CK2 protein kinase is stimulated and redistributed by functional herpes simplex virus ICP27 protein

It has been shown previously (S. Wadd, H. Bryant, O. Filhol, J. E. Scott, T.-T. Hsieh, R. D. Everett, and J. B. Clements, J. Biol. Chem. 274:28991-28998, 2000) that ICP27, an essential and multifunctional herpes simplex virus type 1 (HSV-1) protein, interacts with CK2 and with heterogeneous ribonucleoprotein K (hnRNP K). CK2 is a pleiotropic and ubiquitous protein kinase, and the tetrameric holoenzyme consists of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We show here that HSV-1 infection stimulates CK2 activity. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Both CK2 stimulation and redistribution require expression and cytoplasmic accumulation of ICP27. In HSV-1-infected cells, CK2 phosphorylates ICP27 and affects its cytoplasmic accumulation while it also phosphorylates hnRNP K, which is not ordinarily phosphorylated by this kinase, suggesting an alteration of hnRNP K activities. This is the first example of CK2 stimulation by a viral protein in vivo, and we propose that it might facilitate the HSV-1 lytic cycle by, for example, regulating trafficking of ICP27 protein and/or viral RNAs.     

SUMO-1 modification of human cytomegalovirus IE1/IE72

Human cytomegalovirus (HCMV) immediate-early protein IE1/IE72 is involved in undermining many cellular processes including cell cycle regulation, apoptosis, nuclear architecture, and gene expression. The multifunctional nature of IE72 suggests that posttranslational modifications may modulate its activities. IE72 is a phosphoprotein and has intrinsic kinase activity (S. Pajovic, E. L. Wong, A. R. Black, and J. C. Azizkhan, Mol. Cell. Biol. 17:6459-6464, 1997). We now demonstrate that IE72 is covalently conjugated to the small ubiquitin-like modifier (SUMO-1). SUMO-1 is an 11.5-kDa protein that is conjugated to multiple proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. A covalently modified protein migrating at approximately 92 kDa, which is stabilized by a SUMO-1 hydrolase inhibitor, is revealed by Western blotting with anti-IE72 of lysates from cells infected with HCMV or cells expressing IE72. SUMO modification of IE72 was confirmed by immunoprecipitation with anti-IE72 and anti-SUMO-1 followed by Western blotting with anti-SUMO-1 and anti-IE72, respectively. Lysine 450 is within a sumoylation consensus site (I,V,L)KXE; changing lysine 450 to arginine by point mutation abolishes SUMO-1 modification of IE72. Inhibition of protein phosphatase 1 and 2A, which increases the phosphorylation of IE72, suppresses the formation of SUMO-1-IE72 conjugates. Both wild-type IE72 and IE72(K450R) localize to nuclear PML oncogenic domains and disrupt them. Studies of protein stability, transactivation, and complementation of IE72-deficient HCMV (CR208) have revealed no significant differences between wild-type IE72 and IE72(K450R).     

Recombinant monoclonal antibody recognizes a unique epitope on varicella-zoster virus immediate-early 63 protein

We previously constructed a recombinant monoclonal antibody (rec-MAb 63P4) that detects immediate-early protein IE63 encoded by varicella-zoster virus (VZV) in the cytoplasm of productively infected cells. Here, we used ORF63 truncation mutants to map the rec-MAb 63P4 binding epitope to amino acids 141 to 150 of VZV IE63, a region not shared with other widely used anti-IE63 antibodies, and found that the recombinant antibody does not bind to the simian IE63 counterpart.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

Tyrosine phosphorylation of the herpes simplex virus type 1 regulatory protein ICP22 and a cellular protein which shares antigenic determinants with ICP22.

At least eight herpes simplex virus type 1 (HSV-1) and five HSV-2 proteins were tyrosine phosphorylated in infected cells. The first viral tyrosine phosphoprotein identified was the HSV-1 regulatory protein ICP22. Also, two novel phosphotyrosine proteins were bound by anti-ICP22 antibodies. H(R22) is a cellular protein, while the F(R10) protein is observed only in HSV-1-infected cells.     

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.     

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and HCMV infection in immunocompromised patients may trigger devastating disease. Cytotoxic lymphocytes control HCMV by releasing granzymes towards virus-infected cells. In mice, granzyme M (GrM) has a physiological role in controlling murine CMV infection. However, the underlying mechanism remains poorly understood. In this study, we showed that human GrM was expressed by HCMV-specific CD8⁺ T cells both in latently infected healthy individuals and in transplant patients during primary HCMV infection. We identified host cell heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a physiological GrM substrate. GrM most efficiently cleaved hnRNP K in the presence of RNA at multiple sites, thereby likely destroying hnRNP K function. Host cell hnRNP K was essential for HCMV replication not only by promoting viability of HCMV-infected cells but predominantly by regulating viral immediate-early 2 (IE2) protein levels. Furthermore, hnRNP K interacted with IE2 mRNA. Finally, GrM decreased IE2 protein expression in HCMV-infected cells. Our data suggest that targeting of hnRNP K by GrM contributes to the mechanism by which cytotoxic lymphocytes inhibit HCMV replication. This is the first evidence that cytotoxic lymphocytes target host cell proteins to control HCMV infections.     

Protein kinase B/Akt is present in activated form throughout the entire replicative cycle of deltaU(S)3 mutant virus but only at early times after infection with wild-type herpes simplex virus 1

The product of the herpes simplex virus 1 (HSV-1) US3 gene is a multifunctional serine-threonine protein kinase that can block apoptosis induced by proapoptotic cellular proteins, exogenous agents, or replication-defective viruses. Earlier studies showed that the U(S)3 kinase activates and functionally overlaps cellular protein kinase A (PKA). In this study we examined the status of phosphatidylinositol 3-kinase [PI3K] and of its effector, protein kinase B/Akt (PKB/Akt), a component of a major pathway of mammalian antiapoptotic signaling systems. We report the following. (i) Infection of target cells with HSV-1 induces transient phosphorylation of serine 473 of PKB/Akt early in infection, with a mechanism that is dependent on PI3K. Inhibition of PI3K induced apoptosis in mock-infected or deltaU(S)3 mutant-virus-infected but not in wild-type-virus-infected cells and reduced the accumulation of specific viral gene products, including the U(S)3 protein kinase, but had a marginal effect on virus yields. (ii) At later times after infection, the total amounts of PKB/Akt decreased and phosphorylated PKB/Akt forms disappeared in a U(S)3-dependent and protein phosphatase 2A-independent manner. (iii) Activation of PKA by forskolin did not mediate significant dephosphorylation of PKB/Akt. Our results are consistent with the model that PKB/Akt is activated early in infection and acts to block apoptosis in infected cells prior to the accumulation of U(S)3 protein kinase and that it persists and continues to function as an antiapoptotic protein in the absence of U(S)3 but becomes redundant or even inimical once U(S)3 protein kinase accumulates in effective amounts.     

Casein kinase 2, the versatile regulator of cell survival

Casein kinase 2 (CK2), a highly conservative, multifunctional serine/threonine protein kinase, is critically important for the regulation of a plethora of processes in eukaryotes, such as cell proliferation, differentiation and death. CK2 is expressed in all tissues; in particular, its amount and activity are elevated in tumor cells. Unlike many regulatory proteins CK2 permanently adopts an active conformation. Of the utmost importance are the anti-apoptotic functions of CK2. This protein kinase is capable of regulating cell survival at multiple levels including DNA repair, NF-kappaB, Wnt, PI3K/Akt and JAK-STAT signaling cascades, chaperones, activation of anti-apoptotic proteins and down-regulation of pro-apoptotic counterparts, in particular, caspases. The versatility of CK2-mediated phosphorylation ensures the survival of tumor cells exposed to stimuli that differ in the origin and mechanisms of cytotoxicity. This manifold mode of CK2-dependent survival makes this enzyme an important target for antitumor therapy.     

DNA-dependent protein kinase (DNA-PK) phosphorylates nuclear DNA helicase II/RNA helicase A and hnRNP proteins in an RNA-dependent manner

An RNA-dependent association of Ku antigen with nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), was found in nuclear extracts of HeLa cells by immunoprecipitation and by gel filtration chromatography. Both Ku antigen and NDH II were associated with hnRNP complexes. Two-dimensional gel electrophoresis showed that Ku antigen was most abundantly associated with hnRNP C, K, J, H and F, but apparently not with others, such as hnRNP A1. Unexpectedly, DNA-dependent protein kinase (DNA-PK), which comprises Ku antigen as the DNA binding subunit, phosphorylated hnRNP proteins in an RNA-dependent manner. DNA-PK also phosphorylated recombinant NDH II in the presence of RNA. RNA binding assays displayed a preference of DNA-PK for poly(rG), but not for poly(rA), poly(rC) or poly(rU). This RNA binding affinity of DNA-PK can be ascribed to its Ku86 subunit. Consistently, poly(rG) most strongly stimulated the DNA-PK-catalyzed phosphorylation of NDH II. RNA interference studies revealed that a suppressed expression of NDH II altered the nuclear distribution of hnRNP C, while silencing DNA-PK changed the subnuclear distribution of NDH II and hnRNP C. These results support the view that DNA-PK can also function as an RNA-dependent protein kinase to regulate some aspects of RNA metabolism, such as RNA processing and transport.