Yukihiro Nishiyama

Conserved Protein Kinases Encoded by Herpesviruses and Cellular Protein Kinase cdc2 Target the Same Phosphorylation Site in Eukaryotic Elongation Factor 1δ

ABSTRACT Earlier studies have shown that translation elongation factor 1δ (EF-1δ) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1δ associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1δ by alanine precluded the posttranslational processing of EF-1δ, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1δ containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1δ completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1δ. (v) As observed with EF-1δ, the casein kinase II β subunit (CKIIβ) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIβ by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.

Measuring Epstein-Barr virus (EBV) load: the significance and application for each EBV-associated disease.

Because Epstein–Barr virus (EBV) is ubiquitous and persists latently in lymphocytes, simply detecting EBV is insufficient to diagnose EBV‐associated diseases. Therefore, measuring the EBV load is necessary to diagnose EBV‐associated diseases and to explore EBV pathogenesis. Due to the diverse biology of EBV, the significance of measuring EBV DNA and the optimal type of specimen differ among EBV‐associated diseases. Recent advances in molecular technology have enabled the EBV genome to be quantitated rapidly and accurately. Real‐time polymerase chain reaction (PCR) is a rapid and reliable method to quantify DNA and is widely used not only as a diagnostic tool, but also as a management tool for EBV‐associated diseases. However, each laboratory currently measures EBV load with its own “homebrew” system, and there is no consensus on sample type, sample preparation protocol, or assay units. The EBV real‐time PCR assay system must be standardised for large‐scale studies and international comparisons. Copyright

Construction of a US3 lacZ insertion mutant of herpes simplex virus type 2 and characterization of its phenotype in vitro and in vivo.

We have constructed and characterized a mutant of herpes simplex virus type 2 (HSV-2) which was inserted a modified lacZ gene, placed under the control of HSV-1 beta 8 promotor, into the US3 protein kinase gene. The mutant, L1BR1, could not induce the virus-encoded protein kinase activity, but could replicate in Vero cells as efficiently as the parental virus. When the biological properties of L1BR1 were examined in mice by using four routes (footpad, intraperitoneal, corneal, and intracerebral) of infection, the mutant displayed the route-dependent reduction of virulence; after inoculation by footpad and intraperitoneal routes, the mutant was more than 10,000-fold less virulent than the parental virus, but it exhibited only about a 10-fold decrease in virulence following the corneal and intracerebral infection. In the intraperitoneal inoculation into adult mice, the replication of L1BR1 in the liver and spleen was severely restricted, but in newborn mice the mutant could grow as well as the parental virus in these organs. The adoptive transfer of peritoneal macrophages from adult mice resulted in a marked inhibition in the replication of L1BR1 in the liver and spleen of newborn mice, while the transfer exhibited little or no effect on the production of the wild-type virus in these organs. We also found that the mutant, unlike the parental virus, could not replicate in precultured peritoneal macrophages from adult mice. Taking these observations together, it seems likely that L1BR1 lost the ability to overcome the mononuclear-phagocytic defense system and thereby lost its pathogenicity by intraperitoneal and footpad routes. Furthermore, the mutant was shown to be rescued by a 4.8-kb HindIII/Xbal fragment containing the entire US3 open reading frame. However, we could not rule out the possibility that some of the phenotypes of L1BR1 are due to mutations in the US3-neighboring genes, US2 and US4.

Simultaneous Quantification of Epstein-Barr Virus, Cytomegalovirus, and Human Herpesvirus 6 DNA in Samples from Transplant Recipients by Multiplex Real-Time PCR Assay

ABSTRACT We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.

The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1beta, IL-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed 20- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

Reactivation of Lytic Replication from B Cells Latently Infected with Epstein-Barr Virus Occurs with High S-Phase Cyclin-Dependent Kinase Activity while Inhibiting Cellular DNA Replication

ABSTRACT Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G0/G1 by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21WAF-1/CIP-1 and p27KIP-1, followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21WAF-1/CIP-1, and p27KIP-1 were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G1/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G1 to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

Herpes Simplex Virus 1-Encoded Protein Kinase UL13 Phosphorylates Viral Us3 Protein Kinase and Regulates Nuclear Localization of Viral Envelopment Factors UL34 and UL31

ABSTRACT 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 (ΔUL13). 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 ΔUL13-infected cells. (ii) A recombinant protein containing a domain of Us3 was phosphorylated by UL13 in vitro. (iii) The phenotype of ΔUL13 resembles that of a recombinant virus lacking the Us3 gene (ΔUs3) 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 ΔUL13- and ΔUs3-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.

Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

ABSTRACT We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

Detection and direct typing of herpes simplex virus by polymerase chain reaction

A method for the detection and direct typing of herpes simplex virus (HSV) by the polymerase chain reaction (PCR) technique has been developed. One common upstream primer and two type-specific downstream primers were prepared to amplify DNA from the HSV type 1 and type 2 DNA polymerase gene. Using these three primers simultaneously in the PCR reaction mixtures, both types of HSV DNA were amplified to produce products of different sizes. By direct gel analysis, the products of standard HSV type 1 and type 2 strains had the predictive sizes of 469 and 391 base pairs, respectively, and the difference in molecular mass enabled us to type the HSV strain. A total of 24 strains (type 1; 16 and type 2; 8 strains) were examined by PCR, and the results were consistent with those determined by immunofluorescence using type-specific monoclonal antibodies. No specific amplification was observed using other herpes virus or human genomic DNAs. The PCR method was then applied to clinical specimens. Of 15 samples obtained from oral lesions of children with herpetic gingivostomatitis, all (100%) were HSV positive by PCR, compared with 13 (86.7%) using standard cell culture methods. Three specimens from vulvar lesions of women with genital herpes were positive using both PCR and cell cultures. There was complete agreement in the typing of HSV strains using the PCR method or virus isolation. On the basis of these results, it is suggested that DNA amplification and typing by PCR is particularly useful for material from which virus isolation might be difficult.

Correlation between HHV-6 infection and skin rash after allogeneic bone marrow transplantation

We investigated whether a causal relationship exists between human herpesvirus 6 (HHV-6) and skin rash resembling acute graft-versus-host disease (GVHD) following bone marrow transplantation (BMT). Isolation of HHV-6 was used to monitor active HHV-6 infection in this study. We analyzed 25 episodes of skin rash in 22 recipients. All recipients were seropositive for HHV-6 before BMT. The onset of skin rash started prior to 30 days post transplantation (group A) in 15 of 25 cases, but after that (group B) in the remaining 10 cases. Twenty-five skin tissue samples were obtained from 22 recipients. The HHV-6 genome was detected in four of 15 skin samples from group A, but not detected in those from group B. HHV-6 was isolated from 11 of 22 recipients around 2 to 3 weeks after BMT (range 14 to 28 days after BMT). HHV-6 was isolated at a time between 10 days before and after the onset of skin rash (skin rash-related viremia) in nine cases in group A. Meanwhile, no skin rash-related viremia was observed in group B. Of the four recipients with positive detection of HHV-6 genome in their skin tissue (group A), two had HHV-6 viremia at the same time. The association between the timing of HHV-6 infection and the onset of skin rash was analyzed statistically. HHV-6 viremia (skin rash-related viremia) was found in nine of 15 (60%) cases in group A, compared with none of 10 (0%) cases in group B. This difference was statistically significant (P = 0.008). Moreover, HHV-6 infection (skin rash-related viremia and/or positive detection of HHV-6 DNA in skin tissue) was demonstrated in 11 of 15 (73.3%) cases in group A, compared with none of 10 (0%) cases in group B (P = 0.001). Thus, this study suggests that HHV-6 may be involved in the development of skin rash in the first month after allogeneic BMT. Bone Marrow Transplantation (2001) 28, 77–81.