村田 貴之

The Epstein-Barr virus BMRF1 DNA polymerase processivity factor, which is essential for viral genome replication, exists mainly as a C-shaped head-to-head homodimer but partly forms a ring-shaped tetramer through tail-to-tail association. Based on its molecular structure, several BMRF1 mutant viruses were constructed to examine their influence on viral replication. The R256E virus, which has a severely impaired capacity for DNA binding and polymerase processivity, failed to form replication compartments, resulting in interference of viral replication, while the C95E mutation, which impairs head-to-head contact in vitro, unexpectedly hardly affected the viral replication. Also, surprisingly, replication of the C206E virus, which is expected to have impairment of tail-to-tail contact, was severely restricted, although the mutant protein possesses the same in vitro biochemical activities as the wild type. Since the tail-to-tail contact surface is smaller than that of the head-to-head contact area, its contribution to ring formation might be essential for viral replication.

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.

Background: Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 59 mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein. Methodology/Principal Findings: To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth. Conclusions/Significance: While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV-infected cells.

Expression of Epstein-Barr Virus BZLF1, a key protein initiating the switch from latent to lytic infection, is known to cause cell growth arrest by accumulating p53 and p21(WAF1/CIP1) in epithelial cells, but its molecular mechanism remains elusive. We found here that the BZLF1 protein stimulates p53 binding to its recognition sequence. The BZLF1 accelerated the rate of p53-DNA complex formation through the interaction with p53 protein and also enhanced p53-specific transcription in vitro. Furthermore, p53 protein was found to bind to its target promoter regions specifically in the early stages of lytic replication. Overexpression of p53 at the early stages of lytic replication enhanced viral genome replication, supporting the idea that p53 plays an important role in the initiation steps of EBV replication. Taking the independent role of BZLF1 on p53 degradation into consideration, we propose that the BZLF1 protein regulates p53 and its target gene products in two distinctive manners; transient induction of p53 at the early stages for the initiation of viral productive replication and p53 degradation at the later stages for S-phase like environment preferable for viral replication.

The Epstein-Barr virus (EBV) BMRF1 protein is an essential replication protein acting at viral replication forks as a viral DNA polymerase processivity factor, where as the BALF2 protein is a single stranded DNA-binding protein that also acts at replication forks and is most abundantly expressed during viral productive replication. Here we document that the BMRF1 protein evidently enhances viral BZLF1 transcription factor-mediated transactivation of the BALF2 gene promoter. Mutagenesis and electrophoretic mobility shift assays demonstrated the BALF2 promoter to harbor two BZLF1 protein-binding sites (BZLF1-responsive elements). Direct binding of the BZLF1 protein to BZLF1-responsive elements and physical interaction between BZLF1 and BMRF1 proteins are prerequisite for the BMRF1 protein up-regulation of the BALF2 gene promoter. A monomeric mutant, C95E, which is defective in homodimerization, could still interact and enhance BZLF1-mediated transactivation. Furthermore although EBV protein kinase phosphorylates BMRF1 protein extensively, it turned out that phosphorylation of the protein by the kinase is inhibitory to the enhancement of the BZLF1-mediated transactivation of BALF2 promoter. Exogenous expression of BMRF1 protein augmented BALF2 expression in HEK293 cells harboring the EBV genome but lacking BMRF1 and BALF5 genes, demonstrating functions as a transcriptional regulator in the context of viral infection. Overall the BMRF1 protein is a multifunctional protein that cannot only act as a DNA polymerase processivity factor but also enhances BALF2 promoter transcription as a coactivator for the BZLF1 protein, regulating the expression level of viral single-stranded DNA-binding protein.

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.

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.

Epstein-Barr virus (EBV) productive replication occurs in an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. The EBV protein kinase (PK), encoded by the viral BGLF4 gene, is a Ser/Thr protein kinase, which phosphorylates both viral and cellular proteins, modifying the cellular environment for efficient viral productive replication. We here provide evidence that the EBV PK phosphorylates the CDK inhibitor p27(Kip1), resulting in ubiquitination and degradation in a proteasome-dependent manner during EBV productive replication. Experiments with BGLF4 knockdown by small interfering RNA and BGLF4 knock-out viruses clarified that EBV PK is involved in p27(Kip1) degradation upon lytic replication. Transfection of the BGLF4 expression vector revealed that EBV PK alone could phosphorylate the Thr-187 residue of p27(Kip1) and that the ubiquitination and degradation of p27(Kip1) occurred in an SCFSkp2 ubiquitin ligase-dependent manner. In vitro, EBV PK proved capable of phosphorylating p27(Kip1) at Thr-187. Unlike cyclin E-CDK2 activity, the EBV PK activity was not inhibited by p27(Kip1). Overall, EBV PK enhances p27(Kip1) degradation effectively upon EBV productive replication, contributing to establishment of an S-phase-like cellular environment with high CDK activity.

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. (c) 2009 Elsevier Inc. All rights reserved.

The Epstein-Barr virus (EBV) lyric 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 lyric 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. (C) 2009 Elsevier Inc. All rights reserved.

Reactivation of the Epstein-Barr virus from latency is dependent on expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical inducers such as 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We found here that Transducer of Regulated cAMP-response Element-binding Protein (CREB) (TORC) 2 enhances Zp activity 10-fold and more than 100-fold with co-expression of the BZLF1 protein. Mutational analysis of Zp revealed that the activation by TORC is dependent on ZII and ZIII cis elements, binding sites for CREB family transcriptional factors and the BZLF1 protein, respectively. Immunoprecipitation, chromatin immunoprecipitation, and reporter assay using Gal4-luc and Gal4BD-BZLF1 fusion protein indicate that TORC2 interacts with BZLF1, and that the complex is efficiently recruited onto Zp. These observations clearly indicate that TORC2 activates the promoter through interaction with the BZLF1 protein as well as CREB family transcriptional factors. Induction of the lytic replication resulted in the translocation of TORC2 from cytoplasm to viral replication compartments in nuclei, and furthermore, activation of Zp by TORC2 was augmented by calcium-regulated phosphatase, calcineurin. Silencing of endogenous TORC2 gene expression by RNA interference decreased the levels of the BZLF1 protein in response to 12-O-tetradecanoylphorbol-13-acetate/ionophore. Based on these results, we conclude that Epstein-Barr virus exploits the calcineurin-TORC signaling pathway through interactions between TORC and the BZLF1 protein in reactivation from latency.

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. (C) 2008 Elsevier Inc. All rights reserved.

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.

The promoter of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV), also referred to as the CMV promoter, possesses a cis-acting element positioned downstream of the TATA box between positions -14 and -1 relative to the transcription start site (+1). We determined the role of the cis-acting element in viral replication by comparing recombinant viruses with the cis-acting element replaced with other sequences. Recombinant virus with the simian CMV counterpart replicated efficiently in human foreskin fibroblasts, as well as wild-type virus. In contrast, replacement with the murine CMV counterpart caused inefficient MIE gene transcription, RNA splicing, MIE and early viral gene expression, and viral DNA replication. To determine which nucleotides in the cis-acting element are required for efficient MIE gene transcription and splicing, we constructed mutations within the cis-acting element in the context of a recombinant virus. While mutations in the cis-acting element have only a minor effect on in vitro transcription, the effects on viral replication are major. The nucleotides at -10 and -9 in the cis-acting element relative to the transcription start site (+1) affect efficient MIE gene transcription and splicing at early times after infection. The cis-acting element also acts as a cis-repression sequence when the viral IE86 protein accumulates in the infected cell. We demonstrate that the cis-acting element has an essential role in viral replication.

A series of nucleoside analogues whose 5'-hydroxyl groups are masked by various protective groups were synthesized and evaluated to develop novel anti- hepatitis C virus (HCV) agents. Among the several analogues that showed anti-HCV potency, a 5'-O-benzoyl-2'-deoxyribonucleoside analogue exhibited high anti-HCV activity with an EC(50) of 6.1 microM.

On the basis of our previous study on antiviral agents against the severe acute respiratory syndrome (SARS) coronavirus, a series of nucleoside analogues whose 5′-hydroxyl groups are masked by various protective groups such as carboxylate, sulfonate, and ether were synthesized and evaluated to develop novel anti-hepatitis C virus (HCV) agents. Among these, several 5′-O-masked analogues of 6-chloropurine-2′-deoxyriboside (e.g., 5′-O-benzoyl, 5′-O-p-methoxybenzoyl, and 5′-O-benzyl analogues) were found to exhibit effective anti-HCV activity. In particular, the 5′-O-benzoyl analogue exhibited the highest potency with an EC50of 6.1 μM in a cell-based HCV replicon assay. Since the 5′-O-unmasked analogue (i.e., 6-chloropurine-2′-deoxyriboside) was not sufficiently potent (EC50= 47.2 μM), masking of the 5′-hydroxyl group seems to be an effective method for the development of anti-HCV agents. Presently, we hypothesize two roles for the 5′-O-masked analogues: One is the role as an anti-HCV agent by itself, and the other is as a prodrug of its 5′-O-demasked (deprotected) derivative. © 2007 Elsevier Ltd. All rights reserved.

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell-free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

Translation initiation factor 4E ( eIF4E) is a cytoplasmic cap-binding protein that is required for cap-dependent translation initiation. Here, we have shown that eIF4E is ubiquitinated primarily at Lys-159 and incubation of cells with a proteasome inhibitor leads to increased eIF4E levels, suggesting the proteasome-dependent proteolysis of ubiquitinated eIF4E. Ubiquitinated eIF4E retained its cap binding ability, whereas eIF4E phosphorylation and eIF4G binding were reduced by ubiquitination. The W73A mutant of eIF4E exhibited enhanced ubiquitination/degradation, and 4E-BP overexpression protected eIF4E from ubiquitination/degradation. Because heat shock or the expression of the carboxyl terminus of heat shock cognate protein 70-interacting protein ( Chip) dramatically increased eIF4E ubiquitination, Chip may be at least one ubiquitin E3 ligase responsible for eIF4E ubiquitination.

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. We recently discovered that the immunosuppressant cyclosporin A (CsA) and its analogue lacking immunosuppressive function, NIM811, strongly suppress the replication of HCV in cell culture. Inhibition of a cellular replication cofactor, cyclophilin (CyP) B, is critical for its anti-HCV effects. Here, we explored the potential use of CyP inhibitors for HCV treatment by analyzing the HCV replicon system. Treatment with CsA and NIM811 for 7 days reduced HCV RNA levels by 2-3 logs, and treatment for 3 weeks reduced HCV RNA to undetectable levels. NIM811 exerted higher anti-HCV activity than CsA at lower concentrations. Both CyP inhibitors rapidly reduced HCV RNA levels even further in combination with IFN alpha without modifying the IFN alpha signal transduction pathway. In conclusion, CyP inhibitors may provide a novel strategy for anti-HCV treatment, (c) 2006 Elsevier Inc. All rights reserved.

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CNI replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

Increasing evidence has shown that the stem-loop (SL) structures in the NS5B coding region of hepatitis C virus (HCV) function as cis-replicating elements that are indispensable for viral replication. We have investigated whether small RNA molecules analogous to the SL structures could inhibit HCV replication. Reporter assays showed that both in vitro transcribed and pol III-directed transcripts corresponding to 5BSL3.1 and 5BSL3.2 efficiently inhibited HCV replicon-encoded luciferase expression. Mutagenesis studies revealed that mutation in 5BSL3.2 which debilitated its binding to NS5B also abolished the ability of 5BSL3.2 RNA to inhibit HCV replication, suggesting that SL RNA inhibits HCV by sequestering the replication complex. Further, adenoviral-mediated expression of the SL RNAs potently blocked the replication of HCV replicon in Huh-7 cells. Importantly, SL RNAs derived from HCV 2a, an evolutionarily distant genotype, were also shown to suppress the replication of HCV 1b replicon in spite of the genetic heterogeneity between the SL elements of the two viruses, implying the potential of SL RNA-based approach to inhibit a wide range of HCV isolates. These results suggest that SL RNA decoys may prove to be useful in the treatment of hepatitis C, which may be advantageous over other sequence-specific gene therapy modalities (such as antisense RNA and siRNA) in preventing the escape of genetic variants. (c) 2005 Elsevier Inc. All rights reserved.

Translation initiation of hepatitis C virus (HCV) occurs in an internal ribosome entry site (IPES)-dependent manner. We found that HCV IRES-dependent protein synthesis is enhanced by PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) signaling pathway, while cellular cap-dependent translation was relatively unaffected by the compound. Treatment of cells with PD98059 allowed for robust HCV replication following cellular incubation with HCV-positive serum. Though the molecular mechanism underlying IRES enhancement remains elusive, PD98059 is a potent accelerator of HCV RNA replication. (c) 2005 Elsevier Inc. All rights reserved.

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPlase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Hepatitis C virus (HCV) is one of the major causative agents of liver diseases, such as liver inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Using an efficient HCV subgenomic replicon system, we demonstrate that transforming growth factor-beta (TGF-β) suppresses viral RNA replication and protein expression from the HCV replicon. We further show that the anti-viral effect of this cytokine is associated with cellular growth arrest in a manner dependent on Smad signaling, not mitogen-activated protein kinase (MAPK) signaling. These results suggest a novel insight into the mechanisms of liver diseases caused by HCV. © 2004 Elsevier Inc. All rights reserved.

Glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) was excreted from infected cells into the medium. Peptide mapping analysis and lectin binding assays suggested that the gD in the medium is secreted after full glycosylation and cleavage at its C terminus. Release of HSV-2 gD was inhibited by addition of either tunicamycin or brefeldin A, suggesting that the gD in the medium was secreted through the endoplasmic reticulum and Golgi apparatus.

Previously, we established HEp2 cell lines which express the US3 protein kinase of herpes simplex virus type 2 upon induction with IPTG. Using these cells, we examined whether expression of US3 is sufficient to protect cells from apoptotic cell death induced by sorbitol. Cells expressing US3 showed significantly reduced nuclear fragmentation in the decree that DNA fragmentation and caspase-3 activation were suppressed. It is known that stressors such as osmotic shock and UV irradiation induce the activation of the JNK (c-Jun N-terminal kinase), which can lead to apoptotic cell death. Expression of US3 resulted in the suppression of sorbitol-induced phosphorylation of JNK and MKK4/SEK1, suggesting that the suppression of apoptotic cell death was due to the attenuation of JNK activity. (C) 2002 Editions scientifiqucs et medicales Elsevier SAS. All rights reserved.

Herpes simplex virus expresses proteins modulating the process of apoptosis, and the viral US3 gene encodes a unique serine/threonine kinase that protects infected cells from apoptosis. Here, we discuss the biological roles of the US3 protein kinase and the mechanism by which this kinase blocks apoptosis.

We previously reported the establishment of an HEp2 cell line which expresses the US3 protein kinase (PK) of herpes simplex virus type 2 (HSV-2) upon induction with IPTG. Here we report that expression, phosphorylation and ubiquitination of cytokeratin 17 (CK17) are enhanced in US3-expressing HEp2 cells. In vitro kinase and co-immunoprecipitation assays provided evidence that US3 PK directly phosphorylates CK17. Expression of US3 PK caused a significant decrease in filamentous staining of CK17, suggesting that phosphorylation of CK17 by US3 PK causes a disruption of intermediate filaments. Our observations suggest a role for US3 in the regulation of CKs and intermediate filaments in cells. Moreover, we found that infection of a keratinocyte-derived cell line, A431, with a US3-deficient virus, results in cytopathic effects that are morphologically distinct from those induced by wild-type and revertant viruses, suggesting that US3 PK may be important for interaction between HSV-2 and peripheral epithelial cells.

The US11 gene product of herpes simplex virus is an abundant virion structural protein with RNA-binding regulatory activity. Its carboxyl-terminal half consists of tandem tripeptide repeats of the sequence RXP. We demonstrate that the US11 protein has intercellular trafficking activity and accumulates in the nucleolus when singly expressed in cultured cells, and that the RXP repeats are responsible for this activity. These same properties were also observed in cells expressing a fusion protein linking US11 to the green fluorescent protein. Furthermore, exogenous US11 protein was internalized by cells at 4 degreesC, which suggests that US11 protein uptake occurs primarily through an energy-independent pathway. (C) 2001 Academic Press.

Herpes simplex virus type 2 UL34 protein is expressed late in infection and is required for envelopment of nucleocapsids at the nuclear membrane and possibly at the endoplasmic reticulum (ER). It is a type II membrane protein with a C-terminal anchor that localizes mainly to the nuclear membrane in infected cells. However, in single transient expression, UL34 protein localizes predominantly to the ER. Relocation of UL34 protein from the ER to the internal nuclear membrane and the nucleus was observed in the presence of UL31 protein, a phosphoprotein known to interact physically with UL34. It is suggested here that interaction with UL31 protein is important for the nuclear targetting of UL34 protein and also that the trans-membrane region of UL34 protein is responsible for its localization at the internal nuclear membrane. The results also suggest possible sites for the interaction.

Leptomycin B (LMB) is a specific inhibitor of Crm1-dependent nuclear export of proteins. The replication of herpes simplex virus (HSV) is normally highly sensitive to LMB; a resistant HSV variant, however, was isolated by serial passages of the virus. Analysis of marker transfer and viral DNA sequences revealed that a single amino acid substitution within the ICP27 gene is responsible for conferring this resistance.

The UL24 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 281 amino acid protein with a molecular mass of 30.5 kDa. In this study, the HSV-2 UL24 gene product has been identified by using a rabbit polyclonal antiserum produced against a recombinant protein containing the full-length UL24 gene product of HSV-2 fused to glutathione-S-transferase. The antiserum reacted specifically with a 32 kDa protein in HSV-2 186-infected Vero cells and with 31 and 32 kDa proteins in UL24-expressing Cos-7 cells. Accumulation of UL24 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. UL24 protein was found to be associated with purified HSV-2 virions and C capsids. Indirect immunofluorescence analysis demonstrated that the UL24-specific fluorescence was detected in perinuclear regions of the cytoplasm and/or in the nucleus as small discrete granules from 9 h post infection (hpi). Furthermore, the UL24 protein expressed singly was detected predominantly in the nucleus and slightly in the cytoplasm at 24 h after transfection, with branch-like cytoplasmic protruding structures. Strong nucleolus staining was visible in partial cells.

Background: Herpes simplex virus (HSV) possesses a number of accessory genes which are dispensable for replication in cell culture. A previous study showed that the UL21 gene product of HSV type 1 is a virion component that is not necessary for viral replication. The function of the gene product remains unknown. Results: We found that the HSV-1 UL21 gene product, a capsid-associated tegument protein with an apparent molecular mass of 62 kDa, promotes the outgrowth of long cellular processes when it is over-expressed in non-neural cells. The UL21 protein co-localizes and physically associates with microtubules in the long processes. Analysis using mutant proteins implicates a proline-rich region in promotion of the processes. Conclusions: The results suggest that the UL21 protein, like tau and other MAPs, promotes the process by directly or indirectly interacting with microtubules and facilitates the intracellular transport of the virus.

Among members of the subfamily alpha herpesviruses, herpes simplex virus (HSV) is one of the most well-studied and the most important human pathogen. HSV is reported to possess the large, double-stranded DNA genome which contains at least 80 genes. Recent studies have revealed the functions and roles of many gene products in HSV-infected cells. This article briefly reviews the results of these studies relating to HSV replication mechanism in cells and latent infection in central nerve system (CNS).

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.

Background: 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. Results: We found that US3 PK of HSV-2 induced disruption of actin filaments, cell rounding and accumulation of binucleate cells in HEp2 cells. Cell rounding was abrogated by expression of either kinase-dead forms of US3 PK or a mutant protein lacking the acidic cluster in the kinase regulatory domain. Co-expression of dominant active forms of Cdc42/Rac inhibited cell rounding, suggesting that a signal transduction pathway involving Cdc42/Rac may play a role in the morphological changes induced by the kinase. Cdc42 and Rac, members of the Rho family of small GTPases, function as molecular switches changing actin cytoskeletal organization, influencing transcription and controlling apoptotic cell death. By computed homology search, we noticed significant similarities between US3 PK and p21-activated kinase (PAK), which is activated by the Cdc42 or Rac. We also found that the expression of US3 suppressed the activation of c-Jun N-terminal kinase (JNK), a kinase that is downstream of PAK. Conclusions: These observations suggest that the US3 PK affects the Cdc42/Rac pathway and can act as an upstream suppressor of JNK in the stress-activated signalling pathway.

Bovine herpesvirus-1 (BHV-1) can replicate well in bovine-derived cell lines such as Madin Darby bovine kidney (MDBK) but grows poorly in hamster lung (HmLu-1). Virus replication, DNA synthesis, and immediate-early gene expression are severely restricted in HmLu-1. We compared adsorption and penetration of BHV-1 in permissive MDBK and semi-permissive HmLu-1 cells. At a low multiplicity of infection, BHV-1 attached to permissive MDBK cells twice as much as to HmLu-1. The presence of heparin inhibited the attachment of BHV-1 to MDBK cells by about 60%, but over 90% of the attachment was inhibited in HmLu-1. To investigate the penetration of BHV-1, we performed the quantitative measurement of viral DNA by quantitative competitive (QC)PCR in infected cells. In MDBK cells, virions attached to the cell surface, penetrated into the cells and were transported to the nucleus. However in HmLu-1, only a small fraction of the virions attached to the cell: surface were allowed to penetrate. Our results indicated that the replication of BHV-1 in semi-permissive HmLu-1 nias not dramatically restricted at one certain point but at some various stages including adsorption and penetration. (C) 1999 Elsevier Science B.V. All rights reserved.

Bovine herpesvirus-1 (BHV-1) has been used as a vector of live recombinant vaccines for cattle which express the genes of other pathogens. Because of the importance of the choice of the promoter which allows the efficient expression of the foreign genes in the BHV-1 vector, we compared the relative efficacy of various promoters integrated in the BHV-1 genome. The promoter sequences of the BHV-1 thymidine kinase (tk), gB, gC, SV40 early, and pseudorabies virus (PRV) immediate early (IE) genes were placed at the upstream of the open reading frame of the chloramphenycol acetyl transferase (CAT) gene and the promoter-CAT sequences were integrated into the tk gene of BHV-1 by homologous recombination. The promoter activity was assayed by measuring the CAT activity in the extracts of Madin Darby bovine kidney (MDBK) cells infected with the recombinant BHV-1. The PRV IE promoter was activated earlier and maintained at a higher level activity than the BHV-1 gB or gC promoters throughout the most of the growth phase of BHV-1. At the late phase, however, the activities of the BHV-1 gB and gC promoters reached the higher level. The BHV-1 tk promoter activity was low and the SV40 early promoter was hardly activated when integrated into the BHV-1 genome.

The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection.