization. The HBV core protein (HBc) contains three major serine-proline (S-P) phosphorylation sites in its C-terminal domain (CTD) (32). The duck hepatitis B virus (DHBV) core protein (DHBc) contains six known phosphorylation sites, four of which also have the serine/threonine-proline (S/T-P) motifs (43, 68). Mutational analyses indicate that phosphorylation of the core protein at these S/T-P sites is required for RNA packaging and DNA synthesis in HBV (29, 31). For DHBV, dynamic CTD phosphorylation at the S/T-P sites is required for complete DNA synthesis such that the S/T-P phosphorylation is needed for first-strand DNA synthesis and dephosphorylation is required for second-strand DNA s...

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Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation

Abstract: Summary Capsids of hepatitis B virus and other hepadnaviruses contain a cellular

Cited by 42 publications

References 61 publications

Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication

Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

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