Sanae Nakayama

Degradation of Phosphorylated p53 by Viral Protein-ECS E3 Ligase Complex

p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.

Mutational Inactivation of Herpes Simplex Virus 1 MicroRNAs Identifies Viral mRNA Targets and Reveals Phenotypic Effects in Culture

ABSTRACT Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype.

Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

ABSTRACT Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

Identification of phosphorylation sites on transcription factor Sp1 in response to DNA damage and its accumulation at damaged sites

DNA damage induces hyper-phosphorylation of the Sp1 transcriptional factor. We have demonstrated that ionizing radiation-associated DNA double-strand breaks (DSBs) induce phosphorylation of at least Ser-56 and Ser-101 residues on Sp1 in an ATM-dependent manner. UV irradiation- or hydroxyurea (HU)-induced replicative stress results in phosphorylation of only the Ser-101 residue. Furthermore, silencing of the ATM- and Rad3-related protein (ATR) in ATM-deficient cells treated with HU abrogated the Ser-101 phosphorylation. Thus, phosphorylation of Ser-101 on Sp1 appears to be a general response to DNA damage dependent on both ATM and ATR. Although silencing of Sp1 expression by siRNA targeting resulted in an increase in sensitivity to ionizing radiation (IR), the Ser-101 phosphorylation did not affect transcriptional activity from the Sp1 responsive promoter. Confocal laser microscopy analysis revealed co-localization of phosphorylated Sp1 at Ser-101 with phosphorylated ATM at Ser-1981, the affected sites representing DSBs. These observations suggest that phosphorylated Sp1 might play a role in DNA repair at damage sites rather than functioning in transcriptional regulation.

Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase

The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

Expression of Epstein–Barr virus BZLF1 immediate-early protein induces p53 degradation independent of MDM2, leading to repression of p53-mediated transcription

The Epstein-Barr virus (EBV) lytic program elicits ATM-dependent DNA damage response, resulting in phosphorylation of p53 at N-terminus, which prevents interaction with MDM2. Nevertheless, p53-downstream signaling is blocked. We found here that during the lytic infection p53 was actively degraded in a proteasome-dependent manner even with a reduced level of MDM2. BZLF1 protein enhanced the ubiquitination of p53 in SaOS-2 cells. The degradation of p53 was observed even in the presence of Nutlin-3, an inhibitor of p53-MDM2 interaction, and also in mouse embryo fibroblasts lacking mdm2 gene, indicating that the BZLF1 protein-induced degradation of p53 was independent of MDM2. Furthermore, Nutlin-3 increased the level of p53 in the latent phase of EBV infection but not in the lytic phase. Although p53 level is regulated by MDM2 in the latent phase, it might be mediated by the BZLF1 protein-associated E3 ubiquitin ligase in the lytic phase for efficient viral propagation.

Noncanonical TATA Sequence in the UL44 Late Promoter of Human Cytomegalovirus Is Required for the Accumulation of Late Viral Transcripts

ABSTRACT During productive infection, human cytomegalovirus (HCMV) UL44 transcription initiates at three distinct start sites that are differentially regulated. Two of the start sites, the distal and the proximal, are active at early times, whereas the middle start site is active only at late times after infection. The UL44 early viral gene product is essential for viral DNA synthesis. The UL44 gene product from the late viral promoter affects primarily viral gene expression at late times after infection rather than viral DNA synthesis (H. Isomura, M. F. Stinski, A. Kudoh, S. Nakayama, S. Iwahori, Y. Sato, and T. Tsurumi, J. Virol. 81:6197, 2007). The UL44 early viral promoters have a canonical TATA sequence, “TATAA.” In contrast, the UL44 late viral promoter has a noncanonical TATA sequence. Using recombinant viruses, we found that the noncanonical TATA sequence is required for the accumulation of late viral transcripts. The GC boxes that surround the middle TATA element did not affect the kinetics or the start site of UL44 late transcription. Replacement of the distal TATA element with a noncanonical TATA sequence did not affect the kinetics of transcription or the transcription start site, but it did induce an alternative transcript at late times after infection. The data indicate that a noncanonical TATA box is used at late times after HCMV infection.

Enhanced Phosphorylation of Transcription Factor Sp1 in Response to Herpes Simplex Virus Type 1 Infection Is Dependent on the Ataxia Telangiectasia-Mutated Protein

ABSTRACT The ataxia telangiectasia-mutated (ATM) protein, a member of the related phosphatidylinositol 3-like kinase family encoded by a gene responsible for the human genetic disorder ataxia telangiectasia, regulates cellular responses to DNA damage and viral infection. It has been previously reported that herpes simplex virus type 1 (HSV-1) infection induces activation of protein kinase activity of ATM and hyperphosphorylation of transcription factor, Sp1. We show that ATM is intimately involved in Sp1 hyperphosphorylation during HSV-1 infection rather than individual HSV-1-encoded protein kinases. In ATM-deficient cells or cells silenced for ATM expression by short hairpin RNA targeting, hyperphosphorylation of Sp1 was prevented even as HSV-1 infection progressed. Mutational analysis of putative ATM phosphorylation sites on Sp1 and immunoblot analysis with phosphopeptide-specific Sp1 antibodies clarified that at least Ser-56 and Ser-101 residues on Sp1 became phosphorylated upon HSV-1 infection. Serine-to-alanine mutations at both sites on Sp1 considerably abolished hyperphosphorylation of Sp1 upon infection. Although ATM phosphorylated Ser-101 but not Ser-56 on Sp1 in vitro, phosphorylation of Sp1 at both sites was not detected at all upon infection in ATM-deficient cells, suggesting that cellular kinase(s) activated by ATM could be involved in phosphorylation at Ser-56. Upon viral infection, Sp1-dependent transcription in ATM expression-silenced cells was almost the same as that in ATM-intact cells, suggesting that ATM-dependent phosphorylation of Sp1 might hardly affect its transcriptional activity during the HSV-1 infection. ATM-dependent Sp1 phosphorylation appears to be a global response to various DNA damage stress including viral DNA replication.

Transcriptional repression by sumoylation of Epstein-Barr virus BZLF1 protein correlates with association of histone deacetylase

The transition from latent to lytic phases of the Epstein-Barr virus life cycle is triggered by expression of a viral transactivator, BZLF1, that then induces expression of the viral immediate-early and early genes. The BZLF1 protein is post-translationally modified by a small ubiquitin-related modifier-1 (SUMO-1). Here we found that BZLF1 is conjugated at lysine 12 not only by SUMO-1 but also by SUMO-2 and 3. The K12R mutant of BZLF1, which no longer becomes sumoylated, exhibits stronger transactivation than the wild-type BZLF1 in a reporter assay system as well as in the context of virus genome with nucleosomal structures. Furthermore, exogenous supply of a SUMO-specific protease, SENP, caused de-sumoylation of BZLF1 and enhanced BZLF1-mediated transactivation. Immunoprecipitation experiments proved that histone deacetylase 3 preferentially associated with the sumoylated form of BZLF1. Levels of the sumoylated BZLF1 increased as lytic replication progressed. Based on these observations, we conclude that sumoylation of BZLF1 regulates its transcriptional activity through histone modification during Epstein-Barr virus productive replication.

The Late Promoter of the Human Cytomegalovirus Viral DNA Polymerase Processivity Factor Has an Impact on Delayed Early and Late Viral Gene Products but Not on Viral DNA Synthesis

ABSTRACT Transcription of the DNA polymerase processivity factor gene (UL44) of human cytomegalovirus initiates at three distinct start sites, which are differentially regulated during productive infection. Two of these start sites, the distal and proximal sites, are active at early times, and the middle start site is active at only late times after infection (F. Leach and E. S. Mocarski, J. Virol. 63:1783-1791, 1989). Compared to the wild type, UL44 gene expression was lower for recombinant viruses with the distal or the middle TATA element mutated. The transcripts initiating from the distal or middle start site facilitated late viral gene expression. The level of viral DNA synthesis was affected by mutation of the distal TATA element. In contrast, mutation of the middle TATA element did not affect the level of viral DNA synthesis, but it did affect significantly the level of late viral gene expression. Recombinant viruses with the distal or middle TATA element mutated grew more slowly than the wild type at both low and high multiplicities of infection. Reduced expression of the UL44 gene from the late middle viral promoter correlated with decreased late viral protein expression and decreased viral growth.