Impact of human cytomegalovirus latent infection on myeloid progenitor cell gene expression

Abstract

Herpesviruses establish lifelong latent infections in their hosts. Human cytomegalovirus (CMV) targets a population of bone marrow-derived myeloid lineage progenitor cells that serve as a reservoir for reactivation; however, the mechanisms by which latent CMV infection is maintained are unknown. To gain insights into mechanisms of maintenance and reactivation, we employed microarrays of approximately 26,900 sequence-verified human cDNAs to assess global changes in cellular gene expression during experimental CMV latent infection of granulocyte-macrophage progenitors (GM-Ps). This analysis revealed at least 29 host cell genes whose expression was increased and six whose expression was decreased during CMV latency. These changes in transcript levels appeared to be authentic, judging on the basis of further analysis of a subset by semiquantitative reverse transcription-PCR. This study provides a comprehensive snapshot of changes in host cell gene expression that result from latent infection and suggest that CMV regulates genes that encode proteins involved in immunity and host defense, cell growth, signaling, and transcriptional regulation. The host genes whose expression we found altered are likely to contribute to an environment that sustains latent infection.

FIG. 1.

(A) Quality and size range of aRNAs. The mRNA from mock-infected or CMV latently infected cells was amplified using a linear amplification method. aRNAs from mock-infected (lane 2) and CMV latently infected (lane 3) cells were electrophoresed under denaturing conditions and visualized by ethidium bromide staining. The size range of aRNAs was determined by comparison to a single-stranded RNA ladder (lane 1). The majority of aRNAs ranged in size from 200 to 1,800 bp. (B) Scatter plot depicting the median pixel intensities of Cya5 and Cya3 fluorescence on individual human cDNA clone spots after they were hybridized against aRNAs from mock-infected (Cya3-labeled) and latently infected (Cya5-labeled) GM-Ps. Upregulated (Cya5:Cya3 ≥ 2.0) and downregulated (Cya3:Cya5 ≥ 2.0) clones are shaded red and green, respectively. The vast majority of clones did not exhibit significantly altered expression (i.e., ratios ≤ 2.0). These are plotted between the colored trend lines.

FIG. 2.

Validation of microarray data with semiquantitative dilution RT-PCR. Serial threefold dilutions (from 1:15 to 1:3,645) of cDNA derived from mock-infected and latently infected GM-P cultures were amplified using gene-specific primers under limiting PCR cycle conditions. RT-PCR products were then separated by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining. In total, transcripts from 1 housekeeping gene (GAPDH) and 11 other cellular genes were assessed for their relative abundances in mock-infected and latently infected GM-Ps; these data were compared with microarray-derived expression data for the same genes. GAPDH expression was unchanged (=). Upward- and downward-pointing arrows indicate up- and downregulation (as determined by dilution RT-PCR or microarray). In each case, dilution RT-PCR and microarray-derived data were consistent with each other.

FIG. 3.

Diagram depicting a snapshot of CMV latency with respect to upregulated (red) and downregulated (green) genes which could be grouped by a related function(s). The majority of identified genes could be grouped under the following headings: transcription factors, immunity and host cell defense, signaling, and cell growth.