Immediate-early Region Research Articles

Targeting human cytomegalovirus IE genes by CRISPR/Cas9 nuclease effectively inhibits viral replication and reactivation.

Human cytomegalovirus (HCMV) infection causes high morbidity and mortality among immunocompromised patients and can remain in a latent state in host cells. Expression of the immediate-early (IE) genes sustains HCMV replication and reactivation. As a novel genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been extensively utilized to modify and edit genomic DNA. In the present study, the CRISPR/Cas9 system was used to target the IE region of the HCMV genome via specific single-guide RNAs (sgRNAs). Infection with CRISPR/Cas9/sgRNA lentiviral constructs significantly reduced viral gene expression and virion production in HFF primary fibroblasts and inhibited viral DNA production and reactivation in the THP-1 monocytic cell line. Thus, the CRISPR/Cas9/sgRNA system can accurately and efficiently target HCMV replication and reactivation and represents a novel therapeutic strategy against latent HCMV infection.

The Cellular Coactivator HCF-1 Is Required for Glucocorticoid Receptor-Mediated Transcription of Bovine Herpesvirus 1 Immediate Early Genes.

Following productive infection, bovine herpesvirus 1 (BoHV-1) establishes latency in sensory neurons. As in other alphaherpesviruses, expression of BoHV-1 immediate early (IE) genes is regulated by an enhancer complex containing the viral IE activator VP16, the cellular transcription factor Oct-1, and transcriptional coactivator HCF-1, which is assembled on an IE enhancer core element (TAATGARAT). Expression of the IE transcription unit that encodes the viral IE activators bICP0 and bICP4 may also be induced by the activated glucocorticoid receptor (GR) via two glucocorticoid response elements (GREs) located upstream of the enhancer core. Strikingly, lytic infection and reactivation from latency are consistently enhanced by glucocorticoid treatment in vivo As the coactivator HCF-1 is essential for IE gene expression of alphaherpesviruses and recruited by multiple transcription factors, we tested whether HCF-1 is required for glucocorticoid-induced IE gene expression. Depletion of HCF-1 reduced GR-mediated activation of the IE promoter in mouse neuroblastoma cells (Neuro-2A). More importantly, HCF-1-mediated GR activation of the promoter was dependent on the presence of GRE sites but independent of the TAATGARAT enhancer core element. HCF-1 was also recruited to the GRE region of a promoter lacking the enhancer core, consistent with a direct role of the coactivator in mediating GR-induced transcription. Similarly, during productive lytic infection, HCF-1 and GR occupied the IE region containing the GREs. These studies indicate HCF-1 is critical for GR activation of the viral IE genes and suggests that glucocorticoid induction of viral reactivation proceeds via an HCF-1-GR mechanism in the absence of the viral IE activator VP16.IMPORTANCE BoHV-1 transcription is rapidly activated during stress-induced reactivation from latency. The immediate early transcription unit 1 (IEtu1) promoter is regulated by the GR via two GREs. The IEtu1 promoter regulates expression of two viral transcriptional regulatory proteins, infected cell proteins 0 and 4 (bICP0 and bICP4), and thus must be stimulated during reactivation. This study demonstrates that activation of the IEtu1 promoter by the synthetic corticosteroid dexamethasone requires HCF-1. Interestingly, the GRE sites, but not the IE enhancer core element (TAATGARAT), were required for HCF-1-mediated GR promoter activation. The GR and HCF-1 were recruited to the IEtu1 promoter in transfected and infected cells. Collectively, these studies indicate that HCF-1 is critical for GR activation of the viral IE genes and suggest that an HCF-1-GR complex can stimulate the IEtu1 promoter in the absence of the viral IE activator VP16.

Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

Previously, we have reported the use of a recombinant adenovirus (Ad)-based host cell reactivation (HCR) assay to examine nucleotide excision repair (NER) of UVC-induced DNA lesions in several mammalian cell types. The recombinant non-replicating Ad expresses the Escherichia coli β-galactosidase (β-gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG's potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded β-gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass.

The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner

The Epstein-Barr virus (EBV) BRRF1 lytic gene product (Na) is encoded within the same immediate-early region as the BZLF1 (Z) and BRLF1(R) gene products, but its role during EBV infection has not been well defined. We previously showed that Na cooperates with the R protein to induce lytic gene expression in latently infected EBV-positive 293 cells, and in some EBV-negative cell lines it can activate the Z promoter in reporter gene assays. Here we show that overexpression of Na alone is sufficient to induce lytic gene expression in several different latently infected epithelial cell lines (Hone-Akata, CNE2-Akata, and AGS-Akata), while knockdown of endogenous Na expression reduces lytic gene expression. Consistent with its ability to interact with tumor necrosis factor receptor-associated factor 2 (TRAF2) in a yeast two-hybrid assay, we demonstrate that Na interacts with TRAF2 in cells. Furthermore, we show that TRAF2 is required for Na induction of lytic gene expression, that Na induces Jun N-terminal protein kinase (JNK) activation in a TRAF2-dependent manner, and that a JNK inhibitor abolishes the ability of Na to disrupt viral latency. Additionally, we show that Na and the tumor suppressor protein p53 cooperate to induce lytic gene expression in epithelial cells (including the C666-1 nasopharyngeal carcinoma cell line), although Na does not appear to affect p53 function. Together these data suggest that Na plays an important role in regulating the switch between latent and lytic infection in epithelial cells and that this effect requires both the TRAF2 and p53 cellular proteins.

Optimization of the Sleeping Beauty Transposon System to Achieve Stable Transgene Expression in Human CD34+ Hematopoietic Progenitor Cells

Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. Transposon-mediated gene integration may have advantages over viral vectors, with a greater transgene carrying capacity and potentially safer integration site profile. Due to these characteristics of SB, there has been great interest in its potential use in hematopoietic stem cell (HSC) gene therapy. In this study, we optimized the SB transposon-mediated gene transfer system to achieve higher stable transgene expression in K562 human erythroleukemia cells, Jurkat human T-lymphoid cells, and primary human CD34+ hematopoietic progenitor cells. The SB transposon system was optimized by two approaches:to increase the transposition efficacy, a hyperactive mutant of SB, HSB16, was used (Baus et al.; Mol Ther 12:1148, 2005);to optimize the expression of the SB transposase and the transgene cassette carried by the transposon, three different viral and cellular promoters were evaluated, including the modified MPSV long terminal repeat (MNDU3) enhancer-promoter, the human cytomegalovirus (hCMV) immediate-early region enhancer-promoter, and the human elongation factor 1 (hEF1a) promoter.SB components were delivered in trans into the target cells by nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression using fluorescent-activated cell sorting (FACS) analysis over 3–4 weeks. The functional assay showed that HSB16 was a more efficient enzyme compared to the original SB. In purified human cord blood CD34+ cells, HSB16 achieved nearly 7-fold higher long-term transgene expression with 90% less plasmid DNA (from 10 mcg of SB reduced to 1 mcg of HSB16) than the original SB transposase. The highest level of stable transgene integration in all three cell types was achieved using the hEF1a promoter to express HSB16 in comparison to either the hCMV or MND promoter. Our data also suggested that optimal GFP reporter gene expression from the integrated transposon was influenced by the type of promoter and the target cell type. Significantly higher levels of eGFP expression (5-fold) were achieved with the hEF1a promoter in Jurkat human T cells, compared to that achieved with the MND promoter; in contrast the MND promoter expressed GFP at the highest level in K562 myeloid cells. In primary human CD34+ cord blood progenitors, optimal transgene integration and expression was achieved using the hEF1a promoter to express the SB transposase combined with the MND promoter to express GFP reporter, when studied under conditions directing myeloid differentiation. Stable transgene expression was achieved at levels up to 27% for over 4 weeks after optimized gene transfer to CD34+ cells (ave=17%, n=4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34+ progenitor cells are currently in progress. In conclusion, the optimized SB transposon system in primary human CD34+ hematopoietic progenitors reported here has improved the stable gene transfer efficiency by 29-fold, compared to our prior published data (< 1% - Hollis et al.; Exp Hematol 34:1333, 2006). The long-term stable gene expression achieved by our optimized SB transposon system shows promise for further advancement of non-viral based HSC gene therapy.

Internal Deletions of IE2 86 and Loss of the Late IE2 60 and IE2 40 Proteins Encoded by Human Cytomegalovirus Affect the Levels of UL84 Protein but Not the Amount of UL84 mRNA or the Loading and Distribution of the mRNA on Polysomes

The major immediate-early (IE) region of human cytomegalovirus encodes two IE proteins, IE1 72 and IE2 86, that are translated from alternatively spliced transcripts that differ in their 3' ends. Two other proteins that correspond to the C-terminal region of IE2 86, IE2 60 and IE2 40, are expressed at late times. In this study, we used IE2 mutant viruses to examine the mechanism by which IE2 86, IE2 60, and IE2 40 affect the expression of a viral DNA replication factor, UL84. Deletion of amino acids (aa) 136 to 290 of IE2 86 results in a significant decrease in UL84 protein during the infection. This loss of UL84 is both proteasome and calpain independent, and the stability of the protein in the context of infection with the mutant remains unaffected. The RNA for UL84 is expressed to normal levels in the mutant virus-infected cells, as are the RNAs for two other proteins encoded by this region, UL85 and UL86. Moreover, nuclear-to-cytoplasmic transport and the distribution of the UL84 mRNA on polysomes are unaffected. A region between aa 290 and 369 of IE2 86 contributes to the UL84-IE2 86 interaction in vivo and in vitro. IE2 86, IE2 60, and IE2 40 are each able to interact with UL84 in the mutant-infected cells, suggesting that these interactions may be important for the roles of UL84 and the IE2 proteins. Thus, these data have defined the contribution of IE2 86, IE2 60, and IE2 40 to the efficient expression of UL84 throughout the infection.

A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector

A therapeutic CMV vaccine incorporating an antigenic repertoire capable of eliciting a cellular immune response has yet to be successfully implemented for patients who already have acquired an infection. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV. The novelty of this vaccine is the fusion of two adjacent exons from the immediate-early region of CMV, their successful expression in MVA, and robust immunogenicity in both primary and memory response models. Evaluation of the immunogenicity of the viral vaccine in mouse models shows that it can stimulate primary immunity against all three antigens in both the CD4 + and CD8 + T cell subsets. Evaluation of human PBMC from healthy CMV-positive donors or patients within 6 months of receiving hematopoietic cell transplant shows robust stimulation of existing CMV-specific CD4 + and CD8 + T cell subsets.

Pre-Clinical Development of a Subunit Vaccine Expressing an IE1-IE2 Fusion Protein of HCMV.

CMV infection is an important complication of patient recovery from transplantation, and affects a wide variety of individuals including newborns and HIV patients with advanced disease. An effective CMV vaccine for patients who have already acquired an infection has yet to successfully incorporate an antigenic repertoire capable of eliciting a cellular immune response. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV, we have termed CMV-MVA. While other antigens are also immunologically recognized to varying degrees, the evidence for these three antigens to be involved in protective immune responses in a majority of CMV-infected patients is compelling and justifies their inclusion into a vaccine to prevent viremia and control infection. MVA has an extensive history of successful delivery into rodents, Rhesus macaques, and other non-human primates, and more recently as a clinical vaccine in cancer patients and HIV patients in a state of immunosuppression. CMV-MVA is engineered with a bacterial marker to track its purification, which can be removed by recombination, a requirement for clinical development. The novelty of this vaccine is the fusion of the two largest and adjacent protein-coding exons from the immediate-early (IE) region of CMV, their successful expression as a fusion protein in MVA, and robust immunogenicity in both primary and memory response models. The advantages of this approach include placement of all vaccine antigens in one vector, and diminishing the dose of virus needed to attain sufficient immunity simultaneously against all of the included antigens. Evaluation of the immunogenicity of the viral vaccine in transgenic HLA mouse models (A2, B7, A11) shows that it can stimulate primary immunity against all three antigens in both the CD4+ and CD8+ T cell subsets. Evaluation using human PBMC from CMV-positive donors shows robust stimulation of existing CMV-specific T cells in both the CD4+ and CD8+ T cell subset. These results extend to both healthy volunteers and patients within 6 months of receiving hematopoietic cell transplant (HCT). Evaluating PBMC from transplant recipients in all three risk categories (D+/R+,D+/R−, D−R+), we found an equivalently strong recognition of both antigens, in some cases more vigorous than in the PBMC of healthy adults. This candidate vaccine is being developed in partnership with the NCI as a therapeutic for HCT recipients. Strategies of vaccine delivery include vaccinating the transplant donor, and/or the recipient at day 90 or later, if warranted clinically and with sufficient evidence of safety. The ongoing evaluation of a DNA vaccine against CMV suggests a worthwhile strategy of combining MVA with a plasmid DNA vaccine. Our preliminary studies using DNA prime and MVA boost in Rhesus macaques show it to be a more powerful CMV vaccine regimen than either component given separately. Evidence for the capacity of CMV-MVA to modify viremia through immunologic mechanisms from both clinical and monkey studies will be presented.

Murine Cytomegalovirus Major Immediate-Early Enhancer Region Operating as a Genetic Switch in Bidirectional Gene Pair Transcription

Enhancers are defined as DNA elements that increase transcription when placed in any orientation relative to a promoter. The major immediate-early (MIE) enhancer region of murine cytomegalovirus is flanked by transcription units ie1/3 and ie2, which are transcribed in opposite directions. We have addressed the fundamental mechanistic question of whether the enhancer synchronizes transcription of the bidirectional gene pair (synchronizer model) or whether it operates as a genetic switch, enhancing transcription of either gene in a stochastic alternation (switch model). Clonal analysis of cytokine-triggered, transcription factor-mediated MIE gene expression from latent viral genomes provided evidence in support of the switch model.

Exon 3 of the Human Cytomegalovirus Major Immediate-Early Region Is Required for Efficient Viral Gene Expression and for Cellular Cyclin Modulation

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Delta30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Delta30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Delta30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Delta30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Delta30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.

The English strain of rat cytomegalovirus (CMV) contains a novel captured CD200 (vOX2) gene and a spliced CC chemokine upstream from the major immediate-early region: further evidence for a separate evolutionary lineage from that of rat CMV Maastricht

Sequence data for eight genes, together with time-course Northern blotting and 3'- and 5'-RACE (rapid amplification of cDNA ends) analysis for some mRNAs from a 12 kb region upstream from the major immediate-early (MIE) genes of the English isolate of rat cytomegalovirus (RCMV), are presented. The results identified important differences compared to both murine cytomegalovirus (MCMV) and the Maastricht isolate of RCMV. A striking finding is the presence of a highly conserved, rightwards-oriented homologue of the rat cellular CD200 (OX2) gene immediately to the right of the MIE region, which replaces either the leftwards-oriented AAV REP gene of RCMV (Maastricht) or the upstream spliced portions of the immediate-early 2 gene (ie2) in MCMV. From the presence of other homologues of MCMV- and RCMV-specific genes, such as the beta-chemokine MCK-2, SGG1 and an Fcgamma receptor gene, as reported here, the basic architecture of the MIE region (reported previously) and the level of IE2 and DNA polymerase (POL) protein conservation in phylogenetic analyses, it is clear that the English strain of RCMV is also a member of the genus Muromegalovirus, but is a beta-herpesvirus species that is very distinct from both MCMV and RCMV (Maastricht). Both the lack of a CD200 homologue in the other two rodent viruses and the depth of sequence divergence of the rodent CMV IE2 and POL proteins suggest that these three viruses have evolved as separate species in the genus Muromegalovirus since very early in the host rodent lineage.

Mouse cytomegalovirus immediate-early protein 1 binds with host cell repressors to relieve suppressive effects on viral transcription and replication during lytic infection.

Herpesviruses start their transcriptional cascade at nuclear domain 10 (ND10). The deposition of virus genomes at these nuclear sites occurs due to the binding of the interferon-inducible repressor protein promyelocytic leukemia protein (PML) and/or Daxx to a viral DNA-protein complex. However, the presence of repressive proteins at the nuclear site of virus transcription has remained unexplained. We investigated the mouse cytomegalovirus (MCMV) immediate-early 1 protein (IE1), which is necessary for productive infection at low multiplicities of infection and therefore likely to be involved in overcoming cellular repression. Temporal analysis of IE1 distribution revealed its initial segregation into ND10 by binding to PML and/or Daxx and IE1-dependent recruitment of the transcriptional repressor histone deacetylase-2 (HDAC-2) to this site. However, these protein aggregates are dissociated in cells producing sufficient IE1 through titration of PML, Daxx, and HDAC-2. Importantly, binding of IE1 to HDAC-2 decreased deacetylation activity. Moreover, inhibition of HDAC by trichostatin-A resulted in an increase in viral protein synthesis, an increase in cells starting the formation of prereplication compartments, and an increase in the total infectious viruses produced. Thus, IE1, like trichostatin-A, reverses the repressive effect of HDAC evident in the presence of acetylated histones in the immediate-early promoter region. Since HDAC also binds to the promoter region of IE1, as determined by the chromatin immunoprecipitation assay, these combined results suggest that IE1 inhibits or reverses HDAC-mediated repression of the infecting viral genomes, possibly by a process akin to activation of heterochromatin. We propose that even permissive cells can repress transcription of infecting viral genomes through repressors, including HDAC, Daxx, and PML, and the segregation of IE1 to ND10 that would inactivate those repressors. The virus can counter this repression by overexpressing IE1 when present in sufficient copy number, thus reducing the availability and effectiveness of these repressors.

Mutant human cytomegalovirus lacking the immediate-early TRS1 coding region exhibits a late defect.

The human cytomegalovirus IRS1 and TRS1 open reading frames encode immediate-early proteins with identical N-terminal domains and divergent C-terminal regions. Both proteins have been shown previously to activate reporter genes in transfection assays in cooperation with other viral gene products. We have constructed two viruses carrying substitution mutations within either the IRS1 or TRS1 open reading frame. ADsubIRS1 failed to produce the related IRS1 and IRS1(263) proteins, but it replicated with normal kinetics to produce a wild-type yield in human fibroblasts. The addition in trans of the IRS1(263) protein, which antagonizes the ability of IRS1 and TRS1 proteins to activate reporter genes, did not inhibit the growth of the mutant virus. ADsubTRS1 failed to produce the TRS1 protein, and it generated an approximately 200-fold-reduced yield of infectious virus in comparison to its wild-type parent. Viral DNA accumulated normally, as did a set of viral mRNAs that were monitored in ADsubTRS1-infected cells. However, two tegument proteins were partially mislocalized and infectious virus particles did not accumulate to normal levels within ADsubTRS1-infected cells. Further, infectious ADsubTRS1 particles sedimented abnormally in a glycerol-tartrate gradient, indicating that the structure of the mutant particles is aberrant. Our analysis of the ADsubTRS1 phenotype indicates that the TRS1 protein is required, either directly or indirectly, for efficient assembly of virus particles.

Transcription Mapping and Expression Patterns of Genes in the Major Immediate-Early Region of Kaposi's Sarcoma-Associated Herpesvirus

Viral immediate-early (IE) genes are the first class of viral genes expressed during primary infection or reactivation from latency. They usually encode regulatory proteins that play crucial roles in viral life cycle. In a previous study, four regions in the KSHV genome were found to be actively transcribed in the immediate-early stage of viral reactivation in primary effusion lymphoma cells. Three immediate-early transcripts were characterized in these regions, as follows: mRNAs for ORF50 (KIE-1), ORF-45 (KIE-2), and ORF K4.2 (KIE-3) (F. X. Zhu, T. Cusano, and Y. Yuan, 1999, J. Virol. 73, 5556–5567). In the present study, we further analyzed the expression of genes in these IE regions in BC-1 and BCBL-1 cells. One of the immediate-early regions (KIE-1) that encompasses ORF50 and other genes was intensively studied to establish a detailed transcription map and expression patterns of genes in this region. This study led to identification of several novel IE transcripts in this region. They include a 2.6-kb mRNA which encodes ORF48/ORF29b, a family of transcripts that are complementary to ORF50 mRNA and a novel K8 IE mRNA of 1.5 kb. Together with the IE mRNA for ORF50 which was identified previously, four immediate-early genes have been mapped to KIE-1 region. Therefore, we would designate KIE-1 the major immediate-early region of KSHV. In addition, we showed that transcription of K8 gene is controlled by two promoters, yielding two transcripts, an immediate-early mRNA of 1.5 kb and a delayed-early mRNA of 1.3 kb.

Viable human cytomegalovirus recombinant virus with an internal deletion of the IE2 86 gene affects late stages of viral replication.

Using bacterial artificial chromosome (BAC) technology, we have constructed and characterized a human cytomegalovirus recombinant virus with a mutation in the exon specific for the major immediate-early region 2 (IE2) gene product. The resulting IE2 86-kDa protein (IE2 86) has an internal deletion of amino acids 136 to 290 and is fused at the carboxy terminus to enhanced green fluorescent protein (EGFP). The deletion also removes the promoter and initiator methionine for the p40 form of IE2 and initiator methionine for the p60 form of the protein, and therefore, these late gene products are not produced. The mutant virus IE2 86 Delta SX-EGFP is viable but exhibits altered growth characteristics in tissue culture compared with a full-length wild-type (wt) IE2 86-EGFP virus or a revertant virus. When cells are infected with the mutant virus at a low multiplicity of infection (MOI), there is a marked delay in the production of infectious virus. This is associated with slower cell-to-cell spread of the virus. By immunofluorescence and Western blot analyses, we show that the early steps in the replication of the mutant virus are comparable to those for the wt. Although there is significantly less IE2 protein in the cells infected with the mutant, there is only a modest lag in the initial accumulation of IE1 72 and viral early proteins, and viral DNA replication proceeds normally. The mutation also has only a small effect on the synthesis of the viral major capsid protein. The most notable molecular defect in the mutant virus infection is that the steady-state levels of the pp65 (UL83) and pp28 (UL99) matrix proteins are greatly reduced. In the case of UL83, but not UL99, there is also a corresponding decrease in the amount of mRNA present in cells infected with the mutant virus.

Fomivirsen: clinical pharmacology and potential drug interactions.

Fomivirsen sodium is a 21-base phosphorothioate oligodeoxynucleotide complementary to the messenger RNA of the major immediate-early region proteins of human cytomegalovirus, and is a potent and selective antiviral agent for cytomegalovirus retinitis. Following intravitreal administration, fomivirsen is slowly cleared from vitreous with a half-life of approximately 55 hours in humans. Preclinical studies show that fomivirsen distributes to retina and is slowly metabolised by exonuclease digestion. Clearance from retina was shown to be similarly slow following loading from the vitreous. The estimated half-life for clearance of fomivirsen from retina was 78 hours in monkeys following a 115-microg dose. Because of the low doses coupled with slow disposition from the eye, measurable concentrations of drug are not detected in the systemic circulation following intravitreal administration. Systemically administered phosphorothioate oligodeoxynucleotides are highly bound to albumin and alpha2-macroglobulin in blood plasma. Because fomivirsen does not compete for oxidative metabolic processes involved in clearance of many xenobiotics, the most likely mechanism for drug interactions may be altered protein binding of a coadministered drug. The extremely low systemic exposure to this oligodeoxynucleotide following intravitreal administration largely negates its potential ability to interact with systemically administered drugs. Even if fomivirsen were able to access the blood, protein binding assays indicate that drugs that are site I and site II binders of albumin (warfarin, ibuprofen, salicylic acid) are not displaced in the presence of phosphorothioate oligodeoxynucleotides of various sequences at concentrations orders of magnitude higher than that seen for fomivirsen. Administration of fomivirsen with numerous systemically administered antiretrovirals (for example zidovudine and zalcitabine) as well as systemically administered anticytomegalovirus agents such as foscarnet and ganciclovir has been reported to be well tolerated. The only reported warning is a recommendation against administration within 2 to 4 weeks of cidofovir treatment due to an increased risk of ocular inflammation.

Induction of apoptosis in vitro by the 17-kDa nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis.

Infectious bursal disease virus (IBDV) causes severe immunodeficiency in young chickens by destroying the precursors of antibody-producing B cells in the bursa of Fabricius. It has been shown that IBDV infection induces apoptosis in chicken embryo and tissue culture cells. We previously reported that an IBDV mutant lacking the expression of 17-kDa nonstructural (NS) protein exhibited decreased apoptotic effects in cell culture as compared to the parental IBDV, suggesting that the NS protein may be involved in induction of apoptosis. Here, we report that the NS protein of IBDV alone is capable of inducing apoptosis in cell culture. Transfection of chicken B-lymphocyte cell line (RP9) and chicken embryo fibroblast cells with a plasmid DNA, containing the NS protein gene under the control of the immediate-early promoter-enhancer region of human cytomegalovirus, induced programmed cell death in both cell lines. Apoptosis changes, such as chromatin condensation, DNA fragmentation, and the appearance of apoptotic nuclear bodies, were observed in cell cultures 48-h posttransfection. As reported earlier, the mutant IBDV grew to lower titers with slower replication kinetics and lower cytopathogenicity when compared to that of the parental virus. Here, we demonstrate that the mutant virus is closely associated with cells and its yield from the supernatant was approximately 30-fold lower than the wild-type due to increased cell association, indicating a deficiency in lysis of virus-infected cells. Taken together, our results indicate that the NS protein of IBDV is highly cytotoxic, which brings about the release of the viral progeny from cells, and thus play an important role in viral pathogenesis.

Reactivation of the human cytomegalovirus major immediate-early regulatory region and viral replication in embryonal NTera2 cells: role of trichostatin A, retinoic acid, and deletion of the 21-base-pair repeats and modulator.

Inactivity of the human cytomegalovirus (HCMV) major immediate-early regulatory region (MIERR), which is composed of promoter, enhancer, unique region, and modulator, is linked to lack of HCMV replication in latently infected cells and in other nonpermissive cell types, including human embryonal NTera2 carcinoma (NT2) cells. I refined the embryonal NT2 cell model to enable characterization of the unknown mechanistic basis for silencing of HCMV MIERR-dependent transcription and viral replication in nonpermissive cells. These infected NT2 cells contain nonreplicating viral genomes with electrophoretic mobility equivalent to a supercoiled, bacterial artificial chromosome of comparable molecular weight. MIERR-dependent transcription is minimal to negligible. Increasing the availability of positive-acting transcription factors by retinoic acid (RA) treatment after infection is largely insufficient in reactivating the MIERR. In contrast, trichostatin A (TSA), a histone deacetylase inhibitor, reactivates MIERR-dependent transcription. Contrary to prior findings produced from transfected MIERR segments, deletion of the 21-bp repeats and modulator from the MIERR in the viral genome does not relieve MIERR silencing. To demonstrate that MIERR silencing likely results from enhancer inactivity, I examined an HCMV with a heterologous MIERR promoter that is enhancer dependent but exempt from IE2 p86-mediated negative autoregulation. This heterologous promoter, like its neighboring native MIERR promoter, exhibits immediate-early transcriptional kinetics in fibroblasts. In embryonal NT2 cells, the heterologous MIERR promoter is transcriptionally inactive. This silence is relieved by TSA but not by RA. Remarkably, TSA-induced reactivation of MIERR-dependent transcription from quiescent viral genomes is followed by release of infectious virus. I conclude that a mechanism of active repression imposes a block to MIERR-dependent transcription and viral replication in embryonal NT2 cells. Because TSA overcomes the block, viral gene silencing may involve histone deacetylase-based modification of viral chromatin, which might account for the covalently closed circular conformation of quiescent HCMV genomes.

Chromatin Structure Contributes to Latency

Understanding viral latency and reactivation is an important aspect of viral biology and may aid development of strategies to control viral infection. Meier developed a clonal NTera2/D1 (NT2) cell line, which is derived from an embryonal teratocarcinoma and which can be induced to differentiate into neurons in culture. These cells were used to study latency and reactivation of human cytomegalovirus, a virus that replicates in neurons. Based on analysis of the viral DNA structure in infected, but latent, cells, Meier showed that at least a portion of the viral genome had reached the nucleus. Viral replication could be stimulated by pretreatment of the cells with retinoic acid to stimulate differentiation, but retinoic acid had no effect if applied after infection. The histone deacetylase inhibitor trichostatin A stimulated expression of viral major immediate early genes, viral replication, and the production of infectious virus undifferentiated NT2 cells. Thus, differences in chromatin structure in differentiated and undifferentiated cells represents one mechanism by which the immediate early gene transcription can be inhibited, thus inhibiting viral replication and promoting latency.J. L. Meier, Reactivation of the human cytomegalovirus major immediate-early regulatory region and viral replication in embryonal Ntera2 cells: Role of trichostatin A, retinoic acid, and deletion of the 21-base-pair repeats and modulator. J. Virol. 75, 1581-1593 (2001). [Abstract] [Full Text]

Chromatin Structure Contributes to Latency

Understanding viral latency and reactivation is an important aspect of viral biology and may aid development of strategies to control viral infection. Meier developed a clonal NTera2/D1 (NT2) cell line, which is derived from an embryonal teratocarcinoma and which can be induced to differentiate into neurons in culture. These cells were used to study latency and reactivation of human cytomegalovirus, a virus that replicates in neurons. Based on analysis of the viral DNA structure in infected, but latent, cells, Meier showed that at least a portion of the viral genome had reached the nucleus. Viral replication could be stimulated by pretreatment of the cells with retinoic acid to stimulate differentiation, but retinoic acid had no effect if applied after infection. The histone deacetylase inhibitor trichostatin A stimulated expression of viral major immediate early genes, viral replication, and the production of infectious virus undifferentiated NT2 cells. Thus, differences in chromatin structure in differentiated and undifferentiated cells represents one mechanism by which the immediate early gene transcription can be inhibited, thus inhibiting viral replication and promoting latency. J. L. Meier, Reactivation of the human cytomegalovirus major immediate-early regulatory region and viral replication in embryonal Ntera2 cells: Role of trichostatin A, retinoic acid, and deletion of the 21-base-pair repeats and modulator. J. Virol. 75 , 1581-1593 (2001). [Abstract] [Full Text]