Corneal endothelial cells activate innate and acquired arm of anti-viral responses after cytomegalovirus infection.

Infection of the corneal endothelial cells by human cytomegalovirus (CMV) is an important cause of corneal endotheliitis. CMV endotheliitis is difficult to completely cure and relapses are frequent. This can cause blinding corneal bullous keratopathy. However, the pathogenesis of CMV endotheliitis remains undetermined. To understand the immunopathology of endotheliitis, we examined how corneal endothelial cells prime the anti-viral immunity after CMV infection based on global transcriptional responses. To accomplish this, human corneal endothelial (HCEn) cells were infected with CMV, and the global transcriptional responses were determined by microarray analyses for primary anti-viral responses using network analysis. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and protein array analyses were used to examine whether anti-viral cytokines were induced, i.e., to determine whether innate immune responses were activated. To examine whether priming of acquired immune response was activated, CMV-infected HCEn cells were co-cultured with allogeneic CD8+ T cells from CMV seropositive donors and tested for priming activity for the CD8+ effector T cells by measuring interferon-γ secretion. The CMV-induced responses of HCEn cells were characterized by type I interferon and pattern recognition receptor pathways which represent innate immune priming. The global transcriptional activation was specifically associated with antigen presentation with the antimicrobial response functions. Protein array analyses indicated a significant increase in the secretion of anti-viral inflammatory cytokines including CXCL10 as innate immune responses. When HCEn cells were examined to determine whether CMV infection activated anti-viral acquired immunity, CMV-infected HCEn cells directly stimulated the proliferation of CD8+ T cells from CMV-seropositive donors, and pp65 viral epitope induced interferon-γ secretion from the CD8+ T cells. We conclude that CMV-infected HCEn cells induce innate immune priming along with provisions of acquired immune priming of CD8+ effector T cells. This information should help in the development of useful diagnostic procedures and efficacious therapeutic strategy to treat refractory corneal endotheliitis.

Epstein-Barr virus deubiquitinase downregulates TRAF6-mediated NF-κB signaling during productive replication.

Epstein-Barr virus (EBV), a human oncogenic herpesvirus that establishes a lifelong latent infection in the host, occasionally enters lytic infection to produce progeny viruses. The EBV oncogene latent membrane protein 1 (LMP1), which is expressed in both latent and lytic infection, constitutively activates the canonical NF-κB (p65) pathway. Such LMP1-mediated NF-κB activation is necessary for proliferation of latently infected cells and inhibition of viral lytic cycle progression. Actually, canonical NF-κB target gene expression was suppressed upon the onset of lytic infection. TRAF6, which is activated by conjugation of polyubiquitin chains, associates with LMP1 to mediate NF-κB signal transduction. We have found that EBV-encoded BPLF1 interacts with and deubiquitinates TRAF6 to inhibit NF-κB signaling during lytic infection. HEK293 cells with BPLF1-deficient recombinant EBV exhibited poor viral DNA replication compared with the wild type. Furthermore, exogenous expression of BPLF1 or p65 knockdown in cells restored DNA replication of BPLF1-deficient viruses, indicating that EBV BPLF1 deubiquitinates TRAF6 to inhibit NF-κB signal transduction, leading to promotion of viral lytic DNA replication.

Lack of presence of the human cytomegalovirus in human glioblastoma.

Recent reports have indicated human cytomegalovirus (HCMV) to be associated with human glioblastoma carcinogenesis. In established examples of viral carcinogenesis, viral DNA and one or more of its products have been detected in most tumor cells of biopsies in the majority of cases. To test whether HCMV is associated with human glioblastoma based on this criterion, we measured the number of viral DNA molecules per cell in both frozen and paraffin-embedded tumor biopsies from 58 patients using real-time quantitative PCR (QPCR). Immunohistochemical and fluorescence in situ hybridization (FISH) to detect HCMV proteins and genome was performed in 10 cases using formalin-fixed paraffin-embedded glioblastoma tissues. Southern blotting using DNA extracted from four glioblastoma cell lines together with immunoblotting using the four cell lines and five glioblastoma tissue samples were also performed. We further confirmed the immunoblot bands using liquid chromatography-tandem mass spectrometry assay. As a result, HCMV DNA was not detected in the tumor cells from any of the glioblastoma cases by QPCR detecting two different HCMV genes, in clear contrast to samples from patients with HCMV infection. Southern blotting and immunoblotting of cell lines and FISH using paraffin sections were all negative. However, immunoblotting and immunohistochemistry using tissue samples were partly positive, but HCMV proteins were not detected by proteomic analysis, suggesting false positivity of the analyses. As our QPCR analysis could detect 10 copies of HCMV DNA mixed with DNA extracted from 10(4) HCMV-negative cells, we conclude that HCMV is not persistent, at least in the tumor cells, of developed human glioblastoma.

Epigenetic histone modification of Epstein-Barr virus BZLF1 promoter during latency and reactivation in Raji cells.

The Epstein-Barr virus (EBV) predominantly establishes latent infection in B cells, and the reactivation of the virus from latency is dependent on the expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophores, or histone deacetylase (HDAC) inhibitors. In some cell lines latently infected with EBV, an HDAC inhibitor alone can induce BZLF1 transcription, while the treatment does not enhance expression in other cell lines, such as B95-8 or Raji cells, suggesting unknown suppressive mechanisms besides histone deacetylation in those cells. Here, we found the epigenetic modification of the BZLF1 promoter in latent Raji cells by histone H3 lysine 27 trimethylation (H3K27me3), H3K9me2/me3, and H4K20me3. Levels of active markers such as histone acetylation and H3K4me3 were low in latent cells but increased upon reactivation. Treatment with 3-deazaneplanocin A (DZNep), an inhibitor of H3K27me3 and H4K20me3, significantly enhanced the BZLF1 transcription in Raji cells when in combination with an HDAC inhibitor, trichostatin A (TSA). The knockdown of Ezh2 or Suv420h1, histone methyltransferases for H3K27me3 or H4K20me3, respectively, further proved the suppression of Zp by the methylations. Taken together, the results indicate that H3K27 methylation and H4K20 methylation are involved, at least partly, in the maintenance of latency, and histone acetylation and H3K4 methylation correlate with the reactivation of the virus in Raji cells.

Involvement of Jun dimerization protein 2 (JDP2) in the maintenance of Epstein-Barr virus latency.

Reactivation of the Epstein-Barr virus from latency is dependent on expression of the BZLF1 viral immediate-early 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 that Jun dimerization protein 2 (JDP2) plays a significant role in suppressing Zp activity. Reporter, EMSA, and ChIP assays of a Zp mutant virus revealed JDP2 association with Zp at the ZII cis-element, a binding site for CREB/ATF/AP-1. Suppression of Zp activity by JDP2 correlated with HDAC3 association and reduced levels of histone acetylation. Although introduction of point mutations into the ZII element of the viral genome did not increase the level of BZLF1 production, silencing of endogenous JDP2 gene expression by RNA interference increased the levels of viral early gene products and viral DNA replication. These results indicate that JDP2 plays a role as a repressor of Zp and that its replacement by CREB/ATF/AP-1 at ZII is crucial to triggering reactivation from latency to lytic replication.

Identification and characterization of CCAAT enhancer-binding protein (C/EBP) as a transcriptional activator for Epstein-Barr virus oncogene latent membrane protein 1.

Epstein-Barr virus LMP1, a major oncoprotein expressed in latent infection, is critical for primary B cell transformation, functioning as a TNFR family member by aggregation in the plasma membrane resulting in constitutive activation of cellular signals, such as NF-κB, MAPK, JAK/STAT, and AKT. Although transcription of LMP1 in latent type III cells is generally under the control of the viral coactivator EBNA2, little is known about EBNA2-independent LMP1 expression in type II latency. We thus screened a cDNA library for factors that can activate the LMP1 promoter in an EBNA2-independent manner, using a reporter assay system. So far, we have screened >20,000 clones, and here identified C/EBPε as a new transcriptional activator. Exogenous expression of C/EBPα, -ß, or -ε efficiently augmented LMP1 mRNA and protein levels in EBV-positive cell lines, whereas other members of the C/EBP family exhibited modest or little activity. It has been demonstrated that LMP1 gene transcription depends on two promoter regions: proximal (ED-L1) and distal (TR-L1). Interestingly, although we first used the proximal promoter for screening, we found that C/EBP increased transcription from both promoters in latent EBV-positive cells. Mutagenesis in reporter assays and EMSA identified only one functional C/EBP binding site, through which activation of both proximal and distal promoters is mediated. Introduction of point mutations into the identified C/EBP site in EBV-BAC caused reduced LMP1 transcription from both LMP1 promoters in epithelial cells. In conclusion, C/EBP is a newly identified transcriptional activator of the LMP1 gene, independent of the EBNA2 coactivator.

The human cytomegalovirus gene products essential for late viral gene expression assemble into prereplication complexes before viral DNA replication.

The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed.

Spatiotemporally different DNA repair systems participate in Epstein-Barr virus genome maturation.

Productive replication of Epstein-Barr virus occurs in discrete sites in nuclei, called replication compartments, where viral DNA replication proteins and host homologous recombinational repair (HRR) and mismatch repair (MMR) factors are recruited. Three-dimensional (3D) surface reconstruction imaging clarified the spatial arrangements of these factors within the replication compartments. Subnuclear domains, designated BMRF1 cores, which were highly enriched in viral polymerase processivity factor BMRF1 could be identified inside the replication compartments. Pulse-chase experiments revealed that newly synthesized viral genomes organized around the BMRF1 cores were transferred inward. HRR factors could be demonstrated mainly outside BMRF1 cores, where de novo synthesis of viral DNA was ongoing, whereas MMR factors were found predominantly inside. These results imply that de novo synthesis of viral DNA is coupled with HRR outside the cores, followed by MMR inside cores for quality control of replicated viral genomes. Thus, our approach unveiled a viral genome manufacturing plant.

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.

Tetrameric ring formation of Epstein-Barr virus polymerase processivity factor is crucial for viral replication.

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.

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.

The CREB site in the proximal enhancer is critical for cooperative interaction with the other transcription factor binding sites to enhance transcription of the major intermediate-early genes in human cytomegalovirus-infected cells.

One of the two SP1 sites in the proximal enhancer of the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter is essential for transcription in human fibroblast cells (H. Isomura, M. F. Stinski, A. Kudoh, T. Daikoku, N. Shirata, and T. Tsurumi, J. Virol. 79:9597-9607, 2005). Upstream of the two SP1 sites to -223 relative to the +1 transcription start site, there are an additional five DNA binding sites for eukaryotic transcription factors. We determined the effects of the various transcription factor DNA binding sites on viral MIE RNA transcription, viral gene expression, viral DNA synthesis, or infectious virus production. We prepared recombinant HCMV bacterial artificial chromosome (BAC) DNAs with either one site missing or one site present upstream of the two SP1 sites. Infectious recombinant HCMV BAC DNAs were transfected into various cell types to avoid the effect of the virion-associated transactivators. Regardless of the cell type, which included human fibroblast, endothelial, and epithelial cells, the CREB site had the most significant and independent effect on the MIE promoter. The other sites had a minor independent effect. However, the combination of the different transcription factor DNA binding sites was significantly stronger than multiple duplications of the CREB site. These findings indicate that the CREB site in the presence of the other sites has a major role for the replication of HCMV.

Homologous recombinational repair factors are recruited and loaded onto the viral DNA genome in Epstein-Barr virus replication compartments.

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.

Noncanonical TATA sequence in the UL44 late promoter of human cytomegalovirus is required for the accumulation of late viral transcripts.

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.

A cis element between the TATA Box and the transcription start site of the major immediate-early promoter of human cytomegalovirus determines efficiency of viral replication.

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.

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.

Establishment of a novel foreign gene delivery system combining an HSV amplicon with an attenuated replication-competent virus, HSV-1 HF10.

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have been used widely in genetic engineering with many advantages for gene delivery, being easily constructed. An attenuated and replication-competent HSV-1 HF10 clone demonstrating an oncolytic effect on cancer cells in vitro and in vivo has been applied recently for clinical virotherapy of breast cancers and the present studies were conducted to test its efficacy in combination with an HSV-1 amplicon. For this purpose, a new system was developed to produce high titers of the HSV-1 amplicon vector and the results showed that its package efficiency and the titer ratio to HF10 were improved by passage through two cell lines. A high ratio of amplicon/helper virus HF10 (A/H) (>1) was required to express the foreign gene efficiently. Furthermore, in order to express the foreign gene conditionally, an HSV-1 ICP8 promoter was introduced in place of the human cytomegalovirus MIE promoter, this driving expression of the transgene when replication of HF10 progressed. The methodology for simple preparation of mixtures of viruses containing the amplicon with the oncolytic virus is documented. This system should find application for studies of cancer therapy.

Analysis of human cytomegalovirus replication in primary cultured human corneal endothelial cells.

BACKGROUND/AIMS: Since the first case of human cytomegalovirus (HCMV)-induced corneal endotheliitis in which HCMV DNA was detected from the patient's aqueous humour using PCR, the clinical evidence for HCMV endotheliitis has been accumulating. However, it remains to be confirmed whether HCMV can efficiently replicate in corneal endothelial cells. We, therefore, sought to determine whether primary cultured human corneal endothelial cells (HCECs) could support HCMV replication. METHODS: Human foreskin fibroblasts (HFFs) have been shown to be fully permissive for HCMV replication, and are commonly used as an in vitro model for HCMV lytic replication. Therefore, primary cultured HCECs or HFFs were infected with the vascular endotheliotropic HCMV strain TB40/E or laboratory strain Towne. We then compared viral mRNA and protein expression, genome replication and growth between the TB40/E-infected and Towne-infected HCECs and HFFs. RESULTS: When HCECs were infected with TB40/E or Towne, rounded cells resembling owl's eyes as well as viral antigens were detected. Viral mRNA synthesis and protein expression proceeded efficiently in the HCECs and HFFs infected with TB40/E or Towne at a high multiplicity of infection (MOI). Similarly, the viral genome was also effectively replicated, with UL44--a viral DNA polymerase processivity factor--foci observed in the nuclei of HCECs. HCECs produced a substantial number of infectious virions after infection with TB40/E at both a high and low MOI. CONCLUSIONS: Primary cultured HCECs could efficiently support HCMV replication after infection at both a high and low MOI.

Processing bodies accumulate in human cytomegalovirus-infected cells and do not affect viral replication at high multiplicity of infection.

Translationally silenced mRNAs are recruited to two major classes of RNA granules in the cytoplasm, processing bodies (PBs) and stress granules (SGs). We show that PBs accumulated after human cytomegalovirus (HCMV) infection. PB assembly after HCMV infection was also detected in the presence of the protein synthesis inhibitor, cycloheximide, but required active RNA synthesis. UV-inactivated HCMV virions were sufficient to induce PB accumulation in HFF cells treated with cycloheximide. Viral IE1 RNA did not colocalize with PBs, and we could not detect an effect of PB accumulation on viral growth. These results may indicate that HCMV inhibits the colocalization of IE1 mRNA with PBs, preventing IE1 mRNA decay and translational inhibition.

Coordination of late gene transcription of human cytomegalovirus with viral DNA synthesis: recombinant viruses as potential therapeutic vaccine candidates.

INTRODUCTION: During productive infection, human cytomegalovirus (HCMV) genes are expressed in a temporal cascade, with temporal phases designated as immediate-early (IE), early, and late. The major IE (MIE) genes, UL123 and UL122 (IE1/IE2), play a critical role in subsequent viral gene expression and the efficiency of viral replication. The early viral genes encode proteins necessary for viral DNA replication. Following viral DNA replication, delayed-early and late viral genes are expressed which encode structural proteins for the virion. The late genes can be divided into two broad classes. At early times the gamma-1 or leaky-late class are expressed at low levels after infection and are dramatically upregulated at late times. In contrast, the gamma-2 or 'true' late genes are expressed exclusively after viral DNA replication. Expression of true late (gamma-2 class) viral genes is completely prevented by inhibition of viral DNA synthesis. AREAS COVERED: This review addresses the viral genes required for HCMV late gene transcription. Recombinant viruses that are defective for late gene transcription allow for early viral gene expression and viral DNA synthesis, but not infectious virus production. Since current HCMV prophylaxis is limited by several shortcomings, the use of defective recombinant viruses to induce HCMV cell-mediated and humoral immunity is discussed. EXPERT OPINION: HCMV DNA replication and late gene transcription are not completely linked. Viral-encoded trans-acting factors are required. Recombinant viruses proficient in MIE and early viral gene expression and defective in late gene expression may be an alternative therapeutic vaccine candidates for the induction of cell-mediated and humoral immunity.