The herpes simplex virus 1 protein kinase encoded by the US3 gene mediates posttranslational modification of the phosphoprotein encoded by the UL34 gene.

Earlier studies have shown that a herpes simplex virus 1 (HSV-1) open reading frame, US3, encodes a novel protein kinase and have characterized the cognate amino acid sequence which is phosphorylated by this enzyme. This report identifies an apparently essential viral phosphoprotein whose posttranslational processing involves the viral protein kinase. Analyses of viral proteins phosphorylated in the course of productive infection revealed a phosphoprotein whose mobility was viral protein kinase and serotype dependent. Thus, the corresponding HSV-1 and HSV-2 phosphoproteins differ in their electrophoretic mobilities, and the phosphoprotein specified by the HSV-1 mutant deleted in US3 (R7041) differs from that of the corresponding HSV-1 and HSV-2 proteins. Analyses of HSV-1 x HSV-2 recombinants mapped the phosphoprotein between 0.42 and 0.47 map units on the prototype HSV-1 DNA map. Within this region, the UL34 open reading frame was predicted to encode a protein of appropriate molecular weight which would also contain the consensus target site for phosphorylation by the viral protein kinase as previously defined with synthetic peptides. Replacement of the native UL34 gene with a UL34 gene tagged with a 17-amino-acid epitope from the alpha 4 protein identified this gene as encoding the phosphoprotein. Finally, mutagenesis of the predicted phosphorylation site on UL34 in the viral genome, and specifically the substitution of threonine or serine with alanine in the product of the UL34 gene, yielded phosphoproteins whose electrophoretic mobilities could not be differentiated from that of the US3- mutant. We conclude that the posttranslational processing of the UL34 gene product to its wild-type phenotype requires the participation of the viral protein kinase. While the viral protein kinase is not essential for viral replication in cells in culture, the UL34 gene product itself may not be dispensable.     

The product of the UL31 gene of herpes simplex virus 1 is a nuclear phosphoprotein which partitions with the nuclear matrix.

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32Pi.     

The UL25 protein of pseudorabies virus associates with capsids and localizes to the nucleus and to microtubules

The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses. The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PrV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.     

Herpes simplex virus type 1 primary envelopment: UL34 protein modification and the US3-UL34 catalytic relationship

The herpes simplex virus type 1 (HSV-1) US3 kinase is likely important for primary envelopment of progeny nucleocapsids since it localizes to the nuclear envelope of infected cells and largely determines the phosphorylation state and localization of the necessary primary envelopment factor, the UL34 protein. In HEp-2 cells, the production of infectious US3 null progeny is delayed and decreased relative to that of the parental strain, HSV-1(F). Furthermore, the US3 kinase affects the morphology of primary envelopment such that in its absence, UL34 protein-containing enveloped virions accumulate within membrane-bound vesicles. These vesicles are most often found along the interior periphery of the nucleus and may be derived from the inner nuclear membrane. Since the US3 and UL34 proteins comprise a kinase-substrate pair, a reasonable hypothesis is that the US3 kinase influences these replication parameters by direct phosphorylation of the UL34 protein. For this report, recombinant viruses were constructed to determine the significance of UL34 protein phosphorylation and US3 catalytic activity on UL34 protein localization, single-step growth, and envelopment morphology in both HEp-2 and Vero cells. The data presented suggest that the significance of UL34 phosphorylation is cell type dependent and that efficient viral morphogenesis requires US3-mediated phosphorylation of an infected cell protein other than UL34.     

Herpes simplex virus 1-encoded protein kinase UL13 phosphorylates viral Us3 protein kinase and regulates nuclear localization of viral envelopment factors UL34 and UL31

UL13 and Us3 are protein kinases encoded by herpes simplex virus 1. We report here that Us3 is a physiological substrate for UL13 in infected cells, based on the following observations. (i) The electrophoretic mobility, in denaturing gels, of Us3 isoforms from Vero cells infected with wild-type virus was slower than that of isoforms from cells infected with a UL13 deletion mutant virus (DeltaUL13). After treatment with phosphatase, the electrophoretic mobility of the Us3 isoforms from cells infected with wild-type virus changed, with one isoform migrating as fast as one of the Us3 isoforms from DeltaUL13-infected cells. (ii) A recombinant protein containing a domain of Us3 was phosphorylated by UL13 in vitro. (iii) The phenotype of DeltaUL13 resembles that of a recombinant virus lacking the Us3 gene (DeltaUs3) with respect to localization of the viral envelopment factors UL34 and UL31, whose localization has been shown to be regulated by Us3. UL34 and UL31 are localized in a smooth pattern throughout the nuclei of cells infected with wild-type virus, whereas their localization in DeltaUL13- and DeltaUs3-infected cells appeared as nuclear punctate patterns. These results indicate that UL13 phosphorylates Us3 in infected cells and regulates UL34 and UL31 localization, either by phosphorylating Us3 or by a Us3-independent mechanism.     

The herpes simplex virus UL37 protein is phosphorylated in infected cells.

The herpes simplex virus type 1 (HSV-1) UL37 open reading frame encodes a 120-kDa late (gamma 1), nonstructural protein in infected cells. Recent studies in our laboratory have demonstrated that the UL37 protein interacts in the cytoplasm of infected cells with ICP8, the major HSV-1 DNA-binding protein. As a result of this interaction, the UL37 protein is transported to the nucleus and can be coeluted with ICP8 from single-stranded DNA columns. Pulse-labeling and pulse-chase studies of HSV-1-infected cells with [35S]methionine and 32Pi demonstrated that UL37 was a phosphoprotein which did not have a detectable rate of turnover. The protein was phosphorylated soon after translation and remained phosphorylated throughout the viral replicative cycle. UL37 protein expressed from a vaccinia virus recombinant was also phosphorylated during infection, suggesting that the UL37 protein was phosphorylated by a cellular kinase and that interaction with the ICP8 protein was not a prerequisite for UL37 phosphorylation.     

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.     

Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34 gene product

Primary envelopment of several herpesviruses has been shown to occur by budding of intranuclear capsids through the inner nuclear membrane. By subsequent fusion of the primary envelope with the outer nuclear membrane, capsids are released into the cytoplasm and gain their final envelope by budding into vesicles in the trans-Golgi area. We show here that the product of the UL34 gene of pseudorabies virus, an alphaherpesvirus of swine, is localized in transfected and infected cells in the nuclear membrane. It is also detected in the envelope of virions in the perinuclear space but is undetectable in intracytoplasmic and extracellular enveloped virus particles. Conversely, the tegument protein UL49 is present in mature virus particles and absent from perinuclear virions. In the absence of the UL34 protein, acquisition of the primary envelope is blocked and neither virus particles in the perinuclear space nor intracytoplasmic capsids or virions are observed. However, light particles which label with the anti-UL49 serum are formed in the cytoplasm. We conclude that the UL34 protein is required for primary envelopment, that the primary envelope is biochemically different from the final envelope in that it contains the UL34 protein, and that perinuclear virions lack the tegument protein UL49, which is present in mature virions. Thus, we provide additional evidence for a two-step envelopment process in herpesviruses.     

Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture.

The UL7 gene of bovine herpesvirus 1 (BHV-1) strain Schönböken was found at a position and in a context predicted from the gene order in the prototype alphaherpesvirus herpes simplex virus type 1. The gene and flanking regions were sequenced, the UL7 RNA and protein were characterized, and 98.3% of the UL7 open reading frame was deleted from the viral genome without destroying productive virus replication. Concomitant deletion of nine 3' codons from the BHV-1 UL6 ORF and 77 amino acids from the carboxy terminus of the predicted BHV-1 UL8 protein demonstrated that these domains are also not essential for function of the respective proteins. The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa. Comparison with UL7 homologs of other alphaherpesviruses revealed a high degree of homology, the most prominent being to the predicted UL7 polypeptide of varicella-zoster virus, with 43.3% identical amino acids. A monospecific anti-UL7 serum identified the 33-kDa (apparent-molecular-mass) UL7 polypeptide which is translated from an early-expressed 1.7-kb RNA. The UL7 protein was localized in the cytoplasm of infected cells and could not be detected in purified virions. In summary, we describe the first identification of an alphaherpesviral UL7-encoded polypeptide and demonstrate that the UL7 protein is not essential for replication of BHV-1 in cell culture.     

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.     

US3 of herpes simplex virus type 1 encodes a promiscuous protein kinase that phosphorylates and alters localization of lamin A/C in infected cells

The herpes simplex virus type 1 (HSV-1) US3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins UL31 and UL34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins UL34 and UL31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of US3 kinase activity. To determine how US3 alters lamin A/C distribution, US3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative US3 kinase consensus site in the lamin A/C sequence. US3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires US3 kinase activity. These data suggest that US3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.     

The human cytomegalovirus UL44 protein is a substrate for the UL97 protein kinase

The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [(32)P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a UL97 null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.     

Pseudorabies virus UL3 gene codes for a nuclear protein which is dispensable for viral replication

Many of the products of the ca. 80 genes encoded by alphaherpesviruses have already been identified and, at least tentatively, functionally characterized. Among the least characterized proteins are the products of the genes homologous to herpes simplex virus UL3, which are present only in the subfamily Alphaherpesvirinae: To identify the UL3 protein of the porcine alphaherpesvirus pseudorabies virus (PrV), the complete PrV UL3 open reading frame was cloned, expressed in Escherichia coli as a glutathione S-transferase fusion protein, and used for immunization of a rabbit. In Western blots, the generated antiserum specifically detected a 34-kDa protein in PrV-infected cells, which was absent from purified virus preparations, indicating that PrV UL3 encodes a nonstructural protein. In indirect immunofluorescence analysis, the anti-UL3 serum produced predominantly nuclear staining in transfected as well as in infected cells, which was not altered in the absence of other virus-encoded nuclear proteins such as the UL31 and UL34 gene products. To investigate UL3 function, a deletion mutant, PrV-DeltaUL3F2, was constructed and characterized. This mutant replicated and formed plaques on noncomplementing cells indistinguishable from wild-type PrV, demonstrating that PrV UL3 is not required for virus propagation in cultured cells. Moreover, ultrastructural examinations revealed no impairment of capsid formation in the nucleus, nuclear egress of capsids, virion maturation in the cytoplasm, or virus release. Thus, the overall properties of PrV UL3 are similar to those described for the homologous herpes simplex virus proteins which may be indicative of a common, yet hitherto unknown, function in alphaherpesvirus replication. However, based on our studies, an involvement of the UL3 homologs in virion formation appears unlikely.     

U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.     

The U(L)15 gene of herpes simplex virus type 1 contains within its second exon a novel open reading frame that is translated in frame with the U(L)15 gene product.

The U(L)15 gene of herpes simplex virus type 1 is composed of two exons. A mutation previously shown to preclude viral DNA cleavage and packaging at the nonpermissive temperature was identified as a change from a highly conserved serine to proline at codon 653. Separate viral mutants that contained stop codons inserted into exon I of U(L)15 (designated S648) or an insertion of the Escherichia coli lacZ gene into a truncated U(L)15 exon II [designated HSV-1(delta U(L)15ExII)] were constructed. Recombinant viruses derived from S648 and HSV-1(delta U(L)15ExII) and containing restored U(L)15 genes were constructed and designated S648R and HSV-1(delta U(L)15ExIIR), respectively. Unlike HSV-1(delta U(L)15ExIIR) and S648R, the viruses containing mutant U(L)15 genes failed to cleave and package viral DNA when propagated on noncomplementing cells. As revealed by electron microscopy, large numbers of enveloped capsids lacking viral DNA accumulated within the cytoplasm of cells infected with either S648 or HSV-1(delta U(L)15ExII) but not in cells infected with HSV-1(delta U(L)15ExIIR) or S648R. Thus, one function of the U(L)15 gene is to effectively prevent immature particles lacking DNA from exiting the nucleus by envelopment at the inner lamella of the nuclear membrane. Cells infected with HSV-1(delta U(L)15ExII) did not express the 75,000- or 35,000-apparent-Mr proteins previously shown to be products of the U(L)15 open reading frame, whereas the 35,000-apparent-Mr protein was readily detectable in cells infected with S648. We conclude that at least the 75,000-Mr protein is required for viral DNA cleavage and packaging and hypothesize that the 35,000-Mr protein is derived from translation of a novel mRNA located partially or completely within the second exon of U(L)15.     

Herpes simplex virus 1 protein kinase Us3 and major tegument protein UL47 reciprocally regulate their subcellular localization in infected cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We have identified UL47, a major virion protein, as a novel physiological substrate of Us3. In vitro kinase assays and systematic analysis of mutations at putative Us3 phosphorylation sites near the nuclear localization signal of UL47 showed that serine at residue 77 (Ser-77) was required for Us3 phosphorylation of UL47. Replacement of UL47 Ser-77 by alanine produced aberrant accumulation of UL47 at the nuclear rim and impaired the nuclear localization of UL47 in a significant fraction of infected cells. The same defect in UL47 localization was produced by an amino acid substitution in Us3 that inactivated its protein kinase activity. In contrast, a phosphomimetic mutation at UL47 Ser-77 restored wild-type nuclear localization. The UL47 S77A mutation also reduced viral replication in the mouse cornea and the development of herpes stromal keratitis in mice. In addition, UL47 formed a stable complex with Us3 in infected cells, and nuclear localization of Us3 was significantly impaired in the absence of UL47. These results suggested that Us3 phosphorylation of UL47 Ser-77 promoted the nuclear localization of UL47 in cell cultures and played a critical role in viral replication and pathogenesis in vivo. Furthermore, UL47 appeared to be required for efficient nuclear localization of Us3 in infected cells. Therefore, Us3 protein kinase and its substrate UL47 demonstrated a unique regulatory feature in that they reciprocally regulated their subcellular localization in infected cells.     

Comparison between human cytomegalovirus pUL97 and murine cytomegalovirus (MCMV) pM97 expressed by MCMV and vaccinia virus: pM97 does not confer ganciclovir sensitivity

The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056-7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.     

Posttranslational processing of infected cell protein 22 mediated by viral protein kinases is sensitive to amino acid substitutions at distant sites and can be cell-type specific

Infected cell protein 22 (ICP22) is posttranslationally phosphorylated by the viral kinases encoded by U(S)3 and U(L)13 and nucleotidylylated by casein kinase II. In rabbit and rodent cells and in primary human fibroblasts infected with mutants from which the alpha 22 gene encoding ICP22 had been deleted, a subset of late (gamma(2)) gene products exemplified by U(L)38 and U(S)11 proteins are expressed at a reduced level, as measured by the accumulation of both mRNA and protein. The same phenotype was observed in cells infected with mutants lacking the U(L)13 gene. The focus of this report is on three serine- and threonine-rich domains of ICP22. Two of these domains are homologs located between residues 38 to 66 and 300 to 328. The third domain is near the carboxyl terminus and contains the sequence T374SS. The results were as follows. (i) Alanine substitutions in the amino-terminal homolog precluded the posttranslational processing of ICP22 in rabbit skin cells and in Vero cells but had no effect on the accumulation of either U(S)11 or U(L)38 protein. (ii) Alanine substitutions in the carboxyl-terminal homolog had no effect on posttranslational processing of ICP22 accumulating in Vero cells but precluded full processing of ICP22 accumulating in rabbit skin cells. The effect on accumulation of U(L)38 and U(S)11 proteins was insignificant in Vero cells and minimal in rabbit skin cells. (iii) Substitutions of alanine for the threonine and serines in the third domain precluded full processing of ICP22 and caused a reduction of accumulation of U(S)11 and U(L)38 proteins. These results indicate the following. (i) The posttranslational processing of ICP22 is sensitive to mutations within the domains of ICP22 tested and is cell-type dependent. (ii) Posttranslational processing of ICP22 is not required for accumulation of U(L)38 and U(S)11 proteins to the same level as that seen in cells infected with the wild-type virus. (iii) The T374SS sequence shared by ICP22 and the U(S)1.5 proteins is essential for the accumulation of a subset of gamma(2) proteins exemplified by U(S)11 and U(L)38 and is the first step in mapping of the sequences necessary for optimal accumulation of U(S)11 and U(L)38 proteins.