Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejeon 305-701, Korea1
Author for correspondence: Joonho Choe. Fax +82 42 869 5630. e-mail jhchoe{at}sorak.kaist.ac.kr
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All herpesviruses display two modes of replication: lytic and latent. During lytic replication, the host cells are destroyed and viral progeny particles are released. In contrast, during latent replication, viral genomes persist but show restricted gene expression, and viral progeny is not released (Roizman, 1990 ). KSHV persists as a circular episome during latency and is able to reactivate and replicate in response to outside stimuli in cultured B cells. Only a small number of viral genes are transcribed during KSHV latency (Zhong et al., 1996
).
Many herpesviruses encode putative viral protein kinases (Smith & Smith, 1989 ). The protein kinases of herpesviruses were first identified in herpes simplex virus (HSV), varicella-zoster virus (VZV) and EpsteinBarr Virus (EBV). HSV US3 and VZV open reading frame (ORF) 66 encode related protein kinases that are specific to the alphaherpesvirus subfamily. Gammaherpesviruses also have other putative protein kinases. EBV BGLF4, HVS ORF36 and KSHV ORF36 show protein kinase domains in their amino acid sequences, although their kinase activity has not yet been verified. There is mounting evidence that protein phosphorylation is essential for virus replication (Lin & Broyles, 1994
; Lin et al., 1992
). In order to determine whether KSHV ORF36 is a protein kinase, as suggested by Russo et al. (1996)
, we expressed ORF36 as a fusion protein with glutathione S-transferase (GST) in mammalian cells. The purified GSTORF36 fusion protein was shown to display protein kinase activity, and one of its serine residues was preferentially phosphorylated. We also found that the KSHV protein kinase is localized in the nucleus.
Comparative analysis of the deduced amino acid sequence revealed that ORF36 contained a catalytic domain found in serine protein kinases. The domain spans 270 amino acids, from amino acid positions 80 to 350 in KSHV ORF36 (Fig. 1). This domain can be divided into 11 subdomains and contains all of the amino acid residues that are normally conserved in serine/threonine protein kinases (Louise et al., 1998
). Because KSHV ORF36 is known to relate to ORF36 of HVS and BGLF4 of EBV, we aligned the encoded amino acid sequences, obtained from the protein sequence database. These comparisons revealed that the catalytic domain of KSHV ORF36 protein is homologous to those of HVS ORF36 and EBV BGLF4. Conserved subdomain I of KSHV ORF36 contains the glycine loop (GXGXXG), which is important for orienting the phosphates of the ATP substrate. Subdomain VIb has the catalytic loop, which contains a conserved aspartic acid at position 201. Subdomain VII has the conserved sequence DXG, which includes an aspartic acid that is highly conserved in all protein kinases and is important in the binding of an ATP-chelating metal (Louise et al., 1998
).
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To decipher the in vivo functions of the KSHV ORF36 protein, we cloned KSHV ORF36 DNA into pEBG, a GST-containing eukaryotic expression plasmid driven by the elongation factor (EF)-1 promoter, to express an ORF36 fusion protein with GST at its amino terminus (Mayer et al., 1995
); this plasmid was designated GSTKSHV ORF36. Briefly, KSHV total DNA was purified from the BCBL-1 cell line by the Hirt lysis method as previously described (Wilson & Patient, 1991
). The KSHV ORF36 gene was cloned by PCR from purified DNA. The 1·3 kb DNA product was cleaved with EcoRI and XhoI and purified by agarose gel electrophoresis. This fragment was initially inserted into the bacteriophage T7 expression vector pET21a. To obtain a plasmid that allows the GSTORF36 fusion protein to be expressed in mammalian cells, the PCR-amplified DNA was partially digested with BamHI/NotI and the fragment was isolated and inserted into pEBG that had been digested with BamHI and NotI. It is well known that the lysine residue in kinase subdomain II is essential for the kinase activity (Hanks & Quinn, 1991
). We generated a kinase-dead mutant of ORF36 by site-directed mutagenesis that replaced lysine-108 with glutamine. The mutant was cloned into pEBG, and the resulting plasmid was designated GSTKSHV ORF36 (K108Q).
To assess the biochemical functions of KSHV ORF36, we performed in vitro kinase assays using GST affinity-purified ORF36 fusion protein (Lin et al., 1992 ). pEBG, GSTKSHV ORF36 and GSTKSHV ORF36 (K108Q) plasmids were transiently transfected into COS-1 cells by the DEAE-dextran transfection method (Luthman & Magnusson, 1983
). COS-1 cell lysates expressing GST, GSTORF 36 fusion protein and the kinase-dead ORF36 mutant were incubated with glutathioneagarose (35 µl) (Amersham). Before performing the kinase assay, the glutathione-bound complexes were washed once with kinase wash buffer (KWB) [20 mM HEPES (pH 7·5), 5 mM MnCl2, 10 mM
-mercaptoethanol] and resuspended in 25 µl of the same solution supplemented with 10 µCi [
-32P]ATP. The phosphorylated proteins were then separated by 7·5% SDSPAGE, and the gel was subjected to autoradiography. A single prominent band at the expected size of 76 kDa was observed only in the cell lysate transfected with GSTKSHV ORF36 (the wild-type KSHV kinase) (Fig. 3a
, left panel, lane 2). In contrast, phosphorylation of the GSTKSHV ORF36 (K108Q) mutant was dramatically decreased compared to that of wild-type KSHV ORF36 (Fig. 3a
, left panel, lane 3). These results indicated that KSHV ORF36 protein displayed an intrinsic protein kinase activity and was autophosphorylated. Expression of the recombinant ORF36 protein and ORF36 (K108Q) mutant was verified by immunoblotting using a monoclonal antibody to GST (Fig. 3a
, left panel).
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We next sought to determine which amino acid of the KSHV ORF36 protein was autophosphorylated. KSHV ORF36 protein was autophosphorylated with [-32P]ATP and subjected to SDSPAGE. The phosphorylated polypeptide was eluted from the gel. After elution, the protein-containing solution was mixed with 25 µg carrier BSA. The proteins were precipitated with 100% trichloroacetic acid followed by incubation at 4 °C for 16 h. The protein pellet was dissolved in 50 µl of constant-boiling HCl, boiled at 100 °C for 2 h, and dried. The phosphoamino acids were resolved by electrophoresis on thin-layer cellulose (TLC) plates in pH 1·9 buffer. The migration positions of the phosphoserine, phosphothreonine and phosphotyrosine standards were identified by ninhydrin staining. Our results clearly demonstrated that mainly serine was autophosphorylated by the KSHV ORF36 kinase (Fig. 3b
). Phosphorylation of threonine and tyrosine residues could not be detected by this method. The KSHV ORF36 (K108Q) mutant was also tested for its amino acid autophosphorylation specificity. Although the kinase activity of the mutant protein was very low, the pattern of phosphoamino acid specificity was similar to that of wild-type KSHV ORF36. We conclude from these results that the KSHV ORF36 kinase is a serine protein kinase.
In summary, it was predicted from the DNA sequence that the KSHV ORF36 encoded a protein kinase (Russo et al., 1996 ). We expressed the KSHV ORF36 gene product as a GST fusion protein and demonstrated that it did indeed have a protein kinase activity. Phosphoamino acid analysis of the autophosphorylated KSHV ORF36 fusion protein demonstrated that the kinase activity is specific for serine residues. The in vivo function of and physiologically significant protein substrates for KSHV ORF36 remain a matter of conjecture, but we can hypothesize with respect to its function on the basis of its homology with other known herpesvirus protein kinases. Related protein kinases have been identified in genes in three herpesviruses, HSV UL13, VZV ORF47 and EBV BGLF4 (Smith & Smith, 1989
), and we showed that KSHV ORF36 has homology with EBV BGLF4. The HSV UL13 protein kinase has been purified and its activity verified (Daikoku et al., 1997
). It has also been reported that HSV UL13 phosphorylates the regulatory protein
22 (Purves et al., 1993
), which in its phosphorylated form transactivates the alpha 22 promoter (Leopardi et al., 1997
). These reports suggest that the function of UL13 is to provide optimal expression of selected viral proteins (Carter & Roizman, 1996
). In addition, it was reported recently that elongation factor (EF)-1
is hyperphosphorylated by HSV UL13, and EF-1
is the first cellular protein that has been shown to be a target for HSV UL13 (Kawaguchi et al., 1998
). Another viral kinase that is related to UL13 is the VZV ORF47 protein. This was shown to be required for virus growth in human T cells and in skin (Moffat et al., 1998
). From these studies of related viral kinases, we suggest that KSHV ORF36 may function in the virus growth-cycle, infection in host cells or replication of the virus. However, further study is required to decipher the true physiological function(s) of the KSHV ORF36 protein.
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References |
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Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C. & Cesarman, E. (1997). Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation.Nature 385, 347-350.[Medline]
Boshoff, C., Schulz, T. F., Kennedy, M. M., Graham, A. K., Fisher, C., Thomas, A., McGee, J. O., Weiss, R. A. & OLeary, J. J. (1995). Kaposis sarcoma-associated herpesvirus infects endothelial and spindle cells.Nature Medicine 1, 1274-1278.[Medline]
Carter, K. L. & Roizman, B. (1996). The promoter and transcriptional unit of a novel herpes simplex virus 1 alpha gene are contained in, and encode a protein in frame with, the open reading frame of the alpha 22 gene.Journal of Virology 70, 172-178.[Abstract]
Cesarman, E., Chang, Y., Moore, P. S., Said, J. W. & Knowles, D. M. (1995). Kaposis sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.New England Journal of Medicine 332, 1186-1191.
Cesarman, E., Nador, R. G., Bai, E., Bohenzky, R. A., Russo, J. J., Moore, P. S., Chang, Y. & Knowles, D. M. (1996). Kaposis sarcoma-associated herpesvirus contains G protein-coupled receptor and cyclin D homologs which are expressed in Kaposis sarcoma and malignant lymphoma.Journal of Virology 70, 8218-8223.[Abstract]
Chang, Y., Cesarman, E., Pessin, M. S., Lee, F., Culpepper, J., Knowles, D. M. & Moore, P. S. (1994). Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposis sarcoma.Science 266, 1865-1869.[Medline]
Daikoku, T., Shibata, S., Goshima, F., Oshima, S., Tsurumi, T., Yamada, H., Yamashita, Y. & Nishiyama, Y. (1997). Purification and characterization of the protein kinase encoded by the UL13 gene of herpes simplex virus type 2.Virology 235, 82-93.[Medline]
Hanks, S. K. & Quinn, A. M. (1991). Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members.Methods in Enzymology 200, 38-62.[Medline]
Kawaguchi, Y., Van Sant, C. & Roizman, B. (1998). Eukaryotic elongation factor 1 is hyperphosphorylated by the protein kinase encoded by the UL13 gene of herpes simplex virus 1.Journal of Virology 72, 1731-1736.
Leopardi, R., Ward, P. L., Ogle, W. O. & Roizman, B. (1997). Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 protein kinase.Journal of Virology 71, 1133-1139.[Abstract]
Li, M., Lee, H., Yoon, D. W., Albrecht, J. C., Fleckenstein, B., Neipel, F. & Jung, J. U. (1997). Kaposis sarcoma-associated herpesvirus encodes a functional cyclin.Journal of Virology 71, 1984-1991.[Abstract]
Li, M., Lee, H., Guo, J., Neipel, F., Fleckenstein, B., Ozato, K. & Jung, J. U. (1998). Kaposis sarcoma-associated herpesvirus viral interferon regulatory factor.Journal of Virology 72, 5433-5440.
Lin, S. & Broyles, S. S. (1994). Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus.Proceedings of the National Academy of Sciences, USA 91, 7653-7657.[Abstract]
Lin, S., Chen, W. & Broyles, S. S. (1992). The vaccinia virus B1R gene product is a serine/threonine protein kinase.Journal of Virology 66, 2717-2723.[Abstract]
Louise, N. J., Lowe, E. D., Martin, E. M. & David, J. O. (1998). The structural basis for substrate recognition and control by protein kinases.FEBS Letters 430, 1-11.[Medline]
Luthman, H. & Magnusson, G. (1983). High efficiency polyoma DNA transfection of chloroquine treated cells.Nucleic Acids Research 11, 1295-1308.[Abstract]
Mayer, B. J., Hirai, H. & Sakai, R. (1995). Evidence that SH2 domains promote processive phosphorylation by protein kinase.Current Biology 5, 296-305.[Medline]
Mesri, E. A., Cesarman, E., Arvanitakis, L., Rafii, S., Moore, M. A., Posnett, D. N., Knowles, D. M. & Asch, A. S. (1996). Human herpesvirus-8/Kaposis sarcoma-associated herpesvirus is a new transmissible virus that infects B cells.Journal of Experimental Medicine 183, 2385-2390.[Abstract]
Moffat, J. F., Zerboni, L., Sommer, M. H., Heineman, T. C., Cohen, J. I., Kaneshima, H. & Arvin, A. M. (1998). The ORF47 and ORF66 putative protein kinases of varicella-zoster virus determine tropism for human T cells and skin in the SCID-hu mouse.Proceedings of the National Academy of Sciences, USA 95, 11969-11974.
Moore, P. S., Boshoff, C., Weiss, R. A. & Chang, Y. (1996). Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV.Science 274, 1739-1744.
Neipel, F., Albrecht, J. C., Ensser, A., Huang, Y. Q., Li, J. J., Friedman-Kien, A. E. & Fleckenstein, B. (1997a). Human herpesvirus 8 encodes a homolog of interleukin-6.Journal of Virology 71, 839-842.[Abstract]
Neipel, F., Albrecht, J. C. & Fleckenstein, B. (1997b). Cell-homologous genes in the Kaposis sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity?Journal of Virology 71, 4187-4192.
Purves, F. C., Ogle, W. O. & Roizman, B. (1993). Processing of the herpes simplex virus regulatory protein alpha 22 mediated by the UL13 protein kinase determines the accumulation of a subset of alpha and gamma mRNAs and proteins in infected cells.Proceedings of the National Academy of Sciences, USA 90, 6701-6705.[Abstract]
Renne, R., Zhong, W., Herndier, B., McGrath, M., Abbey, N., Kedes, D. & Ganem, D. (1996). Lytic growth of Kaposis sarcoma-associated herpesvirus (human herpesvirus 8) in culture.Nature Medicine 2, 342-346.[Medline]
Roizman, B. (1990). Herpesviridae: a brief introduction. In Virology, pp. 1787-1794. Edited by B. F. Fields & D. M. Knipe. New York: Raven Press.
Russo, J. J., Bohenzky, R. A., Chien, M. C., Chen, J., Yan, M., Maddalena, D., Parry, J. P., Peruzzi, D., Edelman, I. S., Chang, Y. & Moore, P. S. (1996). Nucleotide sequence of the Kaposis sarcoma associated herpesvirus (HHV8).Proceedings of the National Academy of Sciences, USA 93, 14862-14867.
Sarid, R., Sato, T., Bohenzky, R. A., Russo, J. J. & Chang, Y. (1997). Kaposis sarcoma-associated herpesvirus encodes a functional bcl-2 homologue.Nature Medicine 3, 293-298.[Medline]
Smith, R. F. & Smith, T. F. (1989). Identification of new protein kinase-related genes in three herpesviruses, herpes simplex virus, varicella-zoster virus, and EpsteinBarr virus.Journal of Virology 63, 450-455.[Medline]
Soulier, J., Grollet, L., Oksenhendler, E., Cacoub, P., Cazals-Hatem, D., Babinet, P., dAgay, M. F., Clauvel, J. P., Raphael, M., Degos, L. & Sigaux, F. (1995). Kaposis sarcoma-associated herpesvirus-like DNA sequences in multicentric Castlemans disease.Blood 86, 1276-1280.
Sun, G. & Budde, R. J. (1997). Expression, purification, and initial characterization of human Yes protein tyrosine kinase from a bacterial expression system.Archives of Biochemistry and Biophysics 345, 135-142.[Medline]
Sun, R., Lin, S. F., Staskus, K., Gradoville, L., Grogan, E., Haase, A. & Miller, G. (1999). Kinetics of Kaposis sarcoma-associated herpesvirus gene expression.Journal of Virology 73, 2232-2242.
Wilson, A. C. & Patient, R. K. (1991). Evaluation of extrachromosomal gene copy number of transiently transfected cell lines. In Gene Transfer and Expression Protocols, pp. 397-404. Edited by E. J. Murray. New Jersey: Humana Press.
Zhong, W., Wang, H., Herndier, B. & Ganem, D. (1996). Restricted expression of Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi sarcoma.Proceedings of the National Academy of Sciences, USA 93, 6641-6646.
Received 14 October 1999;
accepted 3 December 1999.