Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines

Abstract

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed.

Fig. 1.

HCMV CPE at 72 h p.i. in HFFF-2s, RPE-1s and U373MGs infected at 6 p.f.u. per cell. MI, Mock-infected cells; HCMV, HCMV-infected cells. Bar, 500 µm.

Fig. 2.

Single-step growth curves of HCMV in HFFF-2s, RPE-1s and U373MGs infected at 6 p.f.u. per cell: (a) CAV and (b) CRV.

Fig. 3.

Scatter plots showing time-specific comparisons of HCMV gene expression in RPE-1s and U373MGs relative to HFFF-2s. The solid line denotes equivalent expression levels and the broken lines indicate±twofold differences, and dark and light grey points represent ORFs outside these boundaries.

Fig. 4.

Northern blots showing representative HCMV transcripts produced in HFFF-2s, RPE-1s and U373MGs at 12, 24, 48 and 72 h p.i. (a) IRS1 (3.5 and 1.7 kb), (b) UL4 (1.8 kb), (c) UL83 (3.5 kb), (d) UL93 (10.5 kb), UL94 (9.1 kb), UL95 (7.3 kb), UL96 (5.6 kb), UL97 (4.7 kb), UL98 (2.6 kb) and UL99 (1.6 and 1.3 kb) and (e) glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Genome co-ordinates (GenBank accession no. AY446894.2) of the HCMV probe sequences were: IRS1, 198 271–197 985; UL4, 13 936–142 429; UL83, 121 565–121 800; and UL99 145 796–146 085.