No 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...

The hepatitis C virus (HCV) NS5A protein is highly phosphorylated by cellular protein kinases. To study how NS5A might be integrated in cellular kinase signalling, we isolated phosphoproteins from HuH-7 hepatoma cells that specifically interacted with recombinant NS5A protein. Subsequent mass spectrometry identified the adaptor protein amphiphysin II as a novel interaction partner of NS5A. Mutational analysis revealed that complex formation is primarily mediated by a proline-rich region in the C-terminal part of NS5A, which interacts with the amphiphysin II Src homology 3 domain. Importantly, we could further demonstrate specific co-precipitation and cellular colocalization of endogenous amphiphysin II with NS5A in HuH-7 cells carrying a persistently replicating subgenomic HCV replicon. Although the NS5A-amphiphysin II interaction appeared to be dispensable for replication of these HCV RNAs in cell culture, our results indicate that NS5A-amphiphysin II complex formation might be of physiological relevance for the HCV life cycle.Chronic infection with hepatitis C virus (HCV) bears a substantial risk of developing severe liver disease such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The interactions between cellular proteins and HCV gene products may provide clues for novel approaches to interfere with virus propagation and pathogenesis. The HCV non-structural protein 5A (NS5A) became the focus of studies concerning cellular binding partners when it was reported to be involved in HCV resistance to IFN-a (Enomoto et al., 1995. NS5A is presumed to be a component of the membrane-associated complex of HCV proteins that replicates the plus-strand RNA genome via a minus-strand RNA intermediate (Brass et al., 2002; Hijikata et al., 1993;Shirota et al., 2002). Cell culture-adaptive mutations in the NS5A sequence significantly enhance the replication efficiency of HCV replicons, supporting its role in RNA replication (Blight et al., 2000;Krieger et al., 2001;Lohmann et al., 2001).NS5A proteins of some HCV isolates associate with IFNinduced double-stranded RNA-activated protein kinase (PKR) and inhibit PKR activity (Gale et al., 1997(Gale et al., , 1998. In addition, mechanisms for PKR-independent repression of IFN action by NS5A, such as the induction of IL-8 expression by transcriptional stimulation, have been reported (Polyak et al., 2001). Transcriptional activation mediated by NS5A is most pronounced for N-terminally truncated NS5A, which is transported into the nucleus, in contrast to the perinuclear, cytoplasmic full-length protein (Enomoto et al., 1996; Kato et al., 1997;Tanimoto et al., 1997). A cellular transcription factor (Ghosh et al., 2000), as well as a putative nucleoplasmic transporter, karyopherin b3 (Chung et al., 2000), were found as NS5A interaction partners in yeast two-hybrid screens. Ectopically expressed NS5A protein has been described to interact with Grb2 (Tan et al., 1999), p53 (Majumder et al., 2001, Cdk1 (Arima et al., 2001) and TRAF-2 (Park et al., 2002) and to cau...

Targeted inhibition of protein kinases with small molecule drugs has evolved into a viable approach for anticancer therapy. However, the true selectivity of these therapeutic agents has remained unclear. Here, we used a proteomic method to profile the cellular targets of the clinical epidermal growth factor receptor kinase inhibitor gefitinib. Our data suggest alternative cellular modes of action for gefitinib and provide rationales for the development of related drugs.

The development of novel antiviral drugs against hepatitis C is a challenging and competitive area of research. Progress of this research has been hampered due to the quasispecies nature of the hepatitis C virus, the absence of cellular infection models and the lack of easily accessible and highly representative animal models. The current combination therapy consisting of interferon-alpha and ribavirin mainly acts by supporting host cell defence. These therapeutics are the prototypic representatives of indirect antiviral agents as they act on cellular targets. However, the therapy is not a cure, when considered from the long-term perspective, for almost half of the chronically infected patients. This draws attention to the urgent need for more efficient treatments. Novel anti-hepatitis C treatments under study are directed against a number of so-called direct antiviral targets such as polymerases and proteases, which are encoded by the virus. Although such direct antiviral approaches have proven to be successful in several viral indications, there is a risk of resistant viruses developing. In order to avoid resistance, the development of indirect antiviral compounds has to be intensified. These act on host cell targets either by boosting the immune response or by blocking the virus host cell interaction. A particularly interesting approach is the development of inhibitors that interfere with signal transduction, such as protein kinase inhibitors. The purpose of this review is to stress the importance of developing indirect antiviral agents that act on host cell targets. In doing so, a large source of potential targets and mechanisms can be exploited, thus increasing the likelihood of success. Ultimately, combination therapies consisting of drugs against direct and indirect viral targets will most probably provide the solution to fighting and eradicating hepatitis C virus in patients.