Immediate-early Research Articles

A Novel Selective LSD1/KDM1A Inhibitor Epigenetically Blocks Herpes Simplex Virus Lytic Replication and Reactivation from Latency

ABSTRACTCellular processes requiring access to the DNA genome are regulated by an overlay of epigenetic modifications, including histone modification and chromatin remodeling. Similar to the cellular host, many nuclear DNA viruses that depend upon the host cell’s transcriptional machinery are also subject to the regulatory impact of chromatin assembly and modification. Infection of cells with alphaherpesviruses (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) results in the deposition of nucleosomes bearing repressive histone H3K9 methylation on the viral genome. This repressive state is modulated by the recruitment of a cellular coactivator complex containing the histone H3K9 demethylase LSD1 to the viral immediate-early (IE) gene promoters. Inhibition of the activity of this enzyme results in increased repressive chromatin assembly and suppression of viral gene expression during lytic infection as well as reactivation from latency in a mouse ganglion explant model. However, available small-molecule LSD1 inhibitors are not originally designed to inhibit LSD1, but rather monoamine oxidases (MAO) in general. Thus, their specificity for and potency to LSD1 is low. In this study, a novel specific LSD1 inhibitor was identified that potently repressed HSV IE gene expression, genome replication, and reactivation from latency. Importantly, the inhibitor also suppressed primary infection of HSV in vivo in a mouse model. Based on common control of a number of DNA viruses by epigenetic modulation, it was also demonstrated that this LSD1 inhibitor blocks initial gene expression of the human cytomegalovirus and adenovirus type 5.IMPORTANCE Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents.

Tissue and cell-specific transcriptional activity of the human cytomegalovirus immediate early gene promoter (UL123) in zebrafish

The human cytomegalovirus (CMV) is a member of the herpesvirus superfamily and causes different diseases including encephalitis, gastrointestinal diseases, pneumonitis, hepatitis, and retinitis. The immediate early (IE) gene of the human cytomegalovirus is essential to the viral replication. The proximal promoter region of this gene behaves as a strong enhancer and was commonly used to overexpress genes in vitro and in vivo in numerous cell types and species. However, there was no detailed report on the spatial and temporal transcriptional activity of the human CMV-IE gene promoter in zebrafish. In the present study, we generated stable transgenic zebrafish lines carrying the eGFP reporter gene under the control of the human CMV-IE gene promoter (−602/−14). We demonstrated that the hCMV-IE:eGFP transgene was expressed in numerous tissues but transgene expression was either regionalized or restricted to specific cell types as embryo and larval development progressed. In adult, the global expression pattern was similar but not identical to that described for the simian CMV-IE gene promoter in stable zebrafish with high transgene expression in the spinal cord, olfactory organs, central nervous system, neuromasts, retina, and skeletal muscles. However, we describe additional major expression sites in the hepatocytes, the epithelial cells of the intestine, the epithelial cells of the renal tubules, and the oocytes. Interestingly, our study shows that the tissue and cell specific expression pattern of the human CMV-IE gene promoter is rather well conserved in stable transgenic zebrafish compared to that observed in mouse. The major expression sites described in zebrafish are in agreement with the targeted cells and symptoms resulting from CMV infections in human. Finally, the hCMV:eGFP transgenic lines described in the present study will be valuable tools to trace specific cell lineages in adult zebrafish.

Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells.

To understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication. Rabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection. Egr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays. Our results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus. Together these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.

Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells

AimsTo understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication.Study DesignRabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection.MethodologyEgr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays.ResultsOur results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus.ConclusionTogether these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.

Human cytomegalovirus infection in Nigerians living with human immunodeficiency virus

Context : Human immunodeficiency virus (HIV) patients are at higher risk for Human cytomegalovirus (HCMV) infection. Aim: To identify HCMV and HIV co-infection among Nigerian patients for prompt therapeutic interventions. Materials and Methods: The study drew samples from the antiretroviral clinic of Nigerian Institute of Medical Research and patients' informed consent was taken at enrollment. Variables collected included socio-demographic characteristics such as sex, occupation, marital status, educational status, income, and religion, while health-related variables were CD4 counts and HIV viral load. Genomic DNA from whole blood samples of 236 patients, 164 (69.5%) females and 72 (30.5%) males, was subjected to polymerase chain reaction (PCR) amplification of 2 genes within conserved immediate early (IE) and late (LA) transcribed regions of HCMV genome. Statistical Analysis: Statistical Package for Social Sciences was used to determine frequencies of HMCV infection, while Chi square was used to examine associations between patient's characteristics and HMCV infection. Results: A total of 35 (14.8%) patients; 25 (10.6%) females and 10 (4.2%) males were positive for HCMV infection. Although there was variation in prevalence of HCMV in different marital status, it was statistically insignificant (P = 0.734; P > 0.05). Results also showed that 22 (62.9%) of HCMV positive patients had HIV viral load greater than 10,000, prevalence of HCMV decreased as CD4 counts increased, while 12 (34.3%) of HCMV positive patients had CD4 counts between 1 and 200. Conclusions: This study is the first molecular survey of HCMV/HIV co-infection in Nigeria and has provided valuable information for prompt therapeutic intervention to reduce morbidity among HIV patients.

C-Terminal trans-activation sub-region of VP16 is uniquely required for forskolin-induced herpes simplex virus type 1 reactivation from quiescently infected-PC12 cells but not for replication in neuronally differentiated-PC12 cells

The HSV-1 tegument protein VP16 contains a trans-activation domain (TAD) that is required for induction of immediate early (IE) genes during lytic infection and induced reactivation from latency. Here we report the differential contributions of the two sub-regions of the TAD in neuronal and non-neuronal cells during activation of IE gene expression, virus replication, and reactivation from quiescently infected (QIF)-PC12 cells. Our studies show that VP16- and chemical (hexamethylenebisacetamide)-induced IE gene activation is attenuated in neuronal cells. Irrespective of neuronal or non-neuronal cell backgrounds, IE gene activation demonstrated a greater requirement for the N-terminal sub-region of VP16 TAD (VP16N) than the C-terminal sub-region (VP16C). In surprising contrast to these findings, a recombinant virus (RP4) containing the VP16N deletion was capable of modest forskolin-induced reactivation whereas a recombinant (RP3) containing a deletion of VP16C was incapable of stress-induced reactivation from QIF-PC12 cells. These unique process-dependent functions of the VP16 TAD sub-regions may be important during particular stages of the virus life cycle (lytic, entrance, and maintenance of a quiescent state and reactivation) when viral DNA would be expected to be differentially modified.

Niche Perivascular Stromal Cells As a Potential Reservoir and a Source of HCMV Reactivation

Abstract 3464Cytomegalovirus (HCMV) infection or reactivation is an important cause of morbidity and mortality among hematopoietic cell transplantation (HCT) recipients. HCMV infection in these patients may result from a new HCMV infection, reactivation of pre-existing latent virus, or both. Regardless, HCMV causes end organ diseases including pneumonia, gastroenteritis, retinitis, hepatitis, and encephalitis, and can contribute to graft failure. Although the use of antiviral drugs has decreased the incidence, morbidity, and mortality of HCMV disease in transplant recipients, identification of cells serving as the reservoir and/or that are responsible for reactivation of the disease could potentially lead to the development of novel targeted therapies. Although hematopoietic cells, particularly myeloid progenitors, have been identified as reservoirs for HCMV reactivation, it is possible that within the bone marrow environment there are cells that are able to serve as a reservoir for this virus. Perivascular mesenchymal/stromal cells (MSC) are part of the microenvironmental niche and play a role in hematopoietic stem cell (HSC) maintenance and differentiation. Because MSC have an increased resistance to chemo- and radio-therapy, allowing the survival of resident MSC during HCT, they express CD29, a main receptor for HCMV capture and entry, and they directly interact with HSC within the niche, we hypothesized that MSC could serve as a potential reservoir and a source of HCMV reactivation in transplantation patients. To this end, we collected bone marrow (BM) from 10 different healthy adult donors, who had tested positive for CMV antibodies, and investigated whether MSC (phenotypically defined as cells positive for CXCL12, CD90, CD44, CD29, CD73, CD105, and CD146, but negative for both CD34 and CD45) were positive for CMV, by testing for the presence of Immediate Early (IE) HCMV gene products by PCR with subsequent confirmation by sequencing. MSC sorted from 7 out of 10 adult donors tested positive for the presence of IE HCMV DNA, demonstrating persistence of the virus in these cells in the absence of disease. In order to further evaluate the permissivity of these cells to HCMV infection and to determine whether the virus was able to establish a lytic infection, we infected MSC from HCMV-negative donors in vitro, with the clinical isolate Davis strain of HCMV (MOI of 3). At 1, 3, 5, and 7 days post-infection (dpi), the cells were positive for IE HCMV DNA. They were also positive, at one or more of the tested time points, for UL-83, a late gene associated with lytic infection. IE protein expression was also detected by immunohistochemistry. The mock infected cells remained negative for both IE and UL-83 throughout the duration of the experiment. In order to determine whether MSC were able to produce and release infectious viral particles, supernatants were collected at different times post-infection, and evaluated for the presence of infectious units (IU) using HFF. At 1, 3, 5, and 7 dpi, MSC produced 5167±809, 6867±104, 3800±200, and 3467±788 IU, respectively, demonstrating that MSC are able to produce and release infectious particle and infect neighboring cells. We have therefore shown that a highly defined population of human stromal cells (MSC) naturally harbors latent HCMV. Since MSC reside within/comprise the HSC niche, they share an intimate relationship with hematopoietic progenitors, and they participate in homing and retention of HSC, our data support the conclusion that MSC may serve as a previously unrecognized reservoir for this clinically important virus in transplant recipients. Moreover, upon infection with HCMV, MSC are able to produce infectious virus, thereby propagating HCMV infection to adjacent cells. Studies are underway to determine whether productive HCMV infection of this stromal cell population also alters the expression of proteins that regulate HSC maintenance. Disclosures:No relevant conflicts of interest to declare.

Characterization of white spot syndrome virus immediate-early gene promoters

Twenty-one immediate-early (IE) genes of white spot syndrome virus (WSSV) have been identified so far. However, the transcriptional regulation of WSSV IE genes remains largely unknown. In this report, the 5' flanking regions of 18 WSSV IE genes were cloned and eight functional promoter regions were identified. WSSV IE gene promoters normally contained a TATA box approximately 30 bp upstream of the transcriptional initiation site. Also, the cyclic AMP response element (CRE; TGACGTCA) was frequently found within the WSSV IE promoter regions. Mutations of the CREs of WSSV IE promoters P403 and P465 reduced their activity significantly, suggesting that these elements have a role in WSSV IE gene transcription. Our findings provide a more global view of WSSV IE gene promoters and will facilitate the in-depth investigation of viral gene transcriptional regulation.

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and HCMV infection in immunocompromised patients may trigger devastating disease. Cytotoxic lymphocytes control HCMV by releasing granzymes towards virus-infected cells. In mice, granzyme M (GrM) has a physiological role in controlling murine CMV infection. However, the underlying mechanism remains poorly understood. In this study, we showed that human GrM was expressed by HCMV-specific CD8(+) T cells both in latently infected healthy individuals and in transplant patients during primary HCMV infection. We identified host cell heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a physiological GrM substrate. GrM most efficiently cleaved hnRNP K in the presence of RNA at multiple sites, thereby likely destroying hnRNP K function. Host cell hnRNP K was essential for HCMV replication not only by promoting viability of HCMV-infected cells but predominantly by regulating viral immediate-early 2 (IE2) protein levels. Furthermore, hnRNP K interacted with IE2 mRNA. Finally, GrM decreased IE2 protein expression in HCMV-infected cells. Our data suggest that targeting of hnRNP K by GrM contributes to the mechanism by which cytotoxic lymphocytes inhibit HCMV replication. This is the first evidence that cytotoxic lymphocytes target host cell proteins to control HCMV infections.

Suppression of CD8+ T-cell recognition in the immediate-early phase of human cytomegalovirus infection

Human cytomegalovirus (HCMV) interferes with MHC class I-restricted antigen presentation and thereby reduces recognition by CD8(+) T-cells. This interference is mediated primarily by endoplasmic reticulum-resident glycoproteins that are encoded in the US2-11 region of the viral genome. Such a suppression of recognition would be of particular importance immediately after infection, because several immunodominant viral antigens are already present in the cell in this phase. However, which of the evasion proteins gpUS2-11 interfere(s) with antigen presentation to CD8(+) T-cells at this time of infection is not known. Here we address this question, using recombinant viruses (RV) that express only one of the immunoevasins gpUS2, gpUS3 or gpUS11. Infection with RV-US3 had only a limited impact on the presentation of peptides from the CD8(+) T-cell antigens IE1 and pp65 under immediate-early (IE) conditions imposed by cycloheximide/actinomycin D blocking. Unexpectedly, both RV-US2 and RV-US11 considerably impaired the recognition of IE1 and pp65 by CD8(+) T-cells, and both US2 and, to a lesser extent, US11 were transcribed under IE conditions. Thus, gpUS2 and gpUS11 are key effectors of MHC class I immunoevasion immediately after HCMV infection.

Transcription characteristics of the human cytomegalovirus UL13 gene

The human cytomegalovirus (HCMV) UL13 gene is located in the unique long (UL) region of its genome. The transcript structure of UL13 gene has not been investigated to date. By using cDNA library screening, northern blot, and rapid amplification of cDNA ends (RACE), the HCMV UL13 gene was demonstrated to be transcribed from the immediate early (IE) to the late (L) phase of infection, and at least one 1602-nt unspliced transcript was identified in the present study from three clinical isolates.

Chromatin Assembly on Herpes Simplex Virus 1 DNA Early during a Lytic Infection Is Asf1a Dependent

Herpes simplex virus (HSV) is a large DNA virus which is characterized by its ability to form latent infections in neurons of the peripheral nervous system. Although histones are found in the capsids of small DNA viruses (papovaviruses), none are found in the capsids of large HSV. However, after entry into the infected cell nucleus, the HSV genome begins to associate with nucleosomes during the earliest stages of infection. In contrast, late during infection, newly replicated viral DNA does not appear to associate with nucleosomes, suggesting that histones are deposited specifically on input viral DNA. The mechanisms of deposition and removing histones from the viral genome are unclear. Recently, histone chaperones, involved in the assembly and disassembly of nucleosomes, have been identified. Human antisilencing factor 1 (Asf1) is one such factor which is involved in both the assembly and disassembly of nucleosomes in cellular systems. In this study, we have examined the effect of small interfering RNA (siRNA) knockdown of Asf1a on HSV infections in HeLa cells. Both viral replication and growth were found to be decreased. Also, viral DNA was significantly less protected from micrococcal nuclease (MNase) digestion up to 6 h postinfection (hpi). However, transcription of the immediate early (IE) genes ICP0 and ICP4 was significantly upregulated at 3 h postinfection. Also, these genes were found to be less protected from MNase digestion and, therefore, less associated with nucleosomes. These results suggest that Asf1a plays a role in regulating IE genes by assembling chromatin onto histone-free viral DNA by 3 h postinfection.

Rana grylio Virus (RGV) 50L Is Associated with Viral Matrix and Exhibited Two Distribution Patterns

BackgroundThe complete genome of Rana grylio virus (RGV) was sequenced and analyzed recently, which revealed that RGV 50L had homologues in many iridoviruses with different identities; however, the characteristics and functions of 50L have not been studied yet.Methodology/Principal FindingsWe cloned and characterized RGV50L, and revealed 50L functions in virus assembly and gene regulation. 50L encoded a 499-amino acid structural protein of about 85 kDa in molecular weight and contained a nuclear localization signal (NLS) and a helix- extension-helix motif. Drug inhibition assay demonstrated that 50L was an immediate-early (IE) gene. Immuno-fluorescence assay revealed that 50L appeared early and persisted in RGV-infected cells following two distribution patterns. One pattern was that 50L exhibited a cytoplasm-nucleus- viromatrix distribution pattern, and mutagenesis of the NLS motif revealed that localization of 50L in the nucleus was NLS-dependent; the other was that 50L co-localized with viral matrix which plays important roles in virus assembly and the life circle of viruses.Conclusions/SignificanceRGV 50L is a novel iridovirus IE gene encoded structural protein which plays important roles in virus assembly.

A Myeloid Progenitor Cell Line Capable of Supporting Human Cytomegalovirus Latency and Reactivation, Resulting in Infectious Progeny

Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractable in vitro model system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractable in vitro model system with which to study HCMV latency and reactivation.

Inhibition of Human Cytomegalovirus Immediate-Early Gene Expression by Cyclin A2-Dependent Kinase Activity

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G(0)/G(1) phase of the cell division cycle. S/G(2) cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G(2)-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G(1) was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.

Anti-cytomegalovirus effects of tricin are dependent on CXCL11

It has been reported that treatment with tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone), a derivative of Sasa albo-marginata, after human cytomegalovirus (HCMV) infection significantly suppressed both infectious virus production and HCMV replication in the human embryonic fibroblast cell line MRC-5. In this paper, we examined the mechanisms for the anti-HCMV effects of tricin in MRC-5 cells. Exposure of fibroblasts to tricin inhibited infectious HCMV production, with concomitant decreases in levels of transcripts of the CXC chemokine IFN-inducible T cell alpha chemoattractant (I-TAC or CXCL11) gene. We also found that the transcripts of the HCMV immediate early (IE) gene and replication of HCMV were lower in CXCL11 gene-knockdown cells. These results suggest that tricin is a novel compound with potential anti-HCMV activity and that CXCL11 is one of the chemokines involved in HCMV replication. In addition, it is possible that CXCL11 is the one of the targets of tricin.

Entry of Herpes Simplex Virus Type 1 (HSV-1) into the Distal Axons of Trigeminal Neurons Favors the Onset of Nonproductive, Silent Infection

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons.

Mdv1-miR-M7-5p, located in the newly identified first intron of the latency-associated transcript of Marek’s disease virus, targets the immediate-early genes ICP4 and ICP27

Marek's disease virus serotype 1 (MDV-1) is an oncogenic alphaherpesvirus causing fatal T-cell lymphoma in chickens. MDV latency is characterized by the production of latency-associated transcripts (LATs), a family of non-protein-coding spliced RNAs. A cluster of four microRNAs (cluster mdv1-miR-M8-M10) was identified, but not formally mapped, at the predicted LAT 5' end. We established a LAT cDNA library from latently MDV-infected cell line MSB-1. We identified 22 highly variable LATs, which were due to the extensive alternative splicing of a total of 14 introns. RACE PCR confirmed the predicted 3' end and allowed identification of the 5' end, 400 nt upstream of the previously predicted LAT end. The LATs share their transcription start site with the microRNA-expressing transcript described previously, localizing the microRNAs to the first LAT intron and identifying the LATs as the primary transcripts of the microRNAs. We identified MDV immediate-early (IE) genes ICP4 and ICP27 as putative targets of mdv1-miR-M7-5p, the third microRNA of the cluster mdv1-miR-M8-M10. Endogenously expressed mdv1-miR-M7-5p in MSB-1 cells reduced luciferase activity significantly when microRNA-responsive elements from ICP4 or ICP27 were cloned in the 3' UTR of the firefly luciferase gene. ICP27 protein levels were decreased by 70 % when the mdv1-miR-M7-5p precursor was co-expressed with an ICP27 expression plasmid. Additionally, we showed a negative correlation between the decreased expression of mdv1-miR-M7-5p and an increase in ICP27 expression during virus reactivation. Our results suggest that, by targeting two IE genes, MDV microRNAs produced from LAT transcripts may contribute to establish and/or maintain latency.

Resistance to Bombyx mori nucleopolyhedrovirus via overexpression of an endogenous antiviral gene in transgenic silkworms

Transgenic technology is a powerful tool for improving disease-resistant species. Bmlipase-1, purified from the midgut juice of Bombyx mori, showed strong antiviral activity against B. mori nucleopolyhedrovirus (BmNPV). In an attempt to create an antiviral silkworm strain for sericulture, a transgenic vector overexpressing the Bmlipase-1 gene was constructed under the control of a baculoviral immediate early-1 (IE1) promoter. Transgenic lines were generated via embryo microinjection. The mRNA level of Bmlipase-1 in the midguts of the transgenic line was 27.3 % higher than that of the non-transgenic line. After feeding the silkworm with different amounts of BmNPV, the mortality of the transgenic line decreased to approximately 33 % compared with the non-transgenic line when the virus dose was 10(6) OB/larva. These results imply that overexpressing endogenous antiviral genes can enhance the antiviral resistance of silkworms.

The chromatin-tethering domain of human cytomegalovirus immediate-early (IE) 1 mediates associations of IE1, PML and STAT2 with mitotic chromosomes, but is not essential for viral replication

Human cytomegalovirus (HCMV) immediate-early (IE) 1 protein associates with chromosomes in mitotic cells using its carboxyl-terminal 16 aa region. However, the role of this IE1 activity in viral growth has not been evaluated in the context of mutant virus infection. We produced a recombinant HCMV encoding mutant IE1 with the carboxyl-terminal chromosome-tethering domain (CTD) deleted. This IE1(ΔCTD) virus grew like the wild-type virus in fibroblasts, indicating that the CTD is not essential for viral replication in permissive cells. Unlike wild-type virus infections, PML and STAT2, which interact with IE1, did not accumulate at mitotic chromosomes in IE1(ΔCTD) virus-infected fibroblasts, demonstrating that their associations with chromosomes are IE1 CTD-dependent. IE1 SUMOylation did not affect IE1 association with chromosomes. Our results provide genetic evidence that the CTD is required for the associations of IE1, PML and STAT2 with mitotic chromosomes, but that these IE1-related activities are not essential for viral replication in fibroblasts.