Immediate Early Expression Research Articles

Development of a quantitative real-time RT-PCR for kinetic analysis of immediate-early transcripts of Rat cytomegalovirus

One-step real-time RT-PCR assay was developed for quantification of the immediate-early (IE), namely IE1 and IE2 transcripts of Rat cytomegalovirus (RCMV), strain ALL-03 in rat embryonic fibroblast cells (REF). This in-house SYBR Green I based RT-PCR was shown to have higher amplification efficiency and detection limit as compared to a commercially available real-time RT-PCR kit in quantifying these two transcripts. The quantification histogram revealed the divergence of transcription activities of the two IE genes. The IE1 transcript had a concentration peak at 7 hrs post infection (p.i.), whereas IE2 transcript at 20 hrs p.i. Regulation of IE expression is critical for determination, whether the infection is going to be abortive, lytic or latent. Therefore, this in-house developed quantitative RT-PCR assay offers an alternative for diagnosis and monitoring of the acute cytomegalovirus (CMV) infection directed at IE transcript detection.

Recruitment of the Transcriptional Coactivator HCF-1 to Viral Immediate-Early Promoters during Initiation of Reactivation from Latency of Herpes Simplex Virus Type 1

The transcriptional coactivator host cell factor 1 (HCF-1) is critical for the expression of immediate-early (IE) genes of the alphaherpesviruses herpes simplex virus type 1 (HSV-1) and varicella-zoster virus. HCF-1 may also be involved in the reactivation of these viruses from latency as it is sequestered in the cytoplasm of sensory neurons but is rapidly relocalized to the nucleus upon stimulation that results in reactivation. Here, chromatin immunoprecipitation assays demonstrate that HCF-1 is recruited to IE promoters of viral genomes during the initiation of reactivation, correlating with RNA polymerase II occupancy and IE expression. The data support the model whereby HCF-1 plays a pivotal role in the reactivation of HSV-1 from latency.

A historical analysis of herpes simplex virus promoter activation in vivo reveals distinct populations of latently infected neurones.

Herpes simplex virus type 1 (HSV-1) has the capacity to establish a life-long latent infection in sensory neurones and also to periodically reactivate from these cells. Since mutant viruses defective for immediate-early (IE) expression retain the capacity for latency establishment it is widely assumed that latency is the consequence of a block in IE gene expression. However, it is not clear whether viral gene expression can precede latency establishment following wild-type virus infection. In order to address this question we have utilized a reporter mouse model system to facilitate a historical analysis of viral promoter activation in vivo. This system utilizes recombinant viruses expressing Cre recombinase under the control of different viral promoters and the Cre reporter mouse strain ROSA26R. In this model, viral promoter-driven Cre recombinase mediates a permanent genetic change, resulting in reporter gene activation and permanent marking of latently infected cells. The analyses of HSV-1 recombinants containing human cytomegalovirus major immediate-early, ICP0, gC or latency-associated transcript promoters linked to Cre recombinase in this system have revealed the existence of a population of neurones that have experienced IE promoter activation prior to the establishment of latency.

Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes

Vaccinia virus is the prototypic orthopoxvirus and was the vaccine used to eradicate smallpox, yet the expression profiles of many of its genes remain unknown. Using a genome tiling array approach, we simultaneously measured the expression levels of all 223 annotated vaccinia virus genes during infection and determined their kinetics. For 95% of these genes, significant transcript levels were detected. Most remarkably, classification of the genes by their expression profiles revealed 35 genes exhibiting immediate-early expression. Although a similar kinetic class has been described for other virus families, to our knowledge, this is the first demonstration of its existence in orthopoxviruses. Despite expression levels higher than for genes in the other three kinetic classes, the functions of more than half of these remain unknown. Additionally, genes within each kinetic class were spatially grouped together in the genome. This genome-wide picture of transcription alters our understanding of how orthopoxviruses regulate gene expression.

Characterization of a Potent Refractory State and Persistence of Herpes Simplex Virus 1 in Cell Culture

Herpes simplex virus (HSV) normally undergoes productive cytocidal infection in culture and is thought of as relatively resistant to innate immune responses such as interferon. We previously described an unusual pattern of infection in culture in MDBK cells, which after initial productive infection, surprisingly resulted in progressive suppression of replication and cell recovery. The dominance of the refractory state was due to the inability to suppress interferon production and subsequent paracrine signaling. Here, using a wild-type HSV-1 strain expressing green fluorescent protein (GFP)-VP16, we analyze aspects of long-term HSV persistence resulting from this oscillating refractory state. We show that the gradual suppression of GFP-VP16 expression correlated with a biphasic pattern of accumulation of viral DNA and extracellular virus titers. We quantify virus maintenance in a minor subpopulation of cells during subculture, show the reemergence of virus by infectious center assay, and demonstrate that this required intracellular events over a 24- to 48-h time course. We also demonstrate that conditioned medium (cMed) from infected cells induced a profound shutoff of HSV gene expression at the transcriptional level. Finally, we demonstrate that this suppression was extremely rapid, requiring only 1 h of treatment to essentially abolish HSV immediate-early expression, and surprisingly persisted for almost 2 days after removal of the cMed. These combined effects underpin the oscillating effect both in plaque progression, where infection spreads but is overwhelmed by the accumulation of inhibitory components, enabling cell recovery, and virus maintenance in a subpopulation of cells. These results may be relevant to consider in studies of HSV latency in different animal models.

Transcriptional Pausing Caused by NELF Plays a Dual Role in Regulating Immediate-Early Expression of the junB Gene

Human 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) negatively regulate transcription elongation by RNA polymerase II (RNAPII) in vitro. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF- and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position +50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF- and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.

The Neuronal Host Cell Factor-Binding Protein Zhangfei Inhibits Herpes Simplex Virus Replication

During lytic infection in epithelial cells the expression of herpes simplex virus type 1 (HSV-1) immediate-early (IE) genes is initiated by a multiprotein complex comprising the virion-associated protein VP16 and two cellular proteins, host cellular factor (HCF) and Oct-1. Oct-1 directly recognizes TAATGARAT elements in promoters of IE genes. The role of HCF is not clear. HSV-1 also infects sensory neurons innervating the site of productive infection and establishes a latent infection in these cells. It is likely that some VP16 is retained by the HSV-1 nucleocapsid as it reaches the neuronal nucleus. Its activity must therefore be suppressed for successful establishment of viral latency. Recently, we discovered an HCF-binding cellular protein called Zhangfei. Zhangfei, in an HCF-dependent manner, inhibits Luman/LZIP/CREB3, another cellular HCF-binding transcription factor. Here we show that Zhangfei is selectively expressed in human neurons. When delivered to cultured cells that do not normally express the protein, Zhangfei inhibited the ability of VP16 to activate HSV-1 IE expression. The inhibition was specific for HCF-dependent transcriptional activation by VP16, since a Gal4-VP16 chimeric protein was inhibited only on a TAATGARAT-containing promoter and not a on a Gal4-containing promoter. Zhangfei associated with VP16 and inhibited formation of the VP16-HCF-Oct-1 complex on TAATGARAT motifs. Zhangfei also suppressed HSV-1-induced expression of several cellular genes including topoisomerase IIalpha, suggesting that in addition to suppressing IE expression Zhangfei may have an inhibitory effect on HSV-1 DNA replication and late gene expression.

Synergistic inhibition of human cytomegalovirus replication by interferon-alpha/beta and interferon-gamma

BackgroundRecent studies have shown that gamma interferon (IFN-γ) synergizes with the innate IFNs (IFN-α and IFN-β) to inhibit herpes simplex virus type 1 (HSV-1) replication in vitro. To determine whether this phenomenon is shared by other herpesviruses, we investigated the effects of IFNs on human cytomegalovirus (HCMV) replication.ResultsWe have found that as with HSV-1, IFN-γ synergizes with the innate IFNs (IFN-α/β) to potently inhibit HCMV replication in vitro. While pre-treatment of human foreskin fibroblasts (HFFs) with IFN-α, IFN-β or IFN-γ alone inhibited HCMV plaque formation by ~30 to 40-fold, treatment with IFN-α and IFN-γ or IFN-β and IFN-γ inhibited HCMV plaque formation by 163- and 662-fold, respectively. The generation of isobole plots verified that the observed inhibition of HCMV plaque formation and replication in HFFs by IFN-α/β and IFN-γ was a synergistic interaction. Additionally, real-time PCR analyses of the HCMV immediate early (IE) genes (IE1 and IE2) revealed that IE mRNA expression was profoundly decreased in cells stimulated with IFN-α/β and IFN-γ (~5-11-fold) as compared to vehicle-treated cells. Furthermore, decreased IE mRNA expression was accompanied by a decrease in IE protein expression, as demonstrated by western blotting and immunofluorescence.ConclusionThese findings suggest that IFN-α/β and IFN-γ synergistically inhibit HCMV replication through a mechanism that may involve the regulation of IE gene expression. We hypothesize that IFN-γ produced by activated cells of the adaptive immune response may potentially synergize with endogenous type I IFNs to inhibit HCMV dissemination in vivo.

The TATGARAT Box of the HSV-1 ICP27 Gene is Essential for Immediate Early Expression but not Critical for Efficient Replication in vitro or in vivo

We constructed a recombinant virus containing a promoter mutation altering the immediate-early expression of the HSV-1 ICP27 transcript, ICP27DeltaSma, which contains a deletion of the "TATGARAT" and surrounding sequences, but retains the rest of the ICP27 promoter. This mutant does not exhibit immediate-early expression of ICP27 using criteria of expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. While transcript abundance at 1h after infection at 0.1 PFU/cell in mouse embryo fibroblasts was significantly altered compared to infections with wt -rescues, by 4 h after infection these differences were diminished or absent. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue infections at the earliest times tested, but were equivalent by 8-12 h pi. Further, both single and multi-step virus replication was equivalent with both mutants and rescues. Thus, altering the immediate early kinetics of ICP27 leads to a sub-optimal quantitative lag-phase in gene expression but without consequence to replication fitness in vitro . Infections in vivo also revealed the ability of mutant and rescue virus to invade the CNS of mice following footpad injections was equivalent. The nature of the role of immediate-early ICP27 expression is discussed in light of these observations.

Immediate-early expression of the herpes simplex virus type 1 ICP27 transcript is not critical for efficient replication in vitro or in vivo.

We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.

Full resistance of herpes simplex virus type 1-infected primary human cells to alpha interferon requires both the Us11 and gamma(1)34.5 gene products.

The gamma(1)34.5 gene product is important for the resistance of herpes simplex virus type 1 (HSV-1) to interferon. However, since the inhibition of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus results from the combined loss of the gamma(1)34.5 gene product and the failure to translate the late Us11 mRNA, we sought to characterize the relative interferon sensitivity of mutants unable to produce either the Us11 or the gamma(1)34.5 polypeptide. We now demonstrate that primary human cells infected with a Us11 mutant virus are hypersensitive to alpha interferon, arresting translation upon entry into the late phase of the viral life cycle. Furthermore, immediate-early expression of Us11 by a gamma(1)34.5 deletion mutant is sufficient to render translation resistant to alpha interferon. Finally, we establish that the Us11 gene product is required for wild-type levels of replication in alpha interferon-treated cells and, along with the gamma(1)34.5 gene, is an HSV-1-encoded interferon resistance determinant.

PML-nuclear bodies are involved in cellular serum response.

Serum stimulation leads to the activation of various signal transduction pathways in cells, and the resultant signals are integrated into the serum response factor (SRF)-dependent transcription of immediate-early genes such as c-fos. To further characterize this response, we investigated the mechanism which controls serum response transcription in cultured human cells. Frequency of PML (promyelocytic leukaemia)-nuclear bodies (NBs) formation increases shortly after serum stimulation, probably facilitating the interaction of SRF and CBP acetyltransferase at the NBs. PML modulates SRF-mediated c-fos promoter activities upon addition of serum to cells or expression of constitutively active Rho family GTPases. We mapped the region in the SRF that interacts with PML to the C-terminal transactivation domain. An SRF mutant deleted of the transactivation domain neither co-localizes with CBP in NBs nor fulfills its transcriptional role. Under conditions of serum stimulation, the formation of NBs coincides with the immediate-early expression of the endogenous c-fos gene in fibroblasts and in all-trans retinoic acid-treated acute promyelocytic leukaemia NB4 cells. These data provide an insight into the involvement of NBs in modulating the transcription of serum-induced immediate-early genes.

Construction of recombinant pseudorabies viruses optimized for labeling and neurochemical characterization of neural circuitry

In this study we have modified the neuroinvasiveness of pseudorabies virus strain Bartha, a commonly utilized trans-synaptic tract-tracer. In addition, we sought to facilitate detection of cellular mRNAs in neurons infected with the virus. In order to modify spreading characteristics, we inserted the lacZ or the GFP (green fluorescent protein) genes into the genomic loci containing the putative latency-associated transcript promoter (P LAT2), resulting in the disruption of the promoter function. Following rat kidney injection, mutant viruses labeled central autonomic neurons in a slower and much more restricted manner than the parent Bartha strain. Since both reporter genes were controlled by the human cytomegalovirus immediate early (IE) 1 promoter, they exhibited IE expression kinetics. This property proved to be important for the co-detection of reporter proteins with neuronal mRNAs, readily detected at early but not at late stage of infection, as shown in tyrosine-hydroxylase expressing A5 catecholaminergic neurons and in serotonin transporter expressing raphe magnus neurons.

Cellular Proteins Bind to Sequence Motifs in the R1 Element between the HCMV Immune Evasion Genes

The viral US3 and US6 gene products of human cytomegalovirus (CMV) are sequentially expressed at immediate-early and early times after infection, respectively. They downregulate the surface expression of HLA class I molecules. There are two repeat-containing regulatory regions between the US3 promoter and the US6 transcription unit designated R1 and R2. R2 contains repetitions of the NF-κB responsive element and enhances the immediate-early expression of the US3 gene. R1 contains 19 repetitions of a 5′-TRTCG-3′ pentanucleotide arranged as everted repeats, inverted repeats, and variably spaced single pentanucleotides. In the context of the viral genome, R1 also enhances immediate-early US3 gene expression by an unknown mechanism (G. C. Bullock, et al., 2001, Virology 288, 164–174). We report a sequence motif within the R1 element that binds a human cell nuclear protein which is antigenically related to the Drosophila boundary element-associated factor (BEAF). The potential role of a 35-kDa cellular protein that binds to sequence motifs within the R1 element in regulating the expression of the CMV US3 immune evasion gene is discussed.

Effect of the R1 element on expression of the US3 and US6 immune evasion genes of human cytomegalovirus.

Human cytomegalovirus (HCMV) has several gene products that are important for escape from immune surveillance. These viral gene products downregulate the expression of HLA molecules on the cell surface. The viral US3 and US6 gene products are expressed at immediate-early and early times after infection, respectively. There are two regulatory regions between the US3 and the US6 transcription units. The first region is an NF-kappaB responsive enhancer that promotes the immediate-early expression of the US3 gene and is designated the R2 enhancer. Upstream of the R2 enhancer is a region designated the R1 element that in transient transfection assays behaves as a silencer by repressing the effect of the enhancer on downstream gene expression (A. R. Thrower et al., J. Virol. 1996, 70, 91; Y.-J. Chan et al., J. Virol. 1996, 70, 5312). We constructed recombinant viruses with wild-type or mutated R1 elements. The expression of the US3 gene at 6 h after infection and the US6 gene at 24 h was higher when the R1 element was present. The R1 element in the context of the viral genome is not a silencer of US3 or US6 gene expression. The R1 element has multiple effects on the US3 and US6 RNAs. It enhances the level of US3 and US6 mRNA; it determines the 3'-end cleavage and polyadenylation of US6 RNA, and it stabilizes read-through viral RNAs. The potential mechanisms of R1 enhancement of US3 and US6 gene expression are discussed.

Immediate-early protooncogene expression and liver function following various extents of partial hepatectomy in the rat.

Immediate-early protooncogenes (IEP) are thought to play an important role in hepatocyte replication. Whether the extent of their expression correlates with the strength of the proliferative stimulus and subsequent regenerative activity has yet to be documented in vivo. Data are also lacking with respect to the level at which liver disease is associated with biochemical evidence of hepatic dysfunction. Thus, the objectives of this study were to determine whether a correlation exists between IEP gene mRNA expression and varying extents of partial hepatectomy (PHx) and to document the extent of resection required to result in increases in serum bilirubin levels. Eighty-nine adult, male Sprague-Dawley rats underwent either sham surgery or 20%, 35%, 55%, 70% or 90% PHx. Postoperatively, rats were killed (N = 3-6/group) at 15 and 30 mins and 8 and 24 hrs for c-fos, c-jun, and c-myc mRNA expression by northern blot analyses. Rats killed at 24 hrs also had hepatic regenerative activity documented by [3H]thymidine incorporation into hepatic DNA and serum bilirubin determinations. While c-fos mRNA expression at 15 mins and c-myc mRNA expression at 8 hrs after PHx did not correlate with the extent of PHx (r2 = 0.478 and 0.018, respectively), a weak correlation existed between c-jun mRNA expression at 30 mins and the extent of PHx (r2 = 0.662, P < 0.05). In terms of IEP mRNA expression and hepatic regenerative activity, a strong correlation existed between c-fos mRNA expression and [3H]thymidine incorporation (r2 = 0.851, P < 0.01) but not c-jun or c-myc mRNA expression. Compared to sham operated controls, [3H]thymidine incorporation was 2.0x, 3.4x, 3.2x, 7.8x, and 2.2x increased following 20%, 35%, 55%, 70%, and 90% PHx, respectively. Serum bilirubin levels remained unchanged until 70% PHx, when they increased from baseline values of 0.54+/-0.05 mg/dl to 1.02+/-0.15 mg/dl (P < 0.05). A further increase occurred following 90% PHx (1.83+/-0.30 mg/dl, P < 0.01). In conclusion these findings indicate that c-fos mRNA expression 15 mins after PHx correlates with hepatic regenerative activity but not the strength of the regenerative stimulus and that hepatic parenchymal loss of 55-70% must occur prior to the detection of elevated serum bilirubin levels. The results also indicate that relative to a 70% PHx, 90% PHx is associated with decreased rather than increased hepatic regenerative activity.

Cytomegalovirus infection decreases expression of thrombospondin-1 and -2 in cultured human retinal glial cells: effects of antiviral agents.

In fibroblasts, infection with human cytomegalovirus (HCMV) inhibits expression of the extracellular matrix proteins thrombospondin-1 and -2 (TSP-1 and TSP-2). These effects may depend on expression of HCMV immediate-early (IE) genes, which are activated by cellular transcription factor NF-kappaB. The influence of HCMV infection on TSP-1 and TSP-2 expression and the ability of different antiviral drugs to prevent these cellular changes in permissive cultures of human retinal glial cells were observed. Ganciclovir inhibited only HCMV late antigen (LA) expression, whereas antisense oligonucleotide ISIS 2922 and peptide SN50, inhibitors of HCMV IE expression and NF-kappaB activity, respectively, inhibited both IE and LA expression. ISIS 2922 and SN50, but not ganciclovir, prevented down-modulation of TSP-1 and TSP-2. The results showed that HCMV-induced down-modulation of TSP-1 and TSP-2 in retinal glial cells is prevented by inhibition of HCMV IE expression. These findings may be relevant to pathogenesis and treatment of HCMV retinitis.

The antisense oligonucleotide ISIS 2922 prevents cytomegalovirus-induced upregulation of IL-8 and ICAM-1 in cultured human fibroblasts.

Human cytomegalovirus (HCMV) infection is associated with excessive proinflammatory immune responses such as cytokine/chemokine production or upregulation of adhesion molecules on the host cells. It is assumed that these features of HCMV-related immunopathology can not be treated effectively with currently available anti HCMV drugs. In the present study the efficacy of ganciclovir (GCV), foscarnet (PFA), cidofovir (HPMPC), and ISIS 2922, an antisense oligonucleotide complementary to HCMV immediate-early (IE) mRNA, was investigated on HCMV-induced secretion and functional activity of the C-X-C chemokine IL-8 and the expression of the intercellular adhesion molecule-1 (ICAM-1). As compared with mock-infected cells IL-8 production was increased up to 9-fold and ICAM-1 expression up to 4-fold in HCMV-infected fibroblasts. Treatment of infected cells with GCV (40 microM), PFA (200 microM) or HPMPC (2 microM) suppressed completely virus replication as demonstrated by quantification of late (L) antigen expression and infectious virus production. These drugs, however, failed to inhibit IE antigen expression and did not prevent HCMV-induced upregulation of IL-8 and ICAM-1. In contrast, ISIS 2922 (1 microM) suppressed both IE and L antigen expression by 99% and inhibited infectious virus production by 10(4)-fold. Moreover, ISIS 2922 significantly suppressed HCMV-induced upregulation of both IL-8 and ICAM-1 expression on the transcriptional and on the protein level. Our results indicate that ISIS 2922 but not inhibitors of HCMV DNA prevents HCMV-induced upregulation of IL-8 and ICAM-1, both hallmarks of inflammatory processes. Thus, inhibition of HCMV IE expression with ISIS 2922 may be an important strategy for the treatment of HCMV-related immunopathogenesis.

Activation of herpesvirus gene expression by the human cytomegalovirus protein pp71.

The activation of gene expression by the human cytomegalovirus (HCMV) particle was investigated. The HCMV major immediate-early (IE) promoter was cloned upstream of the Escherichia coli lacZ coding sequences, and the resulting cassette was introduced into the genome of a herpes simplex virus type 1 (HSV-1) mutant lacking functional VP16. Upon infection with the HSV-1 recombinant in the presence of cycloheximide, to block de novo protein synthesis, expression of lacZ-specific transcripts was increased by fivefold when HCMV was included in the inoculum. Accumulation of HSV-1 IE RNAs was also stimulated by coinfection with HCMV, as was expression of the adenovirus 5 VAI transcript when the VAI gene was cloned into the HSV-1 genome. Coinfection with HCMV did not alter mRNA stability or uncoating of the HSV-1 genome. The coding sequences for the HCMV phosphoprotein pp71, controlled by the HCMV IE promoter, were cloned into an HSV-1 recombinant impaired for the production of the three major transactivators (VP16, ICP0, and ICP4) to yield a recombinant (in1324) which expressed pp71 but did not cause significant cytotoxicity. Infection with in1324 resulted in stimulation of HCMV IE, HSV-1 IE, and VAI expression, demonstrating that pp71 is responsible for the effects we observed when using the entire HCMV particle. Therefore, HCMV pp71 exhibits novel properties in its ability to stimulate gene expression from a range of promoters present in a herpesvirus genome.

Proinflammatory Potential of Cytomegalovirus Infection

We observed the effects of antiviral therapy on CMV and/or oxidative-stress-induced stimulation of proinflammatory molecules including interleukin-8 (IL-8), melanoma growth stimulatory activity-α (GRO-α) and intercellular adhesion molecule 1 (ICAM-1) using human foreskin fibroblasts. Ganciclovir, foscarnet or cidofovir completely suppressed virus replication, as demonstrated by CMV late (L) antigen production. These drugs did not influence CMV immediate-early (IE) antigen expression and had no effects on CMV-induced cellular changes in IL-8, GRO-α and ICAM-1 levels. Phosphorothioate oligonucleotide (ISIS 2922) suppressed both CMV IE and L antigen by 99%. ISIS 2922 completely suppressed CMV-induced upregulation of both chemokines and ICAM-1. Induction of oxidative stress by H₂O₂ upregulated IL-8 expression. Oxidative stress and CMV infection showed synergistic effects on IL-8 expression. ISIS 2922 only partially inhibited the upregulation of IL-8 in infected cells treated with H₂O₂, whereas cotreatment with ISIS 2922 and antioxidants inhibited the upregulation almost completely. The results showed that inhibition of CMV IE expression alone or in combination with antioxidants is promising for the treatment of CMV disease.