Tohru Daikoku

Activation of Ataxia Telangiectasia-mutated DNA Damage Checkpoint Signal Transduction Elicited by Herpes Simplex Virus Infection

Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection.

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.

Mitochondrial distribution and function in herpes simplex virus-infected cells

In this study, mitochondria migrated to a perinuclear region in the cytoplasm in herpes simplex virus (HSV)-infected cells. HSV infection did not promote the expression of cytochrome c oxidase subunit 2 but did promote that of stress-responsive HSP60, both of which are known to be components of mitochondria. The levels of cellular ATP and lactate and mitochondrial membrane potential were maintained for at least 6 h but decreased at the late stage of infection. It was also found that the UL41 and UL46 gene products, both of which are known to be tegument proteins, accumulated in the perinuclear region. The clustering of mitochondria and the accumulation of tegument proteins were completely blocked by the addition of nocodazole and vinblastine. These results suggest that mitochondria respond to the stimulation of HSV infection, migrating with tegument proteins along microtubules to a site around the nucleus, and maintain function until at least the middle stage of infection.

Expression of herpes simplex virus type 2 US3 affects the Cdc42/Rac pathway and attenuates c-Jun N-terminal kinase activation.

Although the US3 gene product of herpes simplex virus (HSV) has been identified as a serine/threonine protein kinase (PK), the functions are poorly understood.

Architecture of Replication Compartments Formed during Epstein-Barr Virus Lytic Replication

ABSTRACT Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.

The UL34 gene product of herpes simplex virus type 2 is a tail-anchored type II membrane protein that is significant for virus envelopment

The UL34 gene of herpes simplex virus type 2 (HSV-2) is highly conserved in the herpesvirus family. The UL34 gene product was identified In lysates of HSV-2-infected cells as protein species with molecular masses of 31 and 32.5 kDa, the latter being a phosphorylated product. Synthesis of these proteins occurred at late times post-infection and was highly dependent on viral DNA synthesis. Immunofluorescence assays revealed that the UL34 protein was localized in the cytoplasm in a continuous net-like structure, closely resembling the staining pattern of the endoplasmic reticulum (ER), in both HSV-2-infected cells and in cells transiently expressing UL34 protein. Deletion mutant analysis showed that this colocalization required the C terminus of the UL34 protein. The UL34 protein associated with virions but not with A, B or C capsids. We treated virions, HSV-2-infected cells and cells expressing the UL34 protein with a protease in order to examine the topology of the UL34 protein. In addition, we constructed UL34 deletion mutant proteins and examined their intracellular localization. Our data strongly support the hypothesis that the UL34 protein is inserted into the viral envelope as a tail-anchored type II membrane protein and is significant for virus envelopment.

Inhibition of S-phase cyclin-dependent kinase activity blocks expression of Epstein-Barr virus immediate-early and early genes, preventing viral lytic replication.

ABSTRACT The induction of lytic replication of the Epstein-Barr virus (EBV) completely arrests cell cycle progression, in spite of elevation of S-phase cyclin-dependent kinase (CDK) activity, thereby causing accumulation of hyperphosphorylated forms of retinoblastoma (Rb) protein (A. Kudoh, M. Fujita, T. Kiyono, K. Kuzushima, Y. Sugaya, S. Izuta, Y. Nishiyama, and T. Tsurumi, J. Virol. 77:851-861, 2003). Thus, the EBV lytic program appears to promote specific cell cycle-associated activity involved in the progression from G1 to S phase. We have proposed that this provides a cellular environment that is advantageous for EBV productive infection. Purvalanol A and roscovitine, inhibitors of S-phase CDKs, blocked the viral lytic replication when cells were treated at the early stage of lytic infection, while well-characterized inhibitors of enzymes, such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C, known to be involved in BZLF1 gene expression did not. Inhibition of CDK activity resulted in the accumulation of the hypophosphorylated form of Rb protein and inhibition of expression of EBV immediate-early and early proteins. Cycloheximide block-and-release experiments clearly demonstrated that even in the presence of enough amounts of the BZLF1 protein, purvalanol A blocked expression of lytic viral proteins at transcription level. Furthermore, reporter gene experiments confirmed that BZLF1-induced activation of early EBV promoters was impaired in the presence of the CDK inhibitor. We conclude here that the EBV lytic program promotes specific cell cycle-associated activity involved in the progression from G1 to S phase because the S-phase-like cellular environment is essential for the expression of immediate-early and early genes supplying the viral replication proteins and hence for lytic viral replication.

Identification and Characterization of the UL56 Gene Product of Herpes Simplex Virus Type 2

ABSTRACT The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.

Two Sp1/Sp3 Binding Sites in the Major Immediate-Early Proximal Enhancer of Human Cytomegalovirus Have a Significant Role in Viral Replication

ABSTRACT We previously demonstrated that the major immediate early (MIE) proximal enhancer containing one GC box and the TATA box containing promoter are minimal elements required for transcription and viral replication in human fibroblast cells (H. Isomura, T. Tsurumi, M. F. Stinski, J. Virol. 78:12788-12799, 2004). After infection, the level of Sp1 increased while Sp3 remained constant. Here we report that either Sp1 or Sp3 transcription factors bind to the GC boxes located at approximately positions −55 and −75 relative to the transcription start site (+1). Both the Sp1 and Sp3 binding sites have a positive and synergistic effect on the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. There was little to no change in MIE transcription or viral replication for recombinant viruses with one or the other Sp1 or Sp3 binding site mutated. In contrast, mutation of both the Sp1 and Sp3 binding sites caused inefficient MIE transcription and viral replication. These data indicate that the Sp1 and Sp3 binding sites have a significant role in HCMV replication in human fibroblast cells.

Phosphorylation of MCM4 at Sites Inactivating DNA Helicase Activity of the MCM4-MCM6-MCM7 Complex during Epstein-Barr Virus Productive Replication

ABSTRACT Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.