Identification of SRPK1 and SRPK2 as the Major Cellular Protein Kinases Phosphorylating Hepatitis B Virus Core Protein

Abstract

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich Cterminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.Hepatitis B virus (HBV), a small DNA virus belonging to the family Hepadnaviridae, causes acute and chronic hepatitis in humans. Worldwide, an estimated 350 million persons are persistently infected with HBV (4). A significant subset of these HBV carriers progresses to severe liver disease, such as hepatocellular carcinoma, which is assumed to cause up to one million deaths per year (30). Current treatment of chronic HBV infections by the approved therapeutics alpha interferon and lamivudine has its limitations, and there is a clear medical need for new therapeutic strategies (33).The mature HBV virion consists of an enveloped, spherical nucleocapsid which contains the viral DNA genome and is assembled from dimers of a single capsid protein, the 21-kDa HBV core protein (43). During the assembly process, viral polymerase mediates the specific encapsidation of pregenomic RNA and subsequently converts the pregenomic RNA to viral genomic DNA (3, 19; for reviews, see references 13 and 33).Numerous studies have shown that the HBV core protein is phosphorylated in intact cells (27,32,41). The serine residues of three repeated SPRRR motifs in its arginine-rich C-terminal region were identified as phosphoacceptor sites in vivo (S155, S162, and S170 in strain ayw) (25). Core protein becomes phosphorylated prior to nucleocapsid assembly, and mutational analysis strongly suggests that phosphorylation of serines 162 and 170 is critical for subsequent pregenomic RNA packaging to occur (14,24). As none of the viral proteins possesses intrinsic protein kinase activit...

Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

“…The suggested model is in line with the previous observation in the duck model that serum-derived nucleocapsids are non-phosphorylated whereas nucleocapsids from liver tissue are highly phosphorylated (14). It is tempting to speculate that incoming and recycling capsids are phosphorylated by a capsid-associated kinase activity of cellular origin, which has been described in the WHV and HBV system (27)(28)(29)(30)(31)(32)(33).…”

Hepatitis B virus-related insertional mutagenesis in chronic hepatitis B patients as an early drastic genetic change leading to hepatocarcinogenesis

“…SRPK2 is a serine/threonine kinase expressed in mouse brain, lung, and testis. It phosphorylates the HBV core protein and is a possible molecular target for inhibiting HBV replication (Daub et al, 2002). TERF2 is a key component of vertebrate telomeres and plays a protective role in cellular senescence (van Steensel et al, 1998).…”

Proteome-wide Identification of Cellular Targets Affected by Bisindolylmaleimide-type Protein Kinase C Inhibitors

“…COS-7 cells were transiently transfected as previously described (15). Epidermal growth factor (EGF) stimulation of starved HeLa cells was carried out as described (18).…”

“…The DNA sequences coding for full-length Ste20-related kinase (SLK) and full-length adenosine kinase (AK) were PCR-amplified from human spleen and liver cDNA libraries. The SLK cDNA was fused to a N-terminal FLAG-tag, whereas the AK cDNA was fused to a Nterminal myc-tag and cloned into a pRK5 expression vector (15)(16)(17).…”

“…The primary alkylamine group that was used for coupling with epoxy-activated Sepharose is indicated as linker position. C, structure of CDK2 with the co-crystallized inhibitor staurosporine (1AQ1) (15). lobes of the kinase domain.…”