Immediate-early Research Articles

Tightly regulated expression of Autographa californica multicapsid nucleopolyhedrovirus immediate early genes emerges from their interactions and possible collective behaviors.

To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network.

Viperin is induced following toll-like receptor 3 (TLR3) ligation and has a virus-responsive function in human trophoblast cells

IntroductionViperin, a virus-inducible antiviral protein, has been reported to inhibit the replication of a variety of viruses. However, its expression and function in trophoblast cells remains unclear. Toll-like receptor 3 (TLR3) is a key component of the innate immune system that recognizes viral double-stranded RNA and triggers immune reactions by producing type I interferon. We hypothesized that viperin inhibits the replication of human cytomegalovirus (HCMV) in trophoblast cells. MethodsIn situ examinations of viperin expression was conducted in the human first-trimester placenta by immunohistochemical staining. Using a human trophoblast cell culture system, we studied the effect of TLR-3 ligation on viperin expression by treating trophoblasts with polyinosinic-polycytidylic acid [Poly (I: C)] (a synthetic double-stranded RNA, which mimics viral RNA). Viperin mRNA and protein expression were evaluated by real-time PCR and Western blot analysis. In a HCMV infected Swan 71 cell model, HCMV immediate early 1 (IE1) protein mRNA expression was evaluated by real-time PCR after viperin RNA interference. ResultsViperin was localized in trophoblast cells. Poly (I: C) induced viperin expression in a dosage and time-dependent manner. Blocking of TLR3 signaling by neutralizing antibody against IFN-β abolished the stimulation of viperin expression. After HCMV infection, expression of viperin mRNA and protein was unregulated. HCMV IE1 mRNA expression was significantly inhibited by viperin RNA interference. DiscussionViperin is a virus-responsive protein that is constitutively expressed in human trophoblast cells. However, contrary to our hypothesis, viperin facilitates HCMV replication post infection.

Genome wide nucleosome mapping for HSV-1 shows nucleosomes are deposited at preferred positions during lytic infection.

HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes.

Expression of recipient cytomegalovirus in immunosuppressed and xenotransplanted Macaca fascicularis may be related to more severe gastrointestinal lesions.

Xenotransplantation is a potential answer to the current organ shortage, but the risk of infections related to overimmunosuppression is an important parameter that may predict the recipient's long-term survival. Cytomegalovirus (CMV) in xenotransplanted and immunosuppressed primates is a well-known cause of disease particularly affecting the gastrointestinal (GI) tract and a zoonotic concern. Post-mortem sera and tissues from 45 immunosuppressed and xenografted Macaca fascicularis were evaluated for CMV using antisera specific for the immediate early 1 (IE1), anti-RhCMV, and QPCR for virus. Serological analysis showed 100% positivity for the presence of CMV antibodies following the application of a specific test designed for RhCMV. Five of 45 primates showed typical lesions of CMV infection in the GI tract, including neutrophilic enteritis and inclusion bodies. Molecular analysis confirmed the presence of recipient's CMV in the tissues with CMV histopathology. Porcine CMV from the donor animals was not found in any of the CMV-specific IHC-positive recipients. The presence of active CMV infection in animals intended for xenograft experiments can lead to severe gastrointestinal lesions that could impact the overall aims of the study. In such cases, the animals should be investigated using appropriate (non-human primate-specific) diagnostic tools prior to use and treated aggressively with state-of-the-art antiviral therapy.

UL84-independent replication of human cytomegalovirus strains conferred by a single codon change in UL122

The UL84 gene of human cytomegalovirus (HCMV) is thought to be involved in the initiation of viral DNA replication, and is essential for replication of strains AD169 and Towne. Hence, discovery that strain TB40-BAC4 is viable in the absence of UL84 presented an enigma requiring an explanation. Data reported here show that strain TR also tolerated loss of UL84, whereas strains FIX, Merlin, Ph, and Toledo did not. UL84-independent growth required the viral replication origin. The genetic locus in TB40 that controls UL84 dependence was mapped to codon 388 of the UL122 gene, which encodes the immediate early 2 (IE2) 86kD protein. Introduction of this TB40-BAC4 variant (H388D) into FIX and Toledo clones converted these strains to UL84 independence. These results provide genetic evidence in virus-infected cells that supports the hypothesis that UL122 participates in the initiation of viral DNA replication by a mechanism involving transcription-mediated activation of the origin.

Investigation the role of human cytomegalovirus in the invasive ductal breast carcinoma

Background: Breast cancer is the most common type of female malignancy. Increasing evidence in the last 10 years suggests that human cytomegalovirus (HCMV) is associated with several human malignancies including breast cancer. This study aimed to investigate HCMV in invasive ductal breast carcinoma by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). Materials and Methods: A total of 50 samples of the cancer mass and non-neoplastic safe margin (SM) tissues of breast cancer collected then processed for paraffin block to apply IHC and hematoxylin-eosin staining. In addition to that, 30 blood samples collected from patients and healthy women (controls) for detection of anticytomegalovirus (antiCMV) IgG and IgM by ELISA. Results: About 38 samples (76%) of 50 samples diagnosed as invasive ductal carcinoma (IDC). The results showed that the presence of antiCMV antibody IgG in 100% of patients while the IgM presented in 76.7% of patients. There were significant differences (P < 0.05) between the optical densities of the IgG in breast cancer patients when compared healthy women. The positive results of CMV protein comprise 34 (89.4%) for immediate early 1 (IE1) protein, 35 (92.1%) for late protein, and 34 (89.4%) for phosphoprotein 65 (pp65) from 38 sample of IDC. The results also showed the absence of expression to CMV late and pp65 proteins and low percentage (10%) of IE1 protein in the SM tissues. Conclusion: Many studies including our observation indicated to the association of HCMV with breast cancer, but the role of HCMV in the pathogenesis of breast cancer is unclear.

Poly(A) binding protein 1 enhances cap-independent translation initiation of neurovirulence factor from avian herpesvirus.

Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek’s disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of ‘closed loop’ structure of mRNA.

Immediate-Early (IE) gene regulation of cytomegalovirus: IE1- and pp71-mediated viral strategies against cellular defenses.

Three crucial hurdles hinder studies on human cytomegalovirus (HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus-host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host (latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate-early (IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate-early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses.

Abstract 5112: Histone deacetylase and proteasome inhibitors synergistically induce apoptosis in colon cancer, multiple myeloma and CTCL cells through induction of the immediate early genes ATF3 and JUN

Abstract Background/Aim: Histone deacetylase inhibitors (HDACi) are approved for the treatment of cutaneous T-cell lymphoma. We have previously demonstrated that HDACi-mediated apoptosis is linked to the induction of a defined transcriptional response involving up-regulation of the immediate-early (IE) genes FOS, JUN, ATF3, EGR1 and EGR3, in cell lines derived from multiple tumour types. To identify other drugs which induce a similar transcriptional response we compared the HDACi-induced transcriptional response with that induced by &amp;gt;1000 bioactive molecules using the Broad Institute's Connectivity Map database. The proteasome inhibitor, MG-262, was identified as inducing the most comparable transcriptional response to HDACi. Notably, combination treatment with HDAC and proteasome inhibitors has been shown to synergistically induce apoptosis in vitro, and a phase III trial of this combination demonstrated activity in patients multiple myeloma. The aim of this study was to determine whether these effects are mediated through the additive induction of IE genes. Methods: The effect of the HDACi SAHA and the clinically approved proteasome inhibitor Bortezomib, alone, and in combination, on apoptosis induction was determined in colorectal, multiple myeloma and CTCL cell lines PI staining and FACS analysis. Quantitative RT-PCR was performed to determine gene expression changes upon drug treatment. MAPK pathway activation and histone hyper-acetylation was determined by western blot analysis. Results: Single agent treatment of multiple cancer cells with SAHA or Bortezomib induced expression of the IE genes FOS, JUN and ATF3. In both cases IE gene induction was sustained over 24h. Proteasome inhibitor-induced IE gene expression and apoptosis was selectively dependent on activation of the JNK and p38 stress response pathways, while HDACi-mediated effects were selectively dependent upon the Sp1 and Sp3 transcription factors, indicating these agents induce IE gene expression and apoptosis via independent mechanisms. Co-treatment with Bortezomib and SAHA resulted in synergistic induction of IE gene expression and apoptosis in cell lines derived from multiple tumour types. Importantly, apoptosis induction by the combination was partially inhibited by siRNA-mediated downregulation of two IE genes, ATF3 and JUN. Conclusions: This study provides insight into the mechanistic basis by which combination treatment with HDAC and proteasome inhibitors synergistically induces apoptosis in tumour cells. Citation Format: Janson WT Tse, Anderly C. Chueh, Ian Y. Luk, Fiona Chionh, Yvonne Yeung, Georgia A. Corner, Dominic CH Ng, Hoanh Tran, Amardeep S. Dhillon, John M. Mariadason. Histone deacetylase and proteasome inhibitors synergistically induce apoptosis in colon cancer, multiple myeloma and CTCL cells through induction of the immediate early genes ATF3 and JUN. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2014-5112

Quantitative Proteomic Discovery of Dynamic Epigenome Changes that Control Human Cytomegalovirus (HCMV) Infection

This work represents the first comprehensive quantitative analysis of global histone post-translational modifications (PTMs) from a virus infection, namely human cytomegalovirus (HCMV) infection. We used a nanoLC-MS/MS platform to identify and quantify the dynamic histone H3 and H4 PTMs expressed during HCMV replication in primary fibroblasts. Specifically, we examined the changes in histone PTMs over a 96 h time course to sample the immediate early (IE), early (E), and late (L) stages of viral infection. Several changes in histone H3 and H4 PTMs were observed, including a marked increase in H3K79me2 and H3K27me3K36me2, and a decrease in H4K16ac, highlighting likely epigenetic strategies of transcriptional activation and silencing during HCMV lytic infection. Heavy methyl-SILAC (hm-SILAC) was used to further confirm the histone methylation flux (especially for H3K79) during HCMV infection. We evaluated DOT1L (the H3K79 methyltransferase) mRNA levels in mock and HCMV-infected cells over a 96 h time course, and observed a significant increase in this methyltransferase as early as 24 hpi showing that viral infection up-regulates DOT1L expression, which drives H3K79me2. We then used shRNA to create a DOT1L knockdown cell population, and found that HCMV infection of the knockdown cells resulted in a 10-fold growth defect when compared with infected control cells not subjected to knockdown. This work documents multiple histone PTMs that occur in response to HCMV infection of fibroblasts, and provides a framework for evaluation of the role of epigenetic modifications in the virus-host interaction.

Epstein-Barr virus late gene transcription depends on the assembly of a virus-specific preinitiation complex.

During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique cis element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta- and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta- and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses. Control of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin, which allows assembly of the preinitiation complex (PIC) at the core promoter. Fixation of the TATA box-binding protein (TBP) is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a virus-specific PIC (vPIC).

The 6-Aminoquinolone WC5 inhibits different functions of the immediate-early 2 (IE2) protein of human cytomegalovirus that are essential for viral replication.

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional factor essential for viral replication. IE2 modulates both viral and host gene expression, deregulates cell cycle progression, acts as an immunomodulator, and antagonizes cellular antiviral responses. Based on these facts, IE2 has been proposed as an important target for the development of innovative antiviral approaches. We previously identified the 6-aminoquinolone WC5 as a promising inhibitor of HCMV replication, and here, we report the dissection of its mechanism of action against the viral IE2 protein. Using glutathione S-transferase (GST) pulldown assays, mutagenesis, cell-based assays, and electrophoretic mobility shift assays, we demonstrated that WC5 does not interfere with IE2 dimerization, its interaction with TATA-binding protein (TBP), and the expression of a set of cellular genes that are stimulated by IE2. On the contrary, WC5 targets the regulatory activity exerted by IE2 on different responsive viral promoters. Indeed, WC5 blocked the IE2-dependent negative regulation of the major immediate-early promoter by preventing IE2 binding to the crs element. Moreover, WC5 reduced the IE2-dependent transactivation of a series of indicator constructs driven by different portions of the early UL54 gene promoter, and it also inhibited the transactivation of the murine CMV early E1 promoter by the IE3 protein, the murine cytomegalovirus (MCMV) IE2 homolog. In conclusion, our results indicate that the overall anti-HCMV activity of WC5 depends on its ability to specifically interfere with the IE2-dependent regulation of viral promoters. Importantly, our results suggest that this mechanism is conserved in murine CMV, thus paving the way for further preclinical evaluation in an animal model.

Human cytomegalovirus immediate early protein 2 enhances myocardin-mediated survival of rat aortic smooth muscle cells

Human cytomegalovirus (HCMV) may increase the incidence of restenosis and predispose to atherosclerosis. The lesions of restenosis and atherosclerosis often contain smooth muscle cells (SMCs) with high rates of proliferation and apoptosis. One of the immediate early (IE) gene products of HCMV-IE2 affects transcriptional activities of some cellular factors in SMCs, including myocardin. In this study, we studied the effects of IE2 and myocardin on PI3K pathway inducer wortmannin induced apoptosis in rat aortic SMCs. We show that the transcriptional activity of myocardin on Mcl-1 promoter is enhanced by co-expression of HCMV IE2 in rat aortic SMCs; and the expressions of mRNA and protein of antiapoptotic genes-Mcl-1 and Bcl-2 are upregulated by IE2 alone and co-transfection of myocardin and IE2, but decreased by myocardin-specific shRNA in rat aortic SMCs. We further demonstrate that co-expression of myocardin and HCMV IE2 declines apoptotic cell numbers and caspase-3 activities induced by serum starvation plus wortmannin in rat aortic SMCs. The results suggest that HCMV IE2 enhances myocardin-mediated survival of rat aortic SMCs under serum deprivation and PI3-kinase inhibition, partly via activation of Mcl-1's antiapoptosis effect. Our study connects HCMV IE2 to myocardin-induced transcriptional program for rat aortic SMCs survival and proliferation, involving in HCMV related restenosis and atherosclerosis.

Cooperation between Epstein-Barr virus immune evasion proteins spreads protection from CD8+ T cell recognition across all three phases of the lytic cycle.

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE<E<<L). Moreover, double-knockdown experiments showed that BILF1 and BNLF2a co-operate to further inhibit antigen presentation of L epitopes. Together, these data firstly indicate which potential immune-evasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into the immunodominance pattern seen for CD8+ T cell responses to EBV lytic antigens.

The 19S proteasome activator promotes human cytomegalovirus immediate early gene expression through proteolytic and nonproteolytic mechanisms.

Proteasomes are large, multisubunit complexes that support normal cellular activities by executing the bulk of protein turnover. During infection, many viruses have been shown to promote viral replication by using proteasomes to degrade cellular factors that restrict viral replication. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomal degradation of Daxx, a cellular transcriptional repressor that can silence viral immediate early (IE) gene expression. We previously showed that this degradation requires both the proteasome catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP associates with the 20S CP to facilitate protein degradation but also plays a 20S CP-independent role promoting transcription. Here, we present a nonproteolytic role of the 19S RP in HCMV IE gene expression. We demonstrate that 19S RP subunits are recruited to the major immediate early promoter (MIEP) that directs IE transcription. Depletion of 19S RP subunits generated a defect in RNA polymerase II elongation through the MIE locus during HCMV infection. Our results reveal that HCMV commandeers proteasome components for both proteolytic and nonproteolytic roles to promote HCMV lytic infection. Importance: Proteasome inhibitors decrease or eliminate 20S CP activity and are garnering increasing interest as chemotherapeutics. However, an increasing body of evidence implicates 19S RP subunits in important proteolytic-independent roles during transcription. Thus, pharmacological inhibition of the 20S CP as a means to modulate proteasome function toward therapeutic effect is an incomplete capitalization on the potential of this approach. Here, we provide an additional example of nonproteolytic 19S RP function in promoting HCMV transcription. These data provide a novel system with which to study the roles of different proteasome components during transcription, a rationale for previously described shifts in 19S RP subunit localization during HCMV infection, and a potential therapeutic intervention point at a pre-immediate early stage for the inhibition of HCMV infection.

Analysis of human cytomegalovirus-encoded SUMO targets and temporal regulation of SUMOylation of the immediate-early proteins IE1 and IE2 during infection.

Post-translational modification of proteins by members of the small ubiquitin-like modifier (SUMO) is involved in diverse cellular functions. Many viral proteins are SUMO targets and also interact with the cellular SUMOylation system. During human cytomegalovirus (HCMV) infection, the immediate-early (IE) proteins IE1 and IE2 are covalently modified by SUMO. IE2 SUMOylation promotes its transactivation activity, whereas the role of IE1 SUMOylation is not clear. We performed in silico, genome-wide analysis to identify possible SUMOylation sites in HCMV-encoded proteins and evaluated their modification using the E. coli SUMOylation system and in vitro assays. We found that only IE1 and IE2 are substantially modified by SUMO in E. coli, although US34A was also identified as a possible SUMO target in vitro. We also found that SUMOylation of IE1 and IE2 is temporally regulated during viral infection. Levels of SUMO-modified form of IE1 were increased during the early phase of infection, but decreased in the late phase when IE2 and its SUMO-modified forms were expressed at high levels. IE2 expression inhibited IE1 SUMOylation in cotransfection assays. As in IE2 SUMOylation, PIAS1, a SUMO E3 ligase, interacted with IE1 and enhanced IE1 SUMOylation. In in vitro assays, an IE2 fragment that lacked covalent and non-covalent SUMO attachment sites, but was sufficient for PIAS1 binding, effectively inhibited PIAS1-mediated SUMOylation of IE1, indicating that IE2 expression negatively regulates IE1 SUMOylation. We also found that the IE2-mediated downregulation of IE1 SUMOylation correlates with the IE1 activity to repress the promoter containing the interferon stimulated response elements. Taken together, our data demonstrate that IE1 and IE2 are the main viral SUMO targets in HCMV infection and that temporal regulation of their SUMOylation may be important in the progression of this infection.

Rapamycin does not inhibit human cytomegalovirus reactivation from dendritic cells in vitro

Human cytomegalovirus (HCMV) infection and reactivation are a major cause of morbidity in immune-suppressed patients. Interestingly, epidemiological studies have shown that patients administered the mammalian target of rapamycin (mTOR) inhibitor, sirolimus (rapamycin), exhibit more favourable outcomes, suggestive of activity against HCMV in vivo. Given its relative lack of activity against lytic infection, it is postulated that rapamycin inhibits HCMV reactivation. Here, we showed that rapamycin administered acutely or chronically has little impact on induction of immediate early (IE) gene expression in experimentally latent dendritic cells or cells from naturally latent individuals. Furthermore, we extended these observations to include other inhibitors of mTORC1 and mTORC 2, which similarly have minimal effects on induction of IE gene expression from latency. Taken together, these data suggest that favourable outcomes associated with sirolimus are attributable to indirect effects that influence HCMV reactivation, rather than a direct mechanistic action against HCMV itself.

Characterization of immediate early gene ORF62 of live attenuated varicella vaccine Oka strains pro-duced in China

Objective To investigate mutations in immediate early （ IE） gene ORF62 of three varicella vaccine Oka strains （ vOka ） including two strains produced in China and their parental Oka strain （pOka）, and then to further elucidate its possible roles in attenuation mechanism by comparing their ORF 62 promoter sequences and its activities , ORF62 coding regions and its transactivities .Methods ORF62 promoter-reporter plasmids and ORF62-expressing plasmids of pOka and three vOka strains （ vOka-BK from Changchun BCHT Biotechnology Co ., vOka-SH from Shanghai Institute of Biological Product Co .Ltd., and vOka-GSK from GlaxoSmithKline plc, as control） were constructed, respectively.ORF62 promoter regions and coding regions of the four strains were sequenced and then compared with each other .Differences of activities of the ORF62 promoter, and transactivities of the ORF62-encoded IE62 upon immediate early （ORF4）, early （ORF28） and late （ORF67） gene promoters between pOka and vOka strains were assayed with transient transfection technique .Results Compared with pOka strain , three vOka strains had a consistent T deletion mutation at site 110 050 in ORF62 promoters, which did not result in any change of tran-scription factor binding motif .However , activities of ORF62 promoters from three vOka strains were significantly lower than those of pOka strain .Three consistent substitution mutations were observed in ORF 62 coding regions of three vOka strains and three new enzyme restriction sites including SmaⅠ, NaeⅠand BssHⅡwere generated, respectively.Transactivities of IE62 from three vOka strains upon ORF4, ORF28 and ORF67 promoters were significantly higher than those of pOka both in CV-1 and MeWo cells , except that vOka-SH IE62 showed significantly lower transactivities upon ORF 4 promoter than those of pOka strain in CV-1 cells.Conclusion Consistent T deletion mutation at site 110 050 in ORF62 promoters of three vOka strains might be responsible for the reduced promoter activities and the changes of IE 62 transactivities .However , it seemed that cell types have no significant effect on ORF 62 promoter activity or IE 62 transactivity between pOka and vOka strains . Key words: Varicella-zoster virus; Promoter activity; Transactivity

Normalizing ELISPOT responses to T-cell counts: A novel approach for quantification of HCMV-specific CD4+ and CD8+ T-cell responses in kidney transplant recipients

BackgroundHuman cytomegalovirus (HCMV) is the most common opportunistic virus infection in solid organ transplant recipients. The analysis of HCMV-specific T-cell immunity after organ transplant is of relevant clinical interest. ObjectivesTo analyze HCMV-specific CD4+ and CD8+ T-cell responses in healthy subjects and kidney transplant recipients (KTR). Study designHCMV-specific T-cell responses were evaluated by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phosphoprotein 65 (pp65) (for stimulation of both CD4+ and CD8+ T-cell responses) and a pool of 34 short peptides (8–12 amino acids in length, for stimulation of CD8+ T-cell responses). ELISPOT results were normalized to T-cell subset counts and their correlations with a reported dendritic cell (DC)-based assay, which simultaneously quantifies HCMV-specific CD4+ and CD8+ T-cell responses, were analyzed. ResultsHCMV-seropositive KTR showed higher ELISPOT responses compared to HCMV-seropositive healthy subjects. IE-1 and pp65 ELISPOT responses were mediated mainly by CD8+ T-cells and, to a lesser extent, CD4+ T cells; IE-2 peptides appear to stimulate CD56+ cells (natural killer cells). In HCMV-seropositive healthy subjects, ELISPOT results (expressed either as net spots/million cells or normalized to the corresponding T-cell count) significantly correlated with the DC assay. However, in HMCV-seropositive KTR, only normalized ELISPOT responses to overlapping 15-mer peptide pools significantly correlated with DC-assay responses. ConclusionsThe normalized ELISPOT represents a novel and simple approach for quantifying and monitoring HCMV-specific CD4+ and CD8+ T-cell responses in KTR.

Immediate-early protein of equid herpesvirus type 1 as a target for cytotoxic T-lymphocytes in the Thoroughbred horse.

Cytotoxic T-lymphocytes (CTLs) are associated with protective immunity against disease caused by equid herpesvirus type 1 (EHV-1). However, the EHV-1 target proteins for CTLs are poorly defined. This limits the development of vaccine candidates designed to stimulate strong CTL immunity. Here, classical CTL assays using lymphocytes from horses of three defined MHC class I types that experienced natural infection with EHV-1 and a modified vaccinia virus construct containing an EHV-1 gene encoding the immediate-early (IE) protein are reported. Horses homozygous for the equine leukocyte antigen (ELA)-A2 haplotype, but not the ELA-A5 haplotype, produced MHC-restricted CTL responses against the IE protein. Previously, horses homozygous for the ELA-A3 haplotype also mounted CTL responses against the IE protein. Both haplotypes are common in major horse breeds, including the Thoroughbred. Thus, the IE protein is an attractive candidate molecule for future studies of T-cell immunity to EHV-1 in the horse.