IE Gene Research Articles

A human herpesvirus 6A-encoded microRNA: role in viral lytic replication.

Human herpesvirus 6A (HHV-6A), a member of the betaherpesvirus family, is associated with several human diseases. Like all herpesviruses, HHV-6A establishes a lifelong, latent infection in its host. Reactivation of HHV-6A is frequent within the immunosuppressed and immunocompromised populations and results in lytic viral replication within multiple organs, often leading to severe disease. MicroRNAs (miRNAs) are key regulators of multiple cellular processes that regulate the translation of specific transcripts. miRNAs carried by herpesviruses play important roles in modulating the host cell, thereby facilitating a suitable environment for productive viral infection and/or latency. Currently, there are approximately 150 known human herpesvirus-encoded miRNAs, although an miRNA(s) encoded by HHV-6A has yet to be reported. We hypothesized that HHV-6A, like other members of the human herpesvirus family, encodes miRNAs, which function to promote viral infection. We utilized deep sequencing of small RNA species isolated from cells harboring HHV-6A to identify five novel small noncoding RNA species that originate from the viral genome, one of which has the characteristics of a viral miRNA. These RNAs are expressed during productive infection by either bacterial artificial chromosome (BAC)-derived virus in Jjhan cells or wild-type HHV-6A strain U1102 virus in HSB2 cells and are associated with the RNA induced silencing complex (RISC) machinery. Growth analyses of mutant viruses that lack each individual miRNA revealed that a viral miRNA candidate (miR-U86) targets the HHV-6A IE gene U86, thereby regulating lytic replication. The identification and biological characterization of this HHV-6A-specific miRNA is the first step to defining how the virus regulates its life cycle. A majority of the human population is infected with human herpesvirus 6A (HHV-6A), a betaherpesvirus family member. Infections usually occur in young children, and upon resolution, the virus remains in a latent state within the host. Importantly, during times of weakened immune responses, the virus can reactivate and is correlated with significant disease states. Viruses encode many different types of factors that both undermine the host antiviral response and regulate viral replication, including small RNA species called microRNAs (miRNAs). Here we report that HHV-6A encodes at least one miRNA, which we named miR-U86. We have characterized the requirement of this viral miRNA and its impact on the viral life cycle and found that it functions to regulate a viral protein important for efficient viral replication. Our data suggest that viral miRNAs are important for HHV-6A and that they may serve as an important therapeutic target to inhibit the virus.

Domain 2 of a Kazal serine proteinase inhibitor SPIPm2 from Penaeus monodon possesses antiviral activity against WSSV

A 5-domain Kazal type serine proteinase inhibitor SPIPm2 from Penaeus monodon is involved in innate immune defense against white spot syndrome virus (WSSV). To test which domains were involved, the 5 domains of SPIPm2 were over-expressed and tested against WSSV infection. By using hemocyte primary cell culture treated with each recombinant SPIPm2 domain along with WSSV, the expression of WSSV early genes ie1, WSV477 and late gene VP28 were substantially reduced as compared to other domains when the recombinant domain 2, rSPIPm2D2, was used. Injecting the WSSV along with rSPIPm2D2 but not with other domains caused delay in mortality rate of the infected shrimp. The results indicate that the SPIPm2D2 possesses strong antiviral activity and, hence, contributes predominantly to the antiviral activity of SPIPm2.

Interferon gamma-mediated BoHV-4 replication restriction in bovine endometrial stromal cells is host IDO1 gene expression independent and BoHV-4 IE2 gene expression dependent.

In the present work the interaction between bovine herpesvirus 4 (BoHV-4)-infected bovine endometrial stromal cells (BESCs) and interferon gamma (IFNG) was investigated. Starting from the particular tropism of BoHV-4 toward BESCs, a pure population of these cells, free of CD45-positive cells, was prepared and proven to have a bona fide mesenchymal derivation as shown by vimentin-positive and cytokeratin-negative staining. BESCs expressed functional IFNG receptors (IFNGR) 1 and 2 but not IFNG ligand. BESCs transfected with a new reporter construct made by cloning the bovine indoleamine 2, 3-dioxygenase 1 (IDO1) promoter in front of the luciferase reporter gene responded to exogenous IFNG treatment. Further, IFNG-treated or constitutively secreting IFNG BESCs strongly restricted BoHV-4 replication and consequent cytopathic effect. IDO1 expression in BESCs was tightly induced by IFNG and IDO1 was previously shown to be the mediator for some of the IFNG pathogenostatic effects. However, IDO1 inhibitors and IDO1 constitutive expression could not respectively abrogate or recapitulate IFNG effect on BoHV-4-infected BESCs, whereas BoHV-4 immediate early (IE2) gene expression was transcriptionally depressed by IFNG axis activation independently from IDO1 expression; this was further confirmed by revealing a BoHV-4 IE2 gene promoter area containing potential responsive elements interacting with inhibitory transcription factors induced by IFNG in BESCs. The data achieved in this work highlight at least two issues: first, the role of BESCs as target/effector cells for the IFNG; second, the importance of uterine IFNG integrity to control BoHV-4 infection recrudescence from a persistent/latent state to a chronic disease, endometritis.

Insect cell surface expression of hemagglutinin (HA) of Egyptian H5N1 avian influenza virus under transcriptional control of whispovirus immediate early-1 promoter.

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

Type I and type II crustins from Penaeus monodon, genetic variation and antimicrobial activity of the most abundant crustinPm4

An antimicrobial protein, crustin, is involved in the innate immunity of crustacean by defending the host directly against the microbial pathogens. By data mining the Penaeus monodon EST database, two type I crustins, carcininPm1 and 2, and ten type II crustins, crustinPm1–10, were identified. The abundant crustins were crustinPm1, 4 and 7, each with variation in the length of Gly-rich repeat among its members. A few crustinPm1, 4 and 7 with deletion in the Cys-rich region were also observed. Furthermore, the crustinPm4 with the longest N-terminal Gly-rich region was characterized. The crustinPm4 allelic genes were expressed mainly from the hemocytes. Its expression was up-regulated readily by WSSV infection and gradually decreased to normal level afterwards. The recombinant crustinPm4-1 (rcrustinPm4-1) isoform was produced using the Escherichia coli expression system and tested for its antimicrobial activity. The rcrustinPm4-1 was able to inhibit the growth of a Gram-positive bacterium, Bacillus megaterium but not Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus. It also inhibited the growth of two Gram-negative bacteria, E. coli 363 and Vibrio harveyi 639 at lower potency. The rcrustinPm4-1 affected the WSSV infection because the expression of an intermediate early gene ie1 in WSSV-infected hemocyte cell culture was reduced. It was shown further that the rcrustinPm4-1 could delay by about one and a half days the manifestation of disease by WSSV.

Cloning and characterization of cytoplasmic dynein intermediate chain in Fenneropenaeus chinensis and its essential role in white spot syndrome virus infection

To investigate the role of cytoplasmic dynein in white spot syndrome virus (WSSV) infection, the full-length cDNA of cytoplasmic dynein intermediate chain (FcDYNCI) was cloned in Fenneropenaeus chinensis, which consists of 2582 bp and encodes a polypeptide of 660 amino acids. Sequence analysis and multiple sequence alignment displayed that FcDYNCI was a member of cytoplasmic dynein 1 family. The FcDYNCI mRNA was most highly expressed in hemocytes, which was significantly up-regulated post WSSV infection. At 12 h post infection (hpi), confocal microscopic observation showed that WSSV could be co-localized with cytoplasmic dynein in hemocytes. After silencing by specific FcDYNCI dsRNA, the FcDYNCI mRNA level and the protein amount of FcDYNCI in hemocytes both exhibited a significant reduction, and the expression levels of three WSSV genes ie1, wsv477 and vp28 all exhibited the greatest decreases at 24 hpi. These results suggested that cytoplasmic dynein was involved in WSSV infection.

Latent infection of human cytomegalovirus is associated with the development of gastric cancer.

The worldwide contagion, human cytomegalovirus (HCMV), may cause a series of disorders in infected individuals. The aim of the present study was to investigate whether HCMV infection is associated with the development of gastric cancer. In this study, the positive expression of unique long (UL)133-UL138 and immediate-early (IE)1 genes, which are associated with viral latency and replication, respectively, were detected using nested polymerase chain reaction. A χ2 test and logistic regression analysis were performed to further investigate the preliminary data. The data indicated that the positive rate of UL133, UL135 and UL136 expression in cancer tissues was higher than that in paired normal tissues (P=0.01, 0.027 and 0.013, respectively). However, no significant differences were identified in the UL133–138 locus and IE1 gene when associated with clinicopathological features. Furthermore, seven infection patterns were identified, with the UL133 + UL138 infection pattern representing the largest proportion in the cancer (60.34%) and normal tissues (42.11%). In conclusion, it is possible that the UL133–UL138 locus is important in the occurrence of gastric cancer. The mechanism by which UL133–UL138 locus expression differs in human gastric cancer requires further investigation.

Universal neonatal cytomegalovirus screening using saliva – Report of clinical experience

ObjectivesTo analyze the results of a neonatal universal screen for congenital cytomegalovirus (CMV) using saliva real-time polymerase chain reaction (rt-PCR). Study designDuring one year (15/5/2011–15/5/2012), saliva was collected from 9845 infants (97% of 10,137 newborns). Viral DNA was extracted by Magna-Pure LC (Roche) and was tested for the presence of CMV IE and gB genes. Urine culture was collected from positive infants for confirmation. For all infants with congenital CMV maternal data were collected and head ultrasound, blood count, liver enzymes, retinal examination and auditory brainstem response testing were performed. Parents were notified in advance and had the option to avoid screening. The ethical committee approved retrospective analysis of the data. ResultsFifty six infants (0.57%) had a positive saliva assay. Of these, 47 were confirmed by urine rt-PCR and culture, in another one maternal sero-conversion was documented during pregnancy (48 infants). Twenty-eight mothers (28/47, 60%) had primary infection during pregnancy, 14 (30%) had non-primary infection, and no serological data were obtained from five (10%). Four infants (8.5%), two with prenatal diagnosis of CMV and normal fetal brain imaging and two born to mothers sero-positive before pregnancy, exhibited symptoms related to CMV and were offered antivirals. Hearing impairment was diagnosed in two infants (late onset HI in one case). ConclusionsSaliva rt-PCR assay is a feasible and effective means of universal neonatal CMV screening that can detect affected infants who might benefit from treatment and follow-up. The long-term clinical significance of screening and its cost effectiveness are yet to be determined.

The p35 and ie1 of Autographa californica multiple nucleopolyhedrovirus could rescue late gene expression of Plutella xylostella granulovirus in nonpermissive cell lines

There are no stable permissive cell lines available for in vitro replication of PlxyGV. In this study, several PlxyGV bacmids containing egfp and plasmids expressing the luciferase gene (luc) were constructed and used to transfect insect cell lines from Plutella xylostella, Trichoplusia ni (Hi5), and Spodoptera frugiperda. Fluorescence was observed only in the cells transfected with a bacmid with egfp driven by a PlxyGV ie1 promoter, but not by a PlxyGV vp39 or granulin promoter. In transient assays, various levels of LUC activity were detected in the cells transfected with individual reporter plasmids containing the luc driven by the promoters of PlxyGV early genes ie1, exon0, dnapol, lef1, lef9, and orf105, suggesting that the PlxyGV early genes could be activated in the cells independent of virus infection. The addition of a PlxyGV bacmid in the transfections activated luc expression from the promoters of PlxyGV late genes vp39 and granulin only at minimum levels, and caused significant reduction in luc expression from the early promoters, may be due to apoptosis triggered by the PlxyGV bacmid. PlxyGV reporter bacmids containing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes p35 or p35 and ie1 or p35, ie1 and gp64 expressed LUC from a PlxyGV vp39 promoter at levels of 2.6, 8.3, and 23 times higher than those produced by the basic PlxyGV reporter bacmid, respectively, in transfected Hi5 cells. Green fluorescence was present in the cultures of all three cell lines transfected by a PlxyGV bacmid containing egfp with a vp39 promoter and AcMNPV ie1, p35, and gp64 with their native promoters. The fluorescence was also observed in the culture of Hi5 cells inoculated with the supernatant from the transfection. These results suggest that AcMNPV p35 could rescue late gene expression, and the ie1, p35, and gp64 may cooperatively rescue replication of PlxyGV in the cells.

Resistance of transgenic silkworm to BmNPV could be improved by silencing ie-1 and lef-1 genes

RNA interference (RNAi)-mediated viral inhibition has been used in several organisms for improving viral resistance. In the present study, we reported the use of transgenic RNAi in preventing Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in the transgenic silkworm B. mori. We targeted the BmNPV immediate-early-1 (ie-1) and late expression factor-1 (lef-1) genes in the transiently transfected BmN cells, in the stable transformed BmN cell line and in the transgenic silkworms. We generated four piggyBac-based vectors containing short double-stranded ie-1 RNA (sdsie-1), short double-stranded lef-1 RNA (sdslef-1), long double-stranded ie-1 RNA (ldsie-1) and both sdsie-1 and sdslef-1 (sds-ie1-lef1) expression cassettes. Strong viral repression was observed in the transiently transfected cells and in the stable transformed BmN cells transfected with sds-ie-1, sdslef-1, ldsie-1 or sds-ie-lef. The decrease of ie-1 mRNA level in the sds-ie1-lef1 transiently transfected cells was most obvious among the cells transfected with different vectors. The inhibitory effect of viral multiplication was decreased in a viral dose-dependent manner; the infection ratio of transfected cells for sds-ie-1, sdslef-1, ldsie-1 and sds-ie-lef decreased by 18.83%, 13.73%, 6.93% and 30.63%, respectively, compared with control cells 5 days after infection. We generated transgenic silkworms using transgenic vector piggyantiIE-lef1-neo with sds-ie1-lef1 expression cassette; the fourth instar larvae of transgenic silkworms of generation G5 exhibited stronger resistance to BmNPV, the mortalities for the transgenic silkworms and control silkworms were 60% and 100%, respectively, at 11 days after inoculation with BmNPV (10(6) occlusion bodies per ml). These results suggest that double-stranded RNA expression of essential genes of BmNPV is a feasible method for breeding silkworms with a high antiviral capacity.

An Indole Alkaloid from a Tribal Folklore Inhibits Immediate Early Event in HSV-2 Infected Cells with Therapeutic Efficacy in Vaginally Infected Mice

Herpes genitalis, caused by HSV-2, is an incurable genital ulcerative disease transmitted by sexual intercourse. The virus establishes life-long latency in sacral root ganglia and reported to have synergistic relationship with HIV-1 transmission. Till date no effective vaccine is available, while the existing therapy frequently yielded drug resistance, toxicity and treatment failure. Thus, there is a pressing need for non-nucleotide antiviral agent from traditional source. Based on ethnomedicinal use we have isolated a compound 7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole (HM) from the traditional herb Ophiorrhiza nicobarica Balkr, and evaluated its efficacy on isolates of HSV-2 in vitro and in vivo. The cytotoxicity (CC50), effective concentrations (EC50) and the mode of action of HM was determined by MTT, plaque reduction, time-of-addition, immunofluorescence (IFA), Western blot, qRT-PCR, EMSA, supershift and co-immunoprecipitation assays; while the in vivo toxicity and efficacy was evaluated in BALB/c mice. The results revealed that HM possesses significant anti-HSV-2 activity with EC50 of 1.1-2.8 µg/ml, and selectivity index of >20. The time kinetics and IFA demonstrated that HM dose dependently inhibited 50-99% of HSV-2 infection at 1.5-5.0 µg/ml at 2-4 h post-infection. Further, HM was unable to inhibit viral attachment or penetration and had no synergistic interaction with acyclovir. Moreover, Western blot and qRT-PCR assays demonstrated that HM suppressed viral IE gene expression, while the EMSA and co-immunoprecipitation studies showed that HM interfered with the recruitment of LSD-1 by HCF-1. The in vivo studies revealed that HM at its virucidal concentration was nontoxic and reduced virus yield in the brain of HSV-2 infected mice in a concentration dependent manner, compared to vaginal tissues. Thus, our results suggest that HM can serve as a prototype to develop non-nucleotide antiviral lead targeting the viral IE transcription for the management of HSV-2 infections.

Effects of Early or Overexpression of the Autographa californica Multiple Nucleopolyhedrovirus orf94 (ODV-e25) on Virus Replication

odv-e25(e25) is one of the core genes of baculoviruses. To investigate how it functions in the replication cycle of a baculovirus, a number of Autographa californica multiple nucleopolyhedrovirus recombinants with e25 under control of the promoter of immediate early gene ie1, or the promoter of the very late hyperexpressed gene p10, were constructed using a bacmid system, and the effects of early expression or overexpression of e25 on replication of the virus were evaluated. Microscopy and titration assays demonstrated that bacmids with e25 under control of ie1 promoter were unable to produce budded viruses; and that the recombinant viruses with e25 under control of p10 promoter generated budded virus normally, but formation of occlusion bodies were dramatically reduced and delayed in the infected cells. Electron microscopy showed that there were no mature virions or intact nucleocapsids present in the cells transfected with a recombinant bacmid with e25 under control of ie1 promoter. Quantitative real-time PCR analysis demonstrated that alteration of the e25 promoter did not affect viral DNA synthesis. The reporter gene expression from the promoter of the major capsid protein gene vp39 was reduced 63% by early expression of e25. Confocal microscopy revealed that E25 was predominantly localized in nuclei by 24 hours post infection with wild-type virus, but it remained in the cytoplasm in the cells transfected with a recombinant bacmid with e25 under control of the ie1 promoter, suggesting that the transport of E25 into nuclei was regulated in a specific and strict time dependent manner.

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells

BackgroundThe MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood.MethodsWe constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G0-arrested cells.ResultsMCMV replicated to higher titers in G0-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G0-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G0 by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells.ConclusionBy means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells.

Regulation of Human Cytomegalovirus Transcription in Latency: Beyond the Major Immediate-Early Promoter

Lytic infection of differentiated cell types with human cytomegalovirus (HCMV) results in the temporal expression of between 170–200 open reading frames (ORFs). A number of studies have demonstrated the temporal regulation of these ORFs and that this is orchestrated by both viral and cellular mechanisms associated with the co-ordinated recruitment of transcription complexes and, more recently, higher order chromatin structure. Importantly, HCMV, like all herpes viruses, establishes a lifelong latent infection of the host—one major site of latency being the undifferentiated haematopoietic progenitor cells resident in the bone marrow. Crucially, the establishment of latency is concomitant with the recruitment of cellular enzymes that promote extensive methylation of histones bound to the major immediate early promoter. As such, the repressive chromatin structure formed at the major immediate early promoter (MIEP) elicits inhibition of IE gene expression and is a major factor involved in maintenance of HCMV latency. However, it is becoming increasingly clear that a distinct subset of viral genes is also expressed during latency. In this review, we will discuss the mechanisms that control the expression of these latency-associated transcripts and illustrate that regulation of these latency-associated promoters is also subject to chromatin mediated regulation and that the instructive observations previously reported regarding the negative regulation of the MIEP during latency are paralleled in the regulation of latent gene expression.

Human Cytomegalovirus pUL97 Regulates the Viral Major Immediate Early Promoter by Phosphorylation-Mediated Disruption of Histone Deacetylase 1 Binding

Human cytomegalovirus (HCMV) is a common agent of congenital infection and causes severe disease in immunocompromised patients. Current approved therapies focus on inhibiting viral DNA replication. The HCMV kinase pUL97 contributes to multiple stages of viral infection including DNA replication, controlling the cell cycle, and virion maturation. Our studies demonstrate that pUL97 also functions by influencing immediate early (IE) gene expression during the initial stages of infection. Inhibition of kinase activity using the antiviral compound maribavir or deletion of the UL97 gene resulted in decreased expression of viral immediate early genes during infection. Expression of pUL97 was sufficient to transactivate IE1 gene expression from the viral genome, which was dependent on viral kinase activity. We observed that pUL97 associates with histone deacetylase 1 (HDAC1). HDAC1 is a transcriptional corepressor that acts to silence expression of viral genes. We observed that inhibition or deletion of pUL97 kinase resulted in increased HDAC1 and decreased histone H3 lysine 9 acetylation associating with the viral major immediate early (MIE) promoter. IE expression during pUL97 inhibition or deletion was rescued following inhibition of deacetylase activity. HDAC1 associates with chromatin by protein-protein interactions. Expression of active but not inactive pUL97 kinase decreased HDAC1 interaction with the transcriptional repressor protein DAXX. Finally, using mass spectrometry, we found that HDAC1 is uniquely phosphorylated upon expression of pUL97. Our results support the conclusion that HCMV pUL97 kinase regulates viral immediate early gene expression by phosphorylation-mediated disruption of HDAC1 binding to the MIE promoter.

Characteristics and functional roles of UL14 protein of herpesviruses

The UL14 gene of herpesvirus belongs to the long UL region, which is a late expression gene. The protein encoded by the UL14 gene is a minor component of the virion tegument and plays a major role in virus replication by enhancing effects on the envelope and capsid transport to the nucleus. It is also involved in virus maturation, neurovirulence, antiapoptotic activity and regulation of the expression of IE gene. The subcellular localization of the UL14 protein distributed differently at different times. In this review, the characters of the UL14 gene and the characteristics and functions of UL14 protein are described.

VP16‐HCF interaction is required for herpes simplex virus exit from latency in neurons in vivo

A mechanistic understanding of viral and host functions regulating herpes simplex virus (HSV) latency and reactivation remains a major goal. We have shown that the interaction of VP16 and Oct‐1 is required very early during entry into the lytic cycle from the latent state and have proposed a VP16‐dependent model of reactivation in vivo. HCF‐1 has important roles in promoting HSV replication and a model has been proposed in which stress induces translocation of HCF‐1 into the neuronal nucleus and reactivation is regulated by direct recruitment of HCF to viral IE genes. To investigate whether HCF‐1 can precipitate viral gene expression in latently infected neurons in the absence of binding to VP16, a mutation in VP16 at Y364A, shown previously to disrupt VP16‐ HCF interaction (but not interaction with Oct‐1) was engineered into HSV‐1. Three independent isolates and a genomically restored virus were examined in vitro and in the mouse ocular model. Our findings demonstrate that the interaction of VP16 with HCF‐1 is required for efficient infection at low moi, efficient viral replication in vivo, and exit from latency/entry into the lytic cycle in trigeminal ganglion neurons in mice following hyperthermic stress. In the absence of VP16 binding, nuclear translocation of HCF is not sufficient to initiate the viral lytic cycle, demonstrating the key role of VP16 in regulating in vivo reactivation.

Allitridin inhibits human cytomegalovirus replication in vitro

Human cytomegalovirus (HCMV) has been associated with a wide spectrum of diseases. There is currently no effective treatment for eliminating the virus. Garlic bulb extract has been reported to possess anti-viral efficacy. This study aimed to investigate the expression of the immediate‑early (IE; ul122 and ul123), early (E; ul54) and late (L; ul83) genes of HCMV as well as the inhibitory effect of allitridin on the transcription levels of these genes. The results indicated that a HCMV gene expression cascade occurred, and that the deletion of IE72 had no influence on the transcription of the ul122 gene, while it led to significant reductions of ul54 and ul83 mRNA expression levels. Additionally, allitridin effectively suppressed the transcription of the HCMV IE, E and L genes; the inhibition rates of the transcription of the ul122 and ul123 genes were higher compared with those of ul54 and ul83 mRNA expression, while the expression of the IE genes was not significantly reduced by ganciclovir (GCV). Our results indicate that the HCMV IE72 deletion mutant strain affects the transcription of the virus downstream gene, allitridin inhibits HCMV infection in vitro, and that the IE genes may be the key target of allitridin in its action against HCMV.

The ICP22 protein selectively modifies the transcription of different kinetic classes of pseudorabies virus genes

BackgroundPseudorabies virus (PRV), an alpha-herpesvirus of swine, is a widely used model organism in investigations of the molecular pathomechanisms of the herpesviruses. This work is the continuation of our earlier studies, in which we investigated the effect of the abrogation of gene function on the viral transcriptome by knocking out PRV genes playing roles in the coordination of global gene expression of the virus. In this study, we deleted the us1 gene encoding the ICP22, an important viral regulatory protein, and analyzed the changes in the expression of other PRV genes.ResultsA multi-timepoint real-time RT-PCR technique was applied to evaluate the impact of deletion of the PRV us1 gene on the overall transcription kinetics of viral genes. The mutation proved to exert a differential effect on the distinct kinetic classes of PRV genes at the various stages of lytic infection. In the us1 gene-deleted virus, all the kinetic classes of the genes were significantly down-regulated in the first hour of infection. After 2 to 6 h of infection, the late genes were severely suppressed, whereas the early genes were unaffected. In the late stage of infection, the early genes were selectively up-regulated. In the mutant virus, the transcription of the ie180 gene, the major coordinator of PRV gene expression, correlated closely with the transcription of other viral genes, a situation which was not found in the wild-type (wt) virus. A 4-h delay was observed in the commencement of DNA replication in the mutant virus as compared with the wt virus. The rate of transcription from a gene normalized to the relative copy number of the viral genome was observed to decline drastically following the initiation of DNA replication in both the wt and mutant backgrounds. Finally, the switch between the expressions of the early and late genes was demonstrated not to be controlled by DNA replication, as is widely believed, since the switch preceded the DNA replication.ConclusionsOur results show a strong dependence of PRV gene expression on the presence of functional us1 gene. ICP22 is shown to exert a differential effect on the distinct kinetic classes of PRV genes and to disrupt the close correlation between the transcription kinetics of ie180 and other PRV transcripts. Furthermore, DNA replication exerts a severe constraint on the viral transcription.

Dimerization and proper degradation of BmNPV IE2 are required for efficient virus growth in larvae of Bombyx mori (Lepidoptera: Bombycidae)

The baculovirus ie2 gene is one of the immediate early genes, and its product is known to transactivate viral promoters. However, the roles of Bombyx mori nucleopolyhedrovirus (BmNPV) ie2 in insect larvae are poorly understood. Here we investigated the functions of BmNPV IE2 in cultured cells and in insect larvae using two mutant viruses, BmIE2D and BmIE2CS. BmIE2D lacks the IE2 C-terminal coiled-coil domain that is required for IE2 dimerization. The other mutant BmIE2CS expresses an E3 ligase activity-deficient IE2 derivative, which is degraded more slowly compared with wild-type IE2. We found that ie2 mutations had little effect on BmNPV infection in cultured cells, whereas budded virus and occlusion body production was significantly reduced in the hemolymph of B. mori larvae infected with ie2 mutants. These results indicate that both dimerization and proper degradation of BmNPV IE2 are crucial steps for efficient virus growth in B. mori larvae, but not in cultured cells. Oral infection assays also revealed that the infectivity of the occluded form of ie2 mutants was normal in B. mori larvae, which is inconsistent with the results reported from ie2 mutants of Autographa californica NPV. This suggests that loss of IE2 function causes virus-specific effects in host insects.