This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Browne, E. P.
Right arrow Articles by Shenk, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Browne, E. P.
Right arrow Articles by Shenk, T.

 Previous Article  |  Next Article 

Journal of Virology, December 2001, p. 12319-12330, Vol. 75, No. 24
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.24.12319-12330.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Altered Cellular mRNA Levels in Human Cytomegalovirus-Infected Fibroblasts: Viral Block to the Accumulation of Antiviral mRNAs

Edward P. Browne, Bret Wing, David Coleman, and Thomas Shenk*

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014

Received 24 July 2001/Accepted 25 September 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

The effect of human cytomegalovirus (HCMV) infection on cellular mRNA accumulation was analyzed by gene chip technology. During a 48-h time course after infection of human diploid fibroblasts, 1,425 cellular mRNAs were found to be up-regulated or down-regulated by threefold or greater in at least two consecutive time points. Several classes of genes were prominently affected, including interferon response genes, cell cycle regulators, apoptosis regulators, inflammatory pathway genes, and immune regulators. The number of mRNAs that were up-regulated or down-regulated were roughly equal over the complete time course. However, for the first 8 h after infection, the number of up-regulated mRNAs was significantly less than the number of down-regulated mRNAs. By analyzing the mRNA expression profile of cells infected in the presence of cycloheximide, it was found that a minimum of 25 mRNAs were modulated by HCMV in the absence of protein synthesis. These included mRNAs encoded by a small number of interferon-responsive genes, as well as beta interferon itself. Cellular mRNA levels in cytomegalovirus-infected cells were compared to the levels in cells infected with UV-inactivated virus. The inactivated virus caused the up-regulation of a much greater number of mRNAs, many of which encoded proteins with antiviral roles, such as interferon-responsive genes and proinflammatory cytokines. These data argue that one or more newly synthesized viral gene products block the induction of antiviral pathways that are triggered by HCMV binding and entry.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

Human cytomegalovirus (HCMV) is a ubiquitous member of the Herpesviridae family that causes disease and mortality in immunocompromised individuals. The virus expresses its genes in a cascade fashion with immediate-early genes being transcribed to high levels by 4 h postinfection (hpi) in cultured fibroblasts (17). The immediate-early genes act as transcriptional activators for the early genes, many of which code for proteins involved in the replication of the viral genome. Following the onset of viral DNA replication at about 24 hpi, transcription of the late genes begins, and many of these genes code for structural components of the virion (16).

The majority of studies investigating the replication of HCMV have used human fibroblasts. In this cell type, the virus replication cycle spans 48 to 72 h, ultimately resulting in profound morphological alterations and cell death. Many events in infected fibroblasts that would be expected to alter cellular mRNA levels have been described. Binding and entry of HCMV are known to cause Ca2+ influx, inositol triphosphate, and cyclic AMP (cAMP) synthesis (11, 12, 37) as well as the activation of the transcription factors NF-kappa B and Sp1 (20, 40), and mitogen-activated protein kinase is also activated in a process that depends on viral protein synthesis (31). HCMV also modulates cell cycle progression, inducing infected cells to enter the cell cycle but blocking them at the G1/S transition (5, 9, 23). A number of reports have shown that apoptotic pathways are suppressed (15, 41). In vivo, HCMV infects a wide variety of cell types, including fibroblasts, monocytes, and endothelial cells. It is likely that the details of HCMV's interaction with cellular machinery differs somewhat from cell type to cell type, but many conserved pathways are also likely to exist.

Identification of the cellular transcription pathways that are activated and repressed during viral infection could lead to a better understanding of the virus-host interaction and to the identification of targets for antiviral therapy. A virus can potentially modulate the level of a cellular mRNA by a number of different mechanisms. Binding of the viral surface glycoproteins to cellular receptors can activate signal transduction pathways that lead to the activation of cellular transcription factors (3, 40). Components of the virion and immediate-early proteins are known to act as transcriptional regulators with the potential to directly affect the expression of cellular genes. Cellular transcription changes could also be caused by the activation of immune defense pathways used by the cell to limit viral replication. Furthermore, it is likely that there would be secondary effects on gene expression resulting from increased expression of cellular transcription factors, factors that affect the stability of mRNAs, or signaling molecules that activate specific transcriptional programs in cells.

A previous study from our laboratory investigated the effect of HCMV replication on cellular RNA levels at 0.66, 8, and 24 hpi using a microarray with 6,500 probe sets, and 258 genes were identified whose mRNA levels were regulated by a factor of 4 or more by the virus (43). Other viruses that have been studied in a similar manner include human immunodeficiency virus (13), poliovirus (19), echovirus (30), human T-cell leukemia virus type 1 (8), herpes simplex virus type 1 (28), and influenza virus (14). While each of these studies clearly demonstrates changes in cellular gene expression resulting from infection, it is not yet clear which changes are deliberately instigated by the virus to facilitate replication, which are part of the host cell antiviral response, and which are bystanders that do not play a specific role in the infection process.

In this study, we studied the expression profile of cellular mRNAs in HCMV-infected cells using a larger array (12,626 unique probe sets) and with a much greater density of samples (13 time points). We identified 1,425 mRNAs that are up-regulated or down-regulated (threefold or greater) during infection. Many of these genes have potentially significant roles in either the viral replication cycle or pathogenesis. A clustering program was used to organize the genes according to the time after infection at which their mRNA levels change, and distinct classes of expression profiles were observed. For the first 8 h after infection, the number of up-regulated mRNAs was significantly lower than the number of down-regulated mRNAs. By analyzing cells infected with UV-inactivated HCMV (UV-HCMV), we found that this was due to the synthesis of one or more viral proteins during the initial stage of infection that prevents the up-regulation of cellular mRNAs, some of whose products play roles in antiviral pathways, such as interferon response genes and inflammatory cytokines.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

Cells and viruses. The AD169 strain of HCMV was used. Virus particles were partially purified by centrifugation through a sorbitol cushion, resuspended in phosphate-buffered saline, and stored at -80°C. Primary human foreskin fibroblasts (HFFs) at passage 13 to 15 were maintained in medium supplemented with 10% fetal calf serum. Plates (10-cm diameter) of cells that had been confluent for at least 3 days were infected at a multiplicity of infection of 6 PFU/cell with purified virions in conditioned medium. After adsorption, additional conditioned medium was added to the cells. At the time points indicated, the medium was removed and the cells were washed once with phosphate-buffered saline and lysed with 8 ml of TRIZOL reagent (Gibco-BRL). For infections in the presence of cycloheximide, the cells were pretreated for 1 h with 100 µg of cycloheximide per ml in the conditioned medium before infection, and cycloheximide was maintained in the medium throughout the infection. For UV inactivation of viruses, purified virions were suspended in a total of 250 µl of phosphate-buffered saline and placed in a well of a six-well dish. The uncovered suspension was irradiated in a Stratagene UV-crosslinker for a period of time that was sufficient to reduce the infectivity of the virions by >105-fold. For the mixed infection of HCMV with UV-HCMV, a purified virus suspension was divided in two, and one half was irradiated and then combined with the competent virus for an infection at 4 PFU/cell.

Sample preparation and hybridization to gene chips. Frozen TRIZOL suspensions were thawed, and total RNA was purified. Five micrograms of the sample was used as a template for cDNA synthesis in a reaction that was primed with an oligonucleotide containing an oligo(dT) and a T7 RNA polymerase promoter. The cDNA was used to make biotin-labeled cRNA probes with an RNA transcript labeling kit (ENZO). The cRNA was purified to remove unincorporated ribonuclotides, and 15 µg was fragmented at 95°C for 30 min in fragmentation buffer (40 mM Tris-acetate [pH 8.1], 100 mM potassium acetate, 30 mM magnesium acetate). The fragmented cRNA was hybridized to HG-U95A arrays for 16 h at 45°C. The arrays were washed and stained according to the Affymetrix antibody amplification protocol (Affymetix Expression Analysis technical manual, 2000). Scanning was performed using an Agilent GeneChip scanner.

Data analysis. Scanned chip data sets were analyzed using the Affymetrix GeneChip analysis software. Data from the time course experiment were analyzed using mock-infected cell data as a baseline. For each time point, we extracted the fold change and difference call for each probe set and compiled it in a spreadsheet. To generate a filtered data set of genes that were considered to be significantly regulated by HCMV, a Perl script that extracted only genes that had a fold change of at least ±3 at two consecutive time points and had a difference call of I (increased), MI (marginally increased), D (decreased), or MD (marginally decreased) was used. Clustering analysis was performed using CLUSTER (10), a hierarchical clustering program obtained from the Eisen Laboratory at the University of California at Berkeley. Before clustering, the data were normalized by gene. For the time course experiment, a gene was considered to be significantly altered if it changed by threefold or greater in two sequential samples and had a difference call of I, MI, D, or MD. For the experiments performed at 6 hpi, duplicate or triplicate samples were analyzed, and a gene was considered to be significantly altered if it changed by threefold or greater in two replicates and had a difference call of I, MI, D, or MD. Data from infections in the presence of cycloheximide were analyzed using data from mock-infected, cycloheximide-treated cells as a baseline. To assign genes in the data sets to specific functional groups, a Perl script that retrieved information on the molecular function, cellular location, and biological process for each gene from the GeneOntology database (2) was used.


    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

To study cellular mRNA accumulation in HCMV-infected cells, human fibroblasts were infected and RNA was harvested at different times over a 48-h time course. RNA samples were analyzed using the Affymetrix HG-U95A array, which contains probe sets designed to detect 12,626 unique human transcripts, and the results were quantified using Affymetrix software. The GeneChip software calculates a fold change as well as a difference call for each gene; the fold change and difference call are reported as MI, D, or MD. Mock-infected cells were used as a baseline. A mRNA was considered to be significantly regulated if (i) its fold change was threefold or greater and (ii) the difference call was I, D, MI, or MD for at least two consecutive time points. It was recently demonstrated that these criteria are sufficient to keep the number of false positives at an acceptably low level (29).

Levels of numerous cellular RNAs change in response to HCMV infection. The levels of 1,425 cellular mRNAs were significantly altered during the 48-h time period. This data set is available at the website http://www.molbio.princeton.edu/labs/shenk/browneetal2001/. Of these mRNAs, 702 were increased and 723 were decreased. A portion of the genes have been previously reported to be regulated by HCMV, such as several interferon response genes, gas-1, COX-2, and adrenomedullin (43), confirming the reliability of our data set.

By plotting the number of genes from the filtered set whose mRNA levels have changed at each time point, we observed a roughly constant increase in the number of altered genes up to 24 hpi (Fig. 1A). When up-regulated and down-regulated mRNAs are plotted separately, they exhibit markedly different profiles (Fig. 1B). From 0 to 8 hpi, the number of down-regulated mRNAs greatly exceeds the number of up-regulated mRNAs, whereas from 8 to 20 h, the up-regulated mRNAs are more numerous.


View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1.   HCMV alters the mRNA level of many cellular genes over a 48-h time course. A set of 1,425 genes was identified as being altered significantly (threefold or greater) during an HCMV time course. The numbers of genes from this set that were significantly up-regulated or down-regulated (change of threefold or greater and a difference call of I, MI, D, or MD) at each time point were calculated and plotted (A). The numbers of genes up-regulated (black diamonds) and down-regulated (shaded squares) were also plotted separately (B).

Certain classes of genes were prominently affected. Consistent with previous reports (42, 43), several interferon-responsive mRNAs were strongly up-regulated by 4 hpi. It is interesting to note that the mRNA encoding the 58-kDa DNAJ-C3 protein, which inhibits the activity of the interferon-inducible kinase PKR (21), was induced by a factor of 8.6. This could be a means by which HCMV is able to bypass the block to protein synthesis caused by the activation of PKR. Indeed, influenza virus is thought to induce this protein to evade PKR activity as well (21).

Ninety-seven mRNAs encoding proteins with likely roles in cell cycle regulation and oncogenesis were modulated by the virus (Table 1). HCMV infection activates both proliferative and antiproliferative signals in infected cells. HCMV infection is known to induce quiescent (G0) cells to enter the cell cycle (5, 18), and this is reflected by the rapid down-regulation of transcription for G0-specific transcripts such as Gas-1 and p27 (44) (Table 1). Transcription of a number of the cyclins is also affected. Consistent with a previous report (18), cyclin E2 mRNA is strongly induced (6.2-fold at 20 h). Cyclin A1 and D3 mRNAs are also induced, although somewhat less strongly. Cyclins T2 and B1 mRNAs are transiently repressed early in infection, while cyclin G1 mRNA was weakly repressed late in infection. The oncogenic transcription factor c-fos and the growth repressive factor junB were rapidly and strongly, but transiently, induced (10- and 5.7-fold, respectively, at 2 h). Several tumor suppressor mRNAs, such as AIM1, DLC3, and ST5, were down-regulated during the time course, while the ets2 repressor factor was up-regulated.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Cell cycle regulators or oncogenes

CMV is able to inhibit apoptosis (7, 15, 41). We found that 25 mRNAs, encoding proapoptotic and antiapoptotic proteins, were significantly regulated by the virus (Table 2). The proapoptotic caspase 1 enzyme is down-regulated by 14 h into infection, reaching a 10.8-fold reduction by 48 hpi. The proapoptotic cytokine tumor necrosis factor alpha (TNF-alpha ), which has roles in the proliferation of T lymphocytes and activation-induced cell death (1), is greatly up-regulated (up to 130-fold) beginning at 6 hpi---presumably as part of an antiviral response. The APO-1 gene, which encodes a receptor for the apoptosis-inducing FAS protein (35) is strongly down-regulated (6.9-fold), possibly protecting infected cells from cytotoxic T-cell killing.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Apoptosis regulators

HCMV is well-known for its ability to evade normal immune response pathways such as antigen presentation (22, 25, 26, 38). Many genes with a role in the immune system were perturbed by viral infection (Table 3). Consistent with our previous study (43), several mRNAs encoding proteins with roles in inflammation were up-regulated, including COX2 and phospholipase A2. Proinflammatory cytokine mRNAs such as RANTES and interleukin 8 were also moderately induced. Strikingly, interleukin 11 mRNA was massively up-regulated (up to 78-fold) from very early times after infection. Interleukin 11 is an antiinflammatory cytokine that interacts with CD4+ lymphocytes and inhibits the development of a cell-based Th1 response in favor of an antibody-based Th2 response (4). This might be an HCMV strategy to subvert the cell-mediated Th1 response. Infection also up-regulated mRNA encoding the interleukin 1 receptor antagonist IL1RN (17.7-fold), possibly preventing this cytokine from performing its roles in inflammation and immunity. A transcription factor, TCF8, known to repress IL-2 transcription (39), was highly up-regulated (43-fold). Il-2 is secreted by activated Th cells and functions to enhance Th cell proliferation and to stimulate Tc cells to become activated cytotoxic lymphocytes (36). Although there is no evidence that HCMV replicates in lymphocytes, it is possible that virions can either contact a receptor on the cell surface or enter these cells and elicit the up-regulation of this gene. HCMV might hinder Th cell function even without undergoing productive replication.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   Immune system regulators

Modest correlation between time clusters and function-based groupings of cellular mRNAs. The data were imported into the hierarchical clustering program CLUSTER (10). The expression profiles were found to be complex: at least 14 distinct clusters of mRNAs whose levels changed in a coordinated fashion as a function of time were observed (Fig. 2A). We did not observe a strong tendency for particular functional groups to be associated with a specific time-based cluster. Indeed, cluster analysis of a specific functional group such as cell cycle genes (Fig. 2B) or interferon-responsive genes (Fig. 2C) highlighted the fact that even within each functional class, several different patterns of expression were discernible. However, it is important to note that functional groupings are imprecise because the functions of most genes remain incompletely understood. It is likely that many of the genes in the same time-related cluster are coregulated by common transcriptional activators.


View larger version (50K):
[in this window]
[in a new window]
  		FIG. 2.   HCMV induced altered mRNA cellular mRNA levels in infected cells. Expression data for 1,425 genes judged as being significantly altered by HCMV were normalized by gene and clustered using the hierarchical clustering program CLUSTER. The clusters were then visualized using the TREEVIEW program (A). Putative clusters are noted by numbers to the right of the image. Elevated (red) and reduced (green) mRNA levels are indicated. Genes with functions relating to cell cycle, cell proliferation, or oncogenesis were identified by a Perl program that annotated Affymetrix GeneChip data with information about the biological function of the genes using the GeneOntology database. These genes were then clustered separately and visualized using TREEVIEW (B). Alpha-interferon-sensitive genes found to be modulated by HCMV were also clustered separately (C).

Changes in cellular mRNA levels that occur independently of protein synthesis after infection. To identify the changes in cellular mRNA levels that result directly from binding and entry of HCMV in the absence of new protein synthesis, we infected fibroblasts in the presence of cycloheximide and harvested RNA for analysis 6 h later. Cells that were mock infected in the presence of cycloheximide were used as a control. A total of 8 mRNAs were up-regulated and 17 mRNAs were down-regulated (Table 4). This is clearly an underestimate of the changes that occur in infection independently of new protein synthesis, since cycloheximide alone is known to activate inflammatory pathways that HCMV also activates, such as the transcription factor NF-kappa B (33). Indeed, by comparing mock-infected, cycloheximide-treated cells to mock-infected cells that were not treated with the drug, it was observed that 53 mRNAs that change significantly in HCMV infection by 6 hpi were also activated by cycloheximide alone (data not shown). Four interferon-responsive mRNAs were among those up-regulated by HCMV in the presence of cycloheximide, including beta-interferon itself. No known interferon-responsive genes were induced by cycloheximide in the absence of infection.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 4.   Genes modulated by HCMV in the presence of cycloheximid

It has been shown previously that binding of glycoprotein B to the surface of cells is sufficient to induce activation of interferon response genes (3) as well as NF-kappa B (40), suggesting that a signal transduction cascade is activated by the binding of gB or the intact virion to its fusion receptor. Alternatively, it is possible that many or most of these changes are not gB specific but rather are caused by the triggering of an innate defensive pathway that detects the attachment or entry of a variety of different virus particles and leads directly to the transcription of a small subset of interferon response genes, including beta-interferon. A similar pathway was recently reported to be activated by herpes simplex virus (28), although this report did not distinguish between genes whose transcription is directly triggered by the virus and genes that are induced as a secondary consequence of interferon synthesis. This pathway seems to be distinct from the JAK-STAT interferon signaling pathway, since a U2OS cell line that was defective for the induction of interferon response genes during infection with herpes simplex virus, was still responsive to the addition of interferon (28).

More cellular mRNAs are induced by UV-HCMV than by HCMV. To monitor the effect of newly synthesized viral proteins on cellular gene expression, we compared the effects of infection with HCMV to UV-HCMV at 6 hpi. UV-inactivated virus delivers virion proteins to the cell, but virion DNA and mRNAs (6) are damaged and cannot be expressed. The levels of 161 mRNAs were altered by HCMV; UV-HCMV, by contrast, affected 340 (Fig. 3). The large increase in the number of mRNAs affected by UV-HCMV comes predominantly from mRNAs that are up-regulated. It is likely that the accumulation of this set of cellular mRNAs is normally blocked by a newly synthesized viral protein, a protein that is not produced in cells infected with the UV-irradiated virus.


View larger version (13K):
[in this window]
[in a new window]
  		FIG. 3.   HCMV replication represses the up-regulation of cellular genes. HFFs were infected at 6 PFU/cell with purified AD169 virus particles. For UV-irradiated HCMV, virus samples were exposed to UV radiation sufficient to reduce infectivity by 105-fold (data not shown). Infections were carried out in duplicate, and at 6 hpi, the cells were lysed and RNA samples were extracted and analyzed by Affymetrix GeneChip analysis to determine the number of up-regulated or down-regulated genes. Genes that changed by threefold or greater and had a difference call of I, MI, D, or MD in both replicates were considered to be significantly regulated. The total number of genes up and down-regulated is shown. CHX-HCMV, HCMV treated with cycloheximide.

We next identified the cellular mRNAs that changed in response to infection with one or both of the virus preparations (HCMV and UV-HCMV). The numbers of mRNAs in the subsets listed in Tables 5 to 7 are not equal to the total numbers of genes displayed in Fig. 3. This is because in many instances, an unambiguous change in a specific mRNA was evident after infection with one of the virus preparations, say HCMV, but the change was borderline and difficult to call for the other virus, i.e., UV-HCMV. Cellular mRNAs were placed into the categories of Tables 5 to 7 only when the data for both virus preparations allowed a clear determination.

Eighty-two mRNAs (Table 5) changed by a factor of at least 3 after infection with both HCMV and UV-HCMV. Presumably, this set of changes occurs as a result of binding and entry of virus particles and/or the action of virion proteins within the infected cell. This set included mRNAs encoding cell cycle regulators that change early in HCMV infection, such as cyclin E, gas-1, and p27. This indicates that the ability of HCMV to cause G0 cells to reenter the cell cycle is probably mediated by virus binding or a tegument protein. Consistent with this hypothesis, the tegument protein pp71 can induce quiescent cells to reenter the cell cycle (R. Kalejita, J. Bechtel, and T. Shenk, submitted for publication).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 5.   Genes modulated similarly by HCMV and UV-HCMV

Fifteen mRNAs (Table 6) were found to be significantly regulated at 6 hpi by HCMV but not by UV-HCMV, presumably representing a subset of mRNAs whose regulation in infection is dependent on new viral protein synthesis, i.e., they were not modulated by entry or virion proteins. Of these, 1 was up-regulated and 14 were down-regulated.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 6.   Genes modulated by HCMV but not UV-HCMV

One hundred seventeen mRNAs (Table 7) were modulated by UV-HCMV but not HCMV. The majority (82 genes) were up-regulated, representing a set of mRNAs whose up-regulation is normally prevented by a newly synthesized viral protein. This list included many known antiviral genes, including members of the interferon response pathway and proinflammatory cytokines.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 7.   Genes modulated by UV-HCMV but not HCMV

We next wanted to determine whether HCMV modulated the expression of all or a subset of the interferon-responsive genes on the 12,626 gene array. To identify the full set of interferon-responsive genes on the array, uninfected human fibroblasts were treated with alpha-interferon for 6 h, and RNA was prepared and analyzed. Interferon induced 79 mRNAs (Fig. 4A) and reduced the level of only one. Of the 161 genes that change in HCMV-infected cells by 6 hpi, only 17 (Fig. 4A) of these overlap with the set that are activated by alpha-interferon. However, when fibroblasts were infected with UV-HCMV, a much larger subset, 50 genes (Fig. 4A) of the interferon-sensitive genes were found to be significantly up-regulated. To rule out the possibility that the interferon response pathway is being activated by the presence of UV-damaged DNA in infected cells, we carried out a mixed infection of HCMV with UV-HCMV. If interferon-sensitive genes are induced by damaged viral DNA, then the inclusion of functional virus will not block their induction. However, the mixed infection led to the up-regulation of only 25 interferon response genes at 6 hpi (Fig. 4A), and the genes that continued to score were induced to a lesser extent. Since the inclusion of HCMV substantially blocked the induction of interferon-responsive mRNAs, we conclude that the lack of a viral gene product, rather than the presence of damaged DNA, accounts for the larger number of interferon response genes up-regulated by UV-HCMV. It has been reported previously that HCMV infection causes a block in the interferon signaling pathway by inducing degradation of JAK1 and p48 (27). However, this was not observed until 48 hpi. Since the interferon response is substantially blocked at 6 hpi, it is likely that a different mechanism is involved, a possibility that is consistent with our observation that HCMV synthesizes one or more gene products that inhibit cellular mRNA accumulation. Interferon regulatory factor 1 (IRF-1) and IRF-4, two proteins that play a role in beta-interferon transcription (24), were much more greatly up-regulated after infection with UV-HCMV than HCMV (Fig. 4B). Consequently, it is possible that the early block to the accumulation of interferon-responsive mRNAs results primarily from a block to transcription of the beta-interferon gene---indeed, beta-interferon transcription was much more highly induced by UV-HCMV than by HCMV (Fig. 4B). A specific subset of interferon-responsive mRNAs continues to accumulate to high levels in the presence of this block. However, many of these mRNAs appear to be expressed at substantially lower levels during infection with HCMV than with UV-HCMV as well.


View larger version (13K):
[in this window]
[in a new window]
  		FIG. 4.   HCMV replication represses activation of the interferon response pathway. Human foreskin fibroblast cells were treated with 5,000 U of alpha-interferon per ml for 6 h. The cells were then lysed, and RNA was analyzed by Affymetrix GeneChip analysis. Seventy-nine genes were found be up-regulated by interferon in duplicate, and the proportion of these genes that were also up-regulated by HCMV, by HCMV in the presence of cycloheximide (CHX), and by UV-HCMV at 6 hpi were counted and graphed (A). The fold inductions for beta-interferon, IRF1, and IRF4 were averaged for two replicates and then graphed (B). IFN, interferon.

Recently it was reported that the changes in mRNA levels occurring in HCMV-infected fibroblasts at 8 or 24 hpi are not significantly different from those that occur following treatment with interferon or a recombinant soluble form of the HCMV glycoprotein B (34). By contrast, our data reveal that at 6 hpi, many interferon-responsive mRNAs fail to accumulate due to active suppression by the virus (Fig. 4A). Furthermore, many mRNAs that change in infection are not responsive to interferon. We cannot yet explain the difference between the two data sets.

Accumulation of several proinflammatory chemokine mRNAs was also prevented by one or more newly synthesized viral gene products at 6 hpi (Fig. 5). RANTES, interleukin 8, HuMIG, MIP1a, and MIP3b were all strongly induced by UV-HCMV but not HCMV. This repression of inflammatory chemokine expression would conceivably prevent the recruitment of immune cells such as lymphocytes and macrophages to the site of infection.


View larger version (11K):
[in this window]
[in a new window]
  		FIG. 5.   HCMV replication represses activation of proinflammatory chemokines. Human foreskin fibroblast cells were infected for 6 h with HCMV or UV-HCMV at 6 PFU/cell, before RNA was extracted and used for Affymetrix GeneChip analysis. The fold inductions for the genes shown above were averaged for two replicates. IL-8, interleukin 8; HuMIG, human inducer of gamma interferon; MIP1a and MIP3b, macrophage inflammatory protein 1a and 3b, respectively.

HCMV also prevents the accumulation of mRNAs that could potentially block cell cycle progression. The transcription factor junB, which negatively regulates the growth-promoting activities of c-jun (32), is rapidly induced in the infection time course but returns to background levels by 4 to 6 hpi. In cells infected with UV-HCMV, however, junB up-regulation remains high at 6 hpi.

Conclusion. Our array analysis has revealed an active, virus-mediated repression of cellular mRNA accumulation taking place during the first 6 h of the infection. During this time period, the number of up-regulated mRNAs lags significantly behind the number of down-regulated mRNAs (Fig. 1B). The repression is not evident at 6 h after infection of cells with UV-inactivated virus (Fig. 3), i.e., more cellular mRNAs accumulate in UV-HCMV-infected cells than in HCMV-infected cells, allowing us to conclude that the repression results from a newly synthesized viral gene product. This gene product blocks the accumulation of a variety of cellular mRNAs including those encoding antiviral proteins. The number of up-regulated mRNAs rises dramatically from 10 to 16 hpi, presumably preparing the cell for the start of viral DNA replication at about 24 hpi. A similar repressive effect of herpes simplex virus on interferon-responsive genes has been reported recently (28).

The identity of the HCMV protein or proteins that inhibit accumulation of cellular mRNAs with antiviral activities, and the mechanism by which they act remain unknown. Since repression occurs very rapidly after infection, it must result from the activity of an immediate-early gene product or an early gene product that is expressed very rapidly after infection. The viral product might function as a transcriptional repressor, directly affecting the transcription of cellular genes; it could act indirectly, up-regulating a cellular repressor; or it could act posttranscriptionally to influence mRNA levels.


    ACKNOWLEDGMENT

This work was supported by a grant from the National Cancer Institute (CA87661).


    FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014. Phone: (609) 258-5992. Fax: (609) 258-1704. E-mail: tshenk{at}princeton.edu.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

1. Aggarwal, B. B. 2000. Tumour necrosis factor receptor associated signalling molecules and their role in activation of apoptosis, JNK and NF-kappa B. Ann. Rheum. Dis. 59(Suppl. 1):i6-16[Abstract/Free Full Text].
2. Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler, J. M. Cherry, A. P. Davis, K. Dolinski, S. S. Dwight, J. T. Eppig, M. A. Harris, D. P. Hill, L. Issel-Tarver, A. Kasarskis, S. Lewis, J. C. Matese, J. E. Richardson, M. Ringwald, G. M. Rubin, and G. Sherlock. 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25:25-29[CrossRef][Medline].
3. Boyle, K. A., R. L. Pietropaolo, and T. Compton. 1999. Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol. Cell. Biol. 19:3607-3613[Abstract/Free Full Text].
4. Bozza, M., J. L. Bliss, A. J. Dorner, and W. L. Trepicchio. 2001. Interleukin-11 modulates Th1/Th2 cytokine production from activated CD4+ T cells. J. Interferon Cytokine Res. 21:21-30[CrossRef][Medline].
5. Bresnahan, W. A., I. Bolldogh, E. A. Thompson, and T. Albrecht. 1996. Human cytomegalovirus inhibits cellular DNA synthesis and arrests productively infected cells in late G1. Virology 224:150-160[CrossRef][Medline].
6. Bresnahan, W. A., and T. Shenk. 2000. A subset of viral transcripts packaged within human cytomegalovirus particles. Science 288:2373-2376[Abstract/Free Full Text].
7. Brune, W., C. Menard, J. Heesemann, and U. H. Koszinowski. 2001. A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism. Science 291:303-305[Abstract/Free Full Text].
8. de La Fuente, C, L. Deng, F. Santiago, L. Arce, L. Wang, and F. Kashanchi. 2000. Gene expression array of HTLV type 1-infected T cells: up-regulation of transcription factors and cell cycle genes. AIDS Res. Hum. Retroviruses 16:1695-1700[CrossRef][Medline].
9. Dittmer, D., and E. S. Mocarski. 1997. Human cytomegalovirus infection inhibits G1/S transition. J. Virol. 71:1629-1634[Abstract].
10. Eisen, M. B., P. T. Spellman, P. O. Brown, and D. Botstein. 1998. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95:14863-14868[Abstract/Free Full Text].
11. Fortunato, E. A., A. K. McElroy, V. Sanchez, and D. Spector. 2000. Exploitation of cellular signaling and regulatory pathways by human cytomegalovirus. Trends Microbiol. 8:111-119[CrossRef][Medline].
12. Garnett, H. M. 1981. Increased ability of human embryonic fibroblasts to accumulate Ca2+ due to cytomegalovirus infection. Cytobios 31:107-116[Medline].
13. Geiss, G. K., R. E. Bumgarner, M. C. An, M. B. Agy, A. B. van't Wout, E. Hammersmark, V. S. Carter, D. Upchurch, J. I. Mullins, and M. G. Katze. 2000. Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays. Virology 266:8-16[CrossRef][Medline].
14. Geiss, G. K., M. C. An, R. E. Bumgarner, E. Hammersmark, D. Cunningham, and M. G. Katze. 2001. Global impact of influenza virus on cellular pathways is mediated by both replication-dependent and -independent events. J. Virol. 75:4321-4331[Abstract/Free Full Text].
15. Goldmacher, V. S., L. M. Bartle, A. Skaletskaya, C. A. Dionne, N. L. Kedersha, C. A. Vater, J. W. Han, R. J. Lutz, S. Watanabe, E. D. Cahir McFarland, E. D. Kieff, E. S. Mocarski, and T. Chittenden. 1999. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc. Natl. Acad. Sci. USA 96:12536-12541[Abstract/Free Full Text].
16. Huang, E. S. 1975. Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid. J. Virol. 16:1560-1565[Abstract/Free Full Text].
17. Jahn, G., E. Knust, H. Schmolla, T. Sarre, J. A. Nelson, J. K. McDougall, and B. Fleckenstein. 1984. Predominant immediate-early transcripts of human cytomegalovirus AD 169. J. Virol. 49:363-370[Abstract/Free Full Text].
18. Jault, F. M., J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector. 1995. Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest. J. Virol. 69:6697-6704[Abstract].
19. Johannes, G., M. S. Carter, M. B. Eisen, P. O. Brown, and P. Sarnow. 1999. Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray. Proc. Natl. Acad. Sci. USA 96:13118-13123[Abstract/Free Full Text].
20. Kowalik, T. F., B. Wing, J. S. Haskill, J. C. Azizkhan, A. S. Baldwin, Jr., and E. S. Huang. 1993. Multiple mechanisms are implicated in the regulation of NF-kappa B activity during human cytomegalovirus infection. Proc. Natl. Acad. Sci. USA 90:1107-1111[Abstract/Free Full Text].
21. Lee, T. G., N. Tang, S. Thompson, J. Miller, and M. G. Katze. 1994. The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins. Mol. Cell. Biol. 14:2331-2342[Abstract/Free Full Text].
22. Loenen, W. A., C. A. Bruggeman, and E. J. Wiertz. 2001. Immune evasion by human cytomegalovirus: lessons in immunology and cell biology. Semin. Immunol. 13:41-49[CrossRef][Medline].
23. Lu, M., and T. Shenk. 1996. Human cytomegalovirus infection inhibits cell cycle progression at multiple points, including the transition from G1 to S. J. Virol. 70:8850-8857[Abstract].
24. Marecki, S., C. J. Riendeau, M. D. Liang, and M. J. Fenton. 2001. Pu.1 and multiple ifn regulatory factor proteins synergize to mediate transcriptional activation of the human il-1beta gene. J. Immunol. 166:6829-6838[Abstract/Free Full Text].
25. Mocarski, E. S., and C. T. Courcelle. 2001. Cytomegaloviruses and their replication. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Strauss (ed.), Fields virology, 4th ed. Lippincott-Raven, Philadelphia, Pa.
26. Michelson, S. 1999. Human cytomegalovirus escape from immune detection. Intervirology 42:301-307[CrossRef][Medline].
27. Miller, D. M., Y. Zhang, B. M. Rahill, W. J. Waldman, and D. D. Sedmak. 1999. Human cytomegalovirus inhibits IFN-alpha -stimulated antiviral and immunoregulatory responses by blocking multiple levels of IFN-alpha signal transduction. J. Immunol. 162:6107-6113[Abstract/Free Full Text].
28. Mossman, K. L., P. F. Macgregor, J. J. Rozmus, A. B. Goryachev, A. M. Edwards, and J. R. Smiley. 2001. Herpes simplex virus triggers and then disarms a host antiviral response. J. Virol. 75:750-758[Abstract/Free Full Text].
29. Muller, H., A. P. Bracken, R. Vernell, M. C. Moroni, F. Christians, E. Grassilli, E. Prosperini, E. Vigo, J. D. Oliner, and K. Helin. 2001. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev. 15:267-285[Abstract/Free Full Text].
30. Pietiainen, V., P. Huttunen, and T. Hyypia. 2000. Effects of echovirus 1 infection on cellular gene expression. Virology 276:243-250[CrossRef][Medline].
31. Rodems, S. M., and D. H. Spector. 1998. Extracellular signal-regulated kinase activity is sustained early during human cytomegalovirus infection. J. Virol. 72:9173-9180[Abstract/Free Full Text].
32. Schutte, J., J. Viallet, M. Nau, S. Segal, J. Fedorko, and J. Minna. 1989. jun-B inhibits and c-fos stimulates the transforming and trans-activating activities of c-jun. Cell 59:987-997[CrossRef][Medline].
33. Sen, R., and D. Baltimore. 1986. Inducibility of kappa immunoglobulin enhancer-binding protein NF-kappa B by a posttranslational mechanism. Cell 47:921-928[CrossRef][Medline].
34. Simmen, K. A., J. Singh, B. G. Luukkonen, M. Lopper, A. Bittner, N. E. Miller, M. R. Jackson, T. Compton, and K. Fruh. 2001. Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc. Natl. Acad. Sci. USA 98:7140-7145[Abstract/Free Full Text].
35. Smyth, M. J., J. M. Kelly, V. R. Sutton, J. E. Davis, K. A. Browne, T. J. Sayers, and J. A. Trapani. Unlocking the secrets of cytotoxic granule proteins. J. Leukoc. Biol. 70:18-29.
36. Swain, S. L. 1995. CD4 T cell development and cytokine polarization: an overview. J. Leukoc. Biol. 57:795-798[Abstract].
37. Valyi-Nagy, T., Z. Bandi, J. Boldogh, and T. Albrecht. 1988. Hydrolysis of inositol lipids: an early signal of human cytomegalovirus infection. Arch. Virol. 101:199-207[CrossRef][Medline].
38. Wiertz, E., A. Hill, D. Tortorella, and H. Ploegh. 1997. Cytomegaloviruses use multiple mechanisms to elude the host immune response. Immunol. Lett. 57:213-216[CrossRef][Medline].
39. Williams, T. M., G. Montoya, Y. Wu, R. L. Eddy, M. G. Byers, and T. B. Shows. 1992. The TCF8 gene encoding a zinc finger protein (Nil-2-a) resides on human chromosome 10p11.2. Genomics 14:194-196[CrossRef][Medline].
40. Yurochko, A. D., M. W. Mayo, E. E. Poma, A. S. Baldwin, Jr., and E. S. Huang. 1997. Induction of the transcription factor Sp1 during human cytomegalovirus infection mediates upregulation of the p65 and p105/p50 NF-kappa B promoters. J. Virol. 71:4638-4648[Abstract].
41. Zhu, H., Y. Shen, and T. Shenk. 1995. Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J. Virol. 69:7960-7970[Abstract].
42. Zhu, H., J. P. Cong, and T. Shenk. 1997. Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular mRNAs: induction of interferon-responsive RNAs. Proc. Natl. Acad. Sci. USA 94:13985-13990[Abstract/Free Full Text].
43. Zhu, H., J. P. Cong, G. Mamtora, T. Gingeras, and T. Shenk. 1998. Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95:14470-14475[Abstract/Free Full Text].

Journal of Virology, December 2001, p. 12319-12330, Vol. 75, No. 24
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.24.12319-12330.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Kamil, J. P., Hume, A. J., Jurak, I., Munger, K., Kalejta, R. F., Coen, D. M. (2009). Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase. Proc. Natl. Acad. Sci. USA 106: 16823-16828 [Abstract] [Full Text]  
  • Schroer, J., Shenk, T. (2008). Inhibition of cyclooxygenase activity blocks cell-to-cell spread of human cytomegalovirus. Proc. Natl. Acad. Sci. USA 105: 19468-19473 [Abstract] [Full Text]  
  • Machesky, N. J., Zhang, G., Raghavan, B., Zimmerman, P., Kelly, S. L., Merrill, A. H. Jr., Waldman, W. J., Van Brocklyn, J. R., Trgovcich, J. (2008). Human Cytomegalovirus Regulates Bioactive Sphingolipids. J. Biol. Chem. 283: 26148-26160 [Abstract] [Full Text]  
  • Wang, F.-Z., Weber, F., Croce, C., Liu, C.-G., Liao, X., Pellett, P. E. (2008). Human Cytomegalovirus Infection Alters the Expression of Cellular MicroRNA Species That Affect Its Replication. J. Virol. 82: 9065-9074 [Abstract] [Full Text]  
  • Chan, G., Bivins-Smith, E. R., Smith, M. S., Smith, P. M., Yurochko, A. D. (2008). Transcriptome Analysis Reveals Human Cytomegalovirus Reprograms Monocyte Differentiation toward an M1 Macrophage. J. Immunol. 181: 698-711 [Abstract] [Full Text]  
  • Kalejta, R. F. (2008). Tegument Proteins of Human Cytomegalovirus. Microbiol. Mol. Biol. Rev. 72: 249-265 [Abstract] [Full Text]  
  • Juckem, L. K., Boehme, K. W., Feire, A. L., Compton, T. (2008). Differential Initiation of Innate Immune Responses Induced by Human Cytomegalovirus Entry into Fibroblast Cells. J. Immunol. 180: 4965-4977 [Abstract] [Full Text]  
  • Wang, D., Yu, Q.-C., Schroer, J., Murphy, E., Shenk, T. (2007). Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells. Proc. Natl. Acad. Sci. USA 104: 20037-20042 [Abstract] [Full Text]  
  • Djavani, M. M., Crasta, O. R., Zapata, J. C., Fei, Z., Folkerts, O., Sobral, B., Swindells, M., Bryant, J., Davis, H., Pauza, C. D., Lukashevich, I. S., Hammamieh, R., Jett, M., Salvato, M. S. (2007). Early Blood Profiles of Virus Infection in a Monkey Model for Lassa Fever. J. Virol. 81: 7960-7973 [Abstract] [Full Text]  
  • Stanton, R. J., McSharry, B. P., Rickards, C. R., Wang, E. C. Y., Tomasec, P., Wilkinson, G. W. G. (2007). Cytomegalovirus Destruction of Focal Adhesions Revealed in a High-Throughput Western Blot Analysis of Cellular Protein Expression. J. Virol. 81: 7860-7872 [Abstract] [Full Text]  
  • van den Pol, A. N., Robek, M. D., Ghosh, P. K., Ozduman, K., Bandi, P., Whim, M. D., Wollmann, G. (2007). Cytomegalovirus InducesInterferon-Stimulated Gene Expression and Is Attenuated by Interferon in the Developing Brain. J. Virol. 81: 332-348 [Abstract] [Full Text]  
  • Hakki, M., Marshall, E. E., De Niro, K. L., Geballe, A. P. (2006). Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1. J. Virol. 80: 11817-11826 [Abstract] [Full Text]  
  • Montag, C., Wagner, J., Gruska, I., Hagemeier, C. (2006). Human Cytomegalovirus Blocks Tumor Necrosis Factor Alpha- and Interleukin-1{beta}-Mediated NF-{kappa}B Signaling. J. Virol. 80: 11686-11698 [Abstract] [Full Text]  
  • Taylor, R. T., Bresnahan, W. A. (2006). Human Cytomegalovirus IE86 Attenuates Virus- and Tumor Necrosis Factor Alpha-Induced NF{kappa}B-Dependent Gene Expression.. J. Virol. 80: 10763-10771 [Abstract] [Full Text]  
  • Shlapobersky, M., Sanders, R., Clark, C., Spector, D. H. (2006). Repression of HMGA2 Gene Expression by Human Cytomegalovirus Involves the IE2 86-Kilodalton Protein and Is Necessary for Efficient Viral Replication and Inhibition of Cyclin A Transcription.. J. Virol. 80: 9951-9961 [Abstract] [Full Text]  
  • Feng, X., Schroer, J., Yu, D., Shenk, T. (2006). Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle.. J. Virol. 80: 8371-8378 [Abstract] [Full Text]  
  • Tang, Q., Maul, G. G. (2006). Mouse cytomegalovirus crosses the species barrier with help from a few human cytomegalovirus proteins.. J. Virol. 80: 7510-7521 [Abstract] [Full Text]  
  • Peng, T., Kotla, S., Bumgarner, R. E., Gustin, K. E. (2006). Human rhinovirus attenuates the type I interferon response by disrupting activation of interferon regulatory factor 3.. J. Virol. 80: 5021-5031 [Abstract] [Full Text]  
  • Xu, D., Brumm, K., Zhang, L. (2006). The Latent Membrane Protein 1 of Epstein-Barr Virus (EBV) Primes EBV Latency Cells for Type I Interferon Production. J. Biol. Chem. 281: 9163-9169 [Abstract] [Full Text]  
  • Paulus, C., Krauss, S., Nevels, M. (2006). A human cytomegalovirus antagonist of type I IFN-dependent signal transducer and activator of transcription signaling. Proc. Natl. Acad. Sci. USA 103: 3840-3845 [Abstract] [Full Text]  
  • Yuan, J., Cahir-McFarland, E., Zhao, B., Kieff, E. (2006). Virus and Cell RNAs Expressed during Epstein-Barr Virus Replication. J. Virol. 80: 2548-2565 [Abstract] [Full Text]  
  • Paulus, C., Sollars, P. J., Pickard, G. E., Enquist, L. W. (2006). Transcriptome Signature of Virulent and Attenuated Pseudorabies Virus-Infected Rodent Brain. J. Virol. 80: 1773-1786 [Abstract] [Full Text]  
  • Taylor, R. T., Bresnahan, W. A. (2006). Human Cytomegalovirus Immediate-Early 2 Protein IE86 Blocks Virus-Induced Chemokine Expression. J. Virol. 80: 920-928 [Abstract] [Full Text]  
  • DeFilippis, V. R., Robinson, B., Keck, T. M., Hansen, S. G., Nelson, J. A., Fruh, K. J. (2006). Interferon Regulatory Factor 3 Is Necessary for Induction of Antiviral Genes during Human Cytomegalovirus Infection. J. Virol. 80: 1032-1037 [Abstract] [Full Text]  
  • Noyce, R. S., Collins, S. E., Mossman, K. L. (2006). Identification of a Novel Pathway Essential for the Immediate-Early, Interferon-Independent Antiviral Response to Enveloped Virions. J. Virol. 80: 226-235 [Abstract] [Full Text]  
  • Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. (2005). Molecular Biology of Pseudorabies Virus: Impact on Neurovirology and Veterinary Medicine. Microbiol. Mol. Biol. Rev. 69: 462-500 [Abstract] [Full Text]  
  • Hahn, A. M., Huye, L. E., Ning, S., Webster-Cyriaque, J., Pagano, J. S. (2005). Interferon Regulatory Factor 7 Is Negatively Regulated by the Epstein-Barr Virus Immediate-Early Gene, BZLF-1. J. Virol. 79: 10040-10052 [Abstract] [Full Text]  
  • DeFilippis, V., Fruh, K. (2005). Rhesus Cytomegalovirus Particles Prevent Activation of Interferon Regulatory Factor 3. J. Virol. 79: 6419-6431 [Abstract] [Full Text]  
  • Zhang, J., Wang, J., Wood, C., Xu, D., Zhang, L. (2005). Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Replication and Transcription Activator Regulates Viral and Cellular Genes via Interferon-Stimulated Response Elements. J. Virol. 79: 5640-5652 [Abstract] [Full Text]  
  • Britt, W. J., Jarvis, M. A., Drummond, D. D., Mach, M. (2005). Antigenic Domain 1 Is Required for Oligomerization of Human Cytomegalovirus Glycoprotein B. J. Virol. 79: 4066-4079 [Abstract] [Full Text]  
  • Taylor, R. T., Bresnahan, W. A. (2005). Human Cytomegalovirus Immediate-Early 2 Gene Expression Blocks Virus-Induced Beta Interferon Production. J. Virol. 79: 3873-3877 [Abstract] [Full Text]  
  • Reinhardt, B., Schaarschmidt, P., Bossert, A., Luske, A., Finkenzeller, G., Mertens, T., Michel, D. (2005). Upregulation of functionally active vascular endothelial growth factor by human cytomegalovirus. J. Gen. Virol. 86: 23-30 [Abstract] [Full Text]  
  • Reinhardt, J., Smith, G. B., Himmelheber, C. T., Azizkhan-Clifford, J., Mocarski, E. S. (2005). The Carboxyl-Terminal Region of Human Cytomegalovirus IE1491aa Contains an Acidic Domain That Plays a Regulatory Role and a Chromatin-Tethering Domain That Is Dispensable during Viral Replication. J. Virol. 79: 225-233 [Abstract] [Full Text]  
  • Gealy, C., Denson, M., Humphreys, C., McSharry, B., Wilkinson, G., Caswell, R. (2005). Posttranscriptional Suppression of Interleukin-6 Production by Human Cytomegalovirus. J. Virol. 79: 472-485 [Abstract] [Full Text]  
  • Hertel, L., Mocarski, E. S. (2004). Global Analysis of Host Cell Gene Expression Late during Cytomegalovirus Infection Reveals Extensive Dysregulation of Cell Cycle Gene Expression and Induction of Pseudomitosis Independent of US28 Function. J. Virol. 78: 11988-12011 [Abstract] [Full Text]  
  • Zhang, J., Das, S. C., Kotalik, C., Pattnaik, A. K., Zhang, L. (2004). The Latent Membrane Protein 1 of Epstein-Barr Virus Establishes an Antiviral State via Induction of Interferon-stimulated Genes. J. Biol. Chem. 279: 46335-46342 [Abstract] [Full Text]  
  • Abate, D. A., Watanabe, S., Mocarski, E. S. (2004). Major Human Cytomegalovirus Structural Protein pp65 (ppUL83) Prevents Interferon Response Factor 3 Activation in the Interferon Response. J. Virol. 78: 10995-11006 [Abstract] [Full Text]  
  • Wang, S.-K., Duh, C.-Y., Wu, C.-W. (2004). Human Cytomegalovirus UL76 Encodes a Novel Virion-Associated Protein That Is Able To Inhibit Viral Replication. J. Virol. 78: 9750-9762 [Abstract] [Full Text]  
  • Chan, G., Stinski, M.F., Guilbert, L.J. (2004). Human Cytomegalovirus-Induced Upregulation of Intercellular Cell Adhesion Molecule-1 on Villous Syncytiotrophoblasts. Biol. Reprod. 71: 797-803 [Abstract] [Full Text]  
  • Challacombe, J. F., Rechtsteiner, A., Gottardo, R., Rocha, L. M., Browne, E. P., Shenk, T., Altherr, M. R., Brettin, T. S. (2004). Evaluation of the host transcriptional response to human cytomegalovirus infection. Physiol. Genomics 18: 51-62 [Abstract] [Full Text]  
  • Slobedman, B., Stern, J. L., Cunningham, A. L., Abendroth, A., Abate, D. A., Mocarski, E. S. (2004). Impact of Human Cytomegalovirus Latent Infection on Myeloid Progenitor Cell Gene Expression. J. Virol. 78: 4054-4062 [Abstract] [Full Text]  
  • Ray, N., Enquist, L. W. (2004). Transcriptional Response of a Common Permissive Cell Type to Infection by Two Diverse Alphaherpesviruses. J. Virol. 78: 3489-3501 [Abstract] [Full Text]  
  • Eickhoff, J., Hanke, M., Stein-Gerlach, M., Kiang, T. P., Herzberger, K., Habenberger, P., Muller, S., Klebl, B., Marschall, M., Stamminger, T., Cotten, M. (2004). RICK Activates a NF-{kappa}B-dependent Anti-human Cytomegalovirus Response. J. Biol. Chem. 279: 9642-9652 [Abstract] [Full Text]  
  • Lin, R., Noyce, R. S., Collins, S. E., Everett, R. D., Mossman, K. L. (2004). The Herpes Simplex Virus ICP0 RING Finger Domain Inhibits IRF3- and IRF7-Mediated Activation of Interferon-Stimulated Genes. J. Virol. 78: 1675-1684 [Abstract] [Full Text]  
  • Collins, S. E., Noyce, R. S., Mossman, K. L. (2004). Innate Cellular Response to Virus Particle Entry Requires IRF3 but Not Virus Replication. J. Virol. 78: 1706-1717 [Abstract] [Full Text]  
  • Boehme, K. W., Singh, J., Perry, S. T., Compton, T. (2004). Human Cytomegalovirus Elicits a Coordinated Cellular Antiviral Response via Envelope Glycoprotein B. J. Virol. 78: 1202-1211 [Abstract] [Full Text]  
  • Benedict, C. A., Angulo, A., Patterson, G., Ha, S., Huang, H., Messerle, M., Ware, C. F., Ghazal, P. (2004). Neutrality of the Canonical NF-{kappa}B-Dependent Pathway for Human and Murine Cytomegalovirus Transcription and Replication In Vitro. J. Virol. 78: 741-750 [Abstract] [Full Text]  
  • Su, Y., Adair, R., Davis, C. N., DiFronzo, N. L., Colberg-Poley, A. M. (2003). Convergence of RNA cis Elements and Cellular Polyadenylation Factors in the Regulation of Human Cytomegalovirus UL37 Exon 1 Unspliced RNA Production. J. Virol. 77: 12729-12741 [Abstract] [Full Text]  
  • Stilwell, J. L., McCarty, D. M., Negishi, A., Superfine, R., Samulski, R. J. (2003). Development and Characterization of Novel Empty Adenovirus Capsids and Their Impact on Cellular Gene Expression. J. Virol. 77: 12881-12885 [Abstract] [Full Text]  
  • Browne, E. P., Shenk, T. (2003). Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells. Proc. Natl. Acad. Sci. USA 100: 11439-11444 [Abstract] [Full Text]  
  • Minisini, R., Tulone, C., Luske, A., Michel, D., Mertens, T., Gierschik, P., Moepps, B. (2003). Constitutive Inositol Phosphate Formation in Cytomegalovirus-Infected Human Fibroblasts Is due to Expression of the Chemokine Receptor Homologue pUS28. J. Virol. 77: 4489-4501 [Abstract] [Full Text]  
  • Compton, T., Kurt-Jones, E. A., Boehme, K. W., Belko, J., Latz, E., Golenbock, D. T., Finberg, R. W. (2003). Human Cytomegalovirus Activates Inflammatory Cytokine Responses via CD14 and Toll-Like Receptor 2. J. Virol. 77: 4588-4596 [Abstract] [Full Text]  
  • Kalejta, R. F., Shenk, T. (2003). The Human Cytomegalovirus UL82 Gene Product (pp71) Accelerates Progression through the G1 Phase of the Cell Cycle. J. Virol. 77: 3451-3459 [Abstract] [Full Text]  
  • Taddeo, B., Esclatine, A., Roizman, B. (2002). The patterns of accumulation of cellular RNAs in cells infected with a wild-type and a mutant herpes simplex virus 1 lacking the virion host shutoff gene. Proc. Natl. Acad. Sci. USA 99: 17031-17036 [Abstract] [Full Text]  
  • Wieland, S. F., Vega, R. G., Muller, R., Evans, C. F., Hilbush, B., Guidotti, L. G., Sutcliffe, J. G., Schultz, P. G., Chisari, F. V. (2002). Searching for Interferon-Induced Genes That Inhibit Hepatitis B Virus Replication in Transgenic Mouse Hepatocytes. J. Virol. 77: 1227-1236 [Abstract] [Full Text]  
  • Jones, J. O., Arvin, A. M. (2002). Microarray Analysis of Host Cell Gene Transcription in Response to Varicella-Zoster Virus Infection of Human T Cells and Fibroblasts In Vitro and SCIDhu Skin Xenografts In Vivo. J. Virol. 77: 1268-1280 [Abstract] [Full Text]  
  • Tang, Q., Maul, G. G. (2002). Mouse Cytomegalovirus Immediate-Early Protein 1 Binds with Host Cell Repressors To Relieve Suppressive Effects on Viral Transcription and Replication during Lytic Infection. J. Virol. 77: 1357-1367 [Abstract] [Full Text]  
  • Goodrum, F. D., Jordan, C. T., High, K., Shenk, T. (2002). Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: A model for latency. Proc. Natl. Acad. Sci. USA 99: 16255-16260 [Abstract] [Full Text]  
  • McCormick, A. L., Smith, V. L., Chow, D., Mocarski, E. S. (2002). Disruption of Mitochondrial Networks by the Human Cytomegalovirus UL37 Gene Product Viral Mitochondrion-Localized Inhibitor of Apoptosis. J. Virol. 77: 631-641 [Abstract] [Full Text]  
  • Noris, E., Zannetti, C., Demurtas, A., Sinclair, J., De Andrea, M., Gariglio, M., Landolfo, S. (2002). Cell Cycle Arrest by Human Cytomegalovirus 86-kDa IE2 Protein Resembles Premature Senescence. J. Virol. 76: 12135-12148 [Abstract] [Full Text]  
  • O'Farrell, C., Vaghefi, N., Cantonnet, M., Buteau, B., Boudinot, P., Benmansour, A. (2002). Survey of Transcript Expression in Rainbow Trout Leukocytes Reveals a Major Contribution of Interferon-Responsive Genes in the Early Response to a Rhabdovirus Infection. J. Virol. 76: 8040-8049 [Abstract] [Full Text]  
  • Lopper, M., Compton, T. (2002). Disulfide Bond Configuration of Human Cytomegalovirus Glycoprotein B. J. Virol. 76: 6073-6082 [Abstract] [Full Text]  
  • Grandvaux, N., Servant, M. J., tenOever, B., Sen, G. C., Balachandran, S., Barber, G. N., Lin, R., Hiscott, J. (2002). Transcriptional Profiling of Interferon Regulatory Factor 3 Target Genes: Direct Involvement in the Regulation of Interferon-Stimulated Genes. J. Virol. 76: 5532-5539 [Abstract] [Full Text]  
  • Child, S. J., Jarrahian, S., Harper, V. M., Geballe, A. P. (2002). Complementation of Vaccinia Virus Lacking the Double-Stranded RNA-Binding Protein Gene E3L by Human Cytomegalovirus. J. Virol. 76: 4912-4918 [Abstract] [Full Text]  
  • Mocarski, E. S. Jr. (2002). Virus self-improvement through inflammation: No pain, no gain. Proc. Natl. Acad. Sci. USA 99: 3362-3364 [Full Text]  
  • Zhu, H., Cong, J.-P., Yu, D., Bresnahan, W. A., Shenk, T. E. (2002). From the Cover: Inhibition of cyclooxygenase 2 blocks human cytomegalovirus replication. Proc. Natl. Acad. Sci. USA 99: 3932-3937 [Abstract] [Full Text]  
  • Song, Y.-J., Stinski, M. F. (2002). Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: A DNA microarray analysis. Proc. Natl. Acad. Sci. USA 10.1073/pnas.052010099v1 [Abstract] [Full Text]  
  • Song, Y.-J., Stinski, M. F. (2002). Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: A DNA microarray analysis. Proc. Natl. Acad. Sci. USA 99: 2836-2841 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Browne, E. P.
Right arrow Articles by Shenk, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Browne, E. P.
Right arrow Articles by Shenk, T.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»