Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs

Abstract

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.

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).

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).

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.

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.

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.