Characterization of regulatory functions of the varicella-zoster virus gene 63-encoded protein.

Varicella-zoster virus (VZV) gene 63 encodes a protein (IE63) with a predicted molecular mass of 30.5 kDa which has amino acid similarities to the immediate-early (IE) protein 22 (ICP22) of herpes simplex virus type 1. ICP22 is a polypeptide synthesized in herpes simplex virus type 1-infected cells, and as is the case for its VZV counterpart, its regulatory functions are unknown. On the basis of the VZV DNA sequence, it has been shown that IE63 exhibits hydrophilic and acidic properties, suggesting that this protein could play a regulatory role during the infectious cycle. We report in this article cotransfection experiments which demonstrate that the VZV gene 63 protein strongly represses, in a dose-dependent manner, the expression of VZV gene 62. On the other hand, transient expression of the VZV gene 63 protein can promote activation of the thymidine kinase gene but cannot affect the expression of the genes encoding glycoproteins I and II. The results of transient expression experiments strongly suggest that the VZV gene 63 protein could play a pivotal role in the repression of IE gene expression as well as in the activation of early gene expression.     

Phosphorylation and nuclear localization of the hepatitis B virus core protein: significance of serine in the three repeated SPRRR motifs.

Hepatitis B virus core protein (antigen) is an important serologic marker of hepatitis B virus infection. This protein is found in the cytoplasm or the nuclei, or both, of infected hepatocytes. A nuclear localization signal has previously been identified in the core protein sequence. This signal overlaps three repeated SPRRR motifs. In this report, we demonstrate that substitution of all of the serine residues in these three SPRRR motifs with alanine can prevent almost entirely the phosphorylation of the core protein in Huh-7 hepatoma cells, enhance nuclear localization of the core protein in both Huh-7 and nonhepatic cells, and abolish cell cycle regulation of nuclear localization of the core protein. Since the three core protein mutants which retained only one serine residue of each of the three SPRRR motifs could be phosphorylated to similar degrees, these three serine residues likely could serve as the acceptor sites for phosphorylation with equal efficiency. These results, together with the observation that the three SPRRR motifs overlap the nuclear localization signal of the core protein, raise the possibility that nuclear localization of the core protein is negatively regulated by phosphorylation of the serine residues in the SPRRR motifs.     

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.     

Phosphorylation of the Epstein-Barr virus nuclear antigen 2.

A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic peptide corresponding to amino acids 464-476 of EBNA-2 as a substrate led to the incorporation of 0.69 mol phosphate/mol peptide indicating that only one of three potential phosphorylation sites within the peptide was modified. The most likely amino acid residues for phosphorylation by CK-2 are Ser469 and Ser470.     

Dissection of a novel nuclear localization signal in open reading frame 29 of varicella-zoster virus

Open reading frame 29 (ORF29) of varicella-zoster virus (VZV) encodes a 120-kDa single-stranded DNA binding protein (ORF29p) that is not packaged in the virion and is expressed during latency. During lytic infection, ORF29p is localized primarily to infected cell nuclei. In contrast, ORF29p is found exclusively in the cytoplasm in neurons of the dorsal root ganglia obtained at autopsy from seropositive latently infected patients. ORF29p accumulates in the nuclei of neurons in dorsal root ganglia obtained at autopsy from patients with active zoster. The localization of this protein is, therefore, tightly correlated with the proposed VZV lytic/latent switch. In this report, we have investigated the nuclear import mechanism of ORF29p. We identified a novel nuclear targeting domain bounded by amino acids 9 to 154 of ORF29p that functions independent of other VZV-encoded factors. In vitro import assays in digitonin-permeabilized HeLa cells reveal that ORF29p is transported into the nucleus by a Ran-, karyopherin alpha- and beta-dependent mechanism. These data are further supported by the demonstration that a glutathione S-transferase-karyopherin alpha fusion interacts with ORF29p, but not with a protein containing a point mutation in its nuclear localization signal (NLS). Therefore, the region of ORF29p responsible for its nuclear targeting is also involved in the association with karyopherin alpha. As a result of this interaction, this noncanonical NLS appears to hijack the classical cellular nuclear import machinery. Elucidation of the mechanisms governing ORF29p nuclear targeting could shed light on the VZV reactivation process.     

Arginine-rich regions succeeding the nuclear localization region of the herpes simplex virus type 1 regulatory protein ICP27 are required for efficient nuclear localization and late gene expression.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein that localizes to the nuclei of infected cells. The strong nuclear localization signal (NLS) of ICP27 was identified recently and shown to reside in the amino-terminal portion of the polypeptide from residues 110 to 137 (W.E. Mears, V. Lam, and S.A. Rice, J. Virol. 69:935-947, 1995). There are also two arginine-rich regions directly succeeding the NLS. The first of these arginine-rich sequences (residues 141 to 151), together with the NLS, has been shown by Mears et al. to form the nucleolar localization signal. Arginine-rich motifs are common in domains involved in nuclear localization and RNA binding. To analyze the role of the arginine-rich regions in ICP27, we constructed stably transformed cell lines containing ICP27 mutants with deletions of all or parts of the NLS and arginine-rich regions. We also constructed mutants in which these regions were replaced with heterologous NLSs or RNA-binding domains. Characterization of these mutants indicated that the arginine-rich regions were required but not sufficient for wild-type localization of ICP27. More importantly, the NLS and arginine-rich regions were also essential to the function of ICP27. Mutants lacking these sequences were defective in late gene expression during infection even when ICP27 was properly localized to the nucleus by substitution of the NLS from simian virus 40 large T antigen. Further, the defect in late gene expression could not be overcome by replacement with the highly basic RNA-binding domain of human immunodeficiency virus type 1 Tat. The deficiency in late gene expression was independent of ICP27's role in stimulating viral DNA replication. In addition, localization of the HSV-1 proteins ICP4, ICP0, and ICP8 was unaffected by ICP27 mutants in this region. These results suggest that the arginine-rich regions are required for efficient nuclear localization and for the regulatory activity of ICP27 involved in viral late gene expression.     

Phosphorylation by MAPK regulates simian immunodeficiency virus Vpx protein nuclear import and virus infectivity

Transport of the viral genome into the nucleus required phosphorylation of components in the preintegration complex by virion-associated host cellular kinases. In this study, we showed that ERK-2/MAPK is associated with simian immunodeficiency virus (SIV) virions and regulated the nuclear transport of Vpx and virus replication in non-proliferating target cells by phosphorylating Vpx. Suppression of the virion-associated ERK-2 activity by MAPK pathway inhibitors impaired both Vpx nuclear import and viral infectivity without affecting virus particle maturation and release. In addition, mutation analysis indicated that the inactivation of Vpx phosphorylation precluded nuclear import and reduced virus replication in macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral preintegration complex nuclear import are present. In this study, we also showed that co-localization of Vpx with Gag precursor in the cytoplasm is a prerequisite for Vpx incorporation into virus particles. Substitution of hydrophobic Leu-74 and Ile-75 with serines in the helical domain abrogated Vpx nuclear import, and its incorporation into virus particles, despite its localization in the cytoplasm, suggested that the structural integrity of helical domains is critical for Vpx functions. Taken together, these studies demonstrated that the host cell MAPK signal transduction pathway regulated an early step in SIV infection.     

Random mutagenesis of the thymidine kinase gene of varicella-zoster virus.

To understand the relationship between the primary structure and function of varicella-zoster virus thymidine kinase (VZV TK; EC 2.7.1.21), we established rapid screening and phenotypic selection of mutant VZV TK genes in TK-deficient Escherichia coli C600 by using a constitutive pKK223-3 expression plasmid. In this screening system, mutant TK genes generated by random mutagenesis were identified by the sensitivity of E. coli-expressing VZV TKs to 5-bromo-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil. Twenty-four mutant clones with amino acid substitutions were isolated, and their nucleotide sequence and enzymatic activities were determined. Of the 24 clones, 20 had single amino acid substitutions, 2 clones had double amino acid substitutions, and 1 clone had triple amino acid substitutions. In 17 cases of single amino acid substitution, six mutations led to lost enzyme activity, and four of these six mutations centered in the ATP-binding site. The other 11 mutations resulted in reduction of both TK and thymidylate kinase activities or only thymidylate kinase activity and were located in scattered positions in the VZV TK gene, although 5 mutations showed a tendency to cluster in the region between positions 251 and 260.     

Complex nuclear localization signals in the matrix protein of vesicular stomatitis virus

The matrix (M) protein of vesicular stomatitis virus (VSV) functions from within the nucleus to inhibit bi-directional nucleocytoplasmic transport. Here, we show that M protein can be imported into the nucleus by an active transport mechanism, even though it is small enough (approximately 27 kDa) to diffuse through nuclear pore complexes. We map two distinct nuclear localization signal (NLS)-containing regions of M protein, each of which is capable of directing the nuclear localization of a heterologous protein. One of these regions, comprising amino acids 47-229, is also sufficient to inhibit nucleocytoplasmic transport. Two amino acids that are conserved among the matrix proteins of vesiculoviruses are important for nuclear localization, but are not essential for the inhibitory activity of M protein. Thus, different regions of M protein function for nuclear localization and for inhibitory activity.     

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.     

Phosphorylation of varicella-zoster virus open reading frame (ORF) 62 regulatory product by viral ORF 47-associated protein kinase.

Varicella-zoster virus (VZV) encodes within its unique long region a gene product with protein kinase motifs. In a previous study, we demonstrated that immunoprecipitated VZV open reading frame (ORF) 47 protein was associated with a functional protein kinase activity, on the basis of its ability to both autophosphorylate and phosphorylate artificial substrates. To further define potential substrates of ORF 47-associated protein kinase, we analyzed individual viral phosphoproteins to determine whether any were modified by the viral protein kinase. These candidates included gene products of VZV ORFs 4, 61, 62, and 63, which are homologs of herpes simplex virus type 1 (HSV-1) immediate-early proteins. Each of the above VZV proteins was coimmunoprecipitated with ORF 47 kinase, and the immune complex was incubated in a protein kinase assay. Under these conditions, only the VZV immediate-early ORF 62 protein was phosphorylated by ORF 47-associated protein kinase. The specificity of this phosphorylation event was analyzed by a competition assay in which a recombinant ORF 47 protein lacking enzymatic activity was able to reduce the amount of phosphorylation of ORF 62 protein by VZV ORF 47-associated kinase. To provide an additional evaluation of specificity, the experiment was repeated with [32P]GTP instead of [32P]ATP, because the VZV ORF 47 kinase has the distinctive property of using GTP as a phosphate donor. Again the ORF 62 substrate was phosphorylated. In summary, the VZV ORF 47-associated protein kinase (the HSV-1 UL13 homolog) catalyzed the in vitro phosphorylation of the VZV ORF 62 protein, the homolog of the HSV-1 ICP4 regulatory protein.     

Phosphorylation of the Abelson murine leukemia virus transforming protein.

The Abelson murine leukemia virus transforming gene product is a phosphorylated protein encoded by both viral and cellular sequences. This gene product has an amino-terminal region derived from the gag gene of its parent virus and a carboxyl-terminal region of (abl) derived from a normal murine cellular gene. Using a combination of partial proteolytic cleavage techniques and antisera specific for gag and abl sequences, we mapped in vivo phosphorylation sites to different regions of the protein. Phosphoproteins encoded by strain variants and transformation-defective mutants of Abelson murine leukemia virus with defined deletions in the primary sequence of the abl region were compared by two dimensional limit digest peptide mapping. Specific phosphorylation pattern differences for wild-type and mutant proteins probably represented deletions of specific phosphate acceptor sites in the abl region. An in vitro autophosphorylation activity copurified with the Abelson murine leukemia virus protein from transformation-competent strains. A peptide analysis of such in vitro reactions demonstrated that these phosphorylation sites were restricted to the amino-terminal region, and the specific sites appeared to be unrelated to the sites found on proteins phosphorylated in vivo. Thus, the autophosphorylation reaction probably correlates with an activity important in transformation, but the specific end product in vitro bears little resemblance to its function in vivo.     

Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27.

Previous work has shown that the herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 localizes to the cell nucleus and that certain mutant ICP27 polypeptides localize preferentially in nucleoli. To map the signals in ICP27 which mediate its nuclear localization, we identified the portions of ICP27 which can direct a cytoplasmic protein, pyruvate kinase (PK), to nuclei. Our results demonstrate that ICP27 contains multiple nuclear localization signals (NLSs) that function with differing efficiencies. First, ICP27 possesses a strong NLS, mapping to residues 110 to 137, which bears similarity to the bipartite NLSs found in Xenopus laevis nucleoplasmin and other proteins. Second, ICP27 possesses one or more weak NLSs which map to a carboxyl-terminal portion of the protein between residues 140 and 512. Our PK-targeting experiments also demonstrate that ICP27 contains a relatively short sequence, mapping to residues 110 to 152, that can function as a nucleolar localization signal (NuLS). This signal includes ICP27's strong NLS as well as 15 contiguous residues which consist entirely of arginine and glycine. This latter sequence is very similar to an RGG box, a putative RNA-binding motif found in a number of cellular proteins which are involved in nuclear RNA processing. To confirm the results of the PK-targeting experiments, we mutated the ICP27 gene by deleting sequences encoding either the strong NLS or the RGG box. Deletion of the strong NLS (residues 109 to 138) resulted in an ICP27 molecule that was only partially defective for nuclear localization, while deletion of the RGG box (residues 139 to 153) resulted in a molecule that was nuclear localized but excluded from nucleoli. Recombinant HSV-1s bearing either of these deletions were unable to replicate efficiently in Vero cells, suggesting that ICP27's strong NLS and RGG box carry out important in vivo functions.     

Regulation of varicella-zoster virus gene expression in human T lymphocytes.

Varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, is the etiologic agent of chicken pox and shingles (zoster) in humans. Using an in vitro transient expression assay, we have evaluated the ability of the putative immediate early VZV genes, ORF4, ORF61, and ORF62 (the analogs of the herpes simplex virus alpha 27, alpha 0, and alpha 4 genes, respectively), to modulate the expression of VZV genes of different putative kinetic classes in a human T lymphocyte cell line. These cells are of the type in which VZV can be readily detected in the viremic phase of human infection. We present evidence to indicate that, in this system, the gene product of ORF62 (IE62) is a major regulatory protein in VZV and is capable of activating VZV genes of all putative kinetic classes. In addition, we demonstrate that the gene product of ORF4 and, unlike the apparent situation in Vero cells, the gene product of ORF61 may play an accessory regulatory role in synergizing the activation of VZV genes induced by the gene product of ORF62 in human T lymphocytes.