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Comparative Study
. 2007 Apr;81(8):3816-26.
doi: 10.1128/JVI.02425-06. Epub 2007 Jan 24.

Rat cytomegalovirus gene expression in cardiac allograft recipients is tissue specific and does not parallel the profiles detected in vitro

Daniel N Streblow  1 , Koen W R van Cleef, Craig N Kreklywich, Christine Meyer, Patricia Smith, Victor Defilippis, Finn Grey, Klaus Früh, Robert Searles, Cathrien Bruggeman, Cornelis Vink, Jay A Nelson, Susan L Orloff
Affiliations
Comparative Study

Rat cytomegalovirus gene expression in cardiac allograft recipients is tissue specific and does not parallel the profiles detected in vitro

Daniel N Streblow et al. J Virol. 2007 Apr.

Abstract

Rat cytomegalovirus (RCMV) is a beta-herpesvirus with a 230-kbp genome containing over 167 open reading frames (ORFs). RCMV gene expression is tightly regulated in cultured cells, occurring in three distinct kinetic classes (immediate early, early, and late). However, the extent of viral-gene expression in vivo and its relationship to the in vitro expression are unknown. In this study, we used RCMV-specific DNA microarrays to investigate the viral transcriptional profiles in cultured, RCMV-infected endothelial cells, fibroblasts, and aortic smooth muscle cells and to compare these profiles to those found in tissues from RCMV-infected rat heart transplant recipients. In cultured cells, RCMV expresses approximately 95% of the known viral ORFs with few differences between cell types. By contrast, in vivo viral-gene expression in tissues from rat heart allograft recipients is highly restricted. In the tissues studied, a total of 80 viral genes expressing levels twice above background (5,000 to 10,000 copies per mug total RNA) were detected. In each tissue type, there were a number of genes expressed exclusively in that tissue. Although viral mRNA and genomic DNA levels were lower in the spleen than in submandibular glands, the number of individual viral genes expressed was higher in the spleen (60 versus 41). This finding suggests that the number of viral genes expressed is specific to a given tissue and is not dependent upon the viral load or viral mRNA levels. Our results demonstrate that the profiles, as well as the amplitude, of viral-gene expression are tissue specific and are dramatically different from those in infected cultured cells, indicating that RCMV gene expression in vitro does not reflect viral-gene expression in vivo.

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Figures

FIG. 1.
FIG. 1.
Microarray analysis of RCMV gene expression. Each chip contained two unique oligonucleotides for each viral gene, and these oligonucleotides were spotted in duplicate. The RCMV oligonucleotides were distributed over four positions on each chip depicted in grids A to D. The yellow lines outline the positions of the RCMV-specific oligonucleotides. The data shown represent microarray analysis of RNA extracted from RCMV-infected REFs at 4, 8, 16, 24, 36, and 48 h p.i.
FIG. 2.
FIG. 2.
RCMV in vitro gene expression. Fluorescence intensities from microarray analysis of RCMV RNAs isolated from infected REFs (A), SMC (B), and EC (C) harvested at 4, 16, and 48 h p.i. The RNA was amplified, and cDNA samples were labeled and hybridized to the RCMV microarray chips containing oligonucleotides specific for all of the known viral genes.
FIG. 3.
FIG. 3.
RCMV in vivo gene expression. Fluorescence intensities from microarray analysis of RCMV mRNA isolated from infected rat SMG that were harvested at days 7, 14, 21, and 28 days p.i. At each time point, SMG samples were isolated from four individual infected rats and analyzed on separate microarray chips. The data shown include gene intensities acquired from each of these four separate arrays.
FIG. 4.
FIG. 4.
Confirmation of in vivo RCMV gene expression. RT-PCR TaqMan analysis was used to confirm and quantify viral-gene expression in RNA samples from (A) in vivo-infected SMG at 7, 14, 21, and 28 days p.i. and (B) in vitro-infected rat fibroblasts. Primers specific for the RCMV genes R75 (gH), R114, R116, r119.1, r119.2, r119.4, R123 (IE1), and r151 were used to quantify viral-gene expression. RNA samples were normalized to expression of L32 (cellular ribosomal protein), and the relative copy numbers were determined using plasmids containing each viral gene. The error bars indicate standard deviations.
FIG. 5.
FIG. 5.
Microarray analysis of RCMV gene expression in infected rat tissues. Total RNA was extracted from RCMV-infected spleen, lung, kidney, and heart allograft at 7 days p.i.; SMG at 21 days p.i. The RNA was processed and analyzed using our RCMV microarray chips (n = 4). The mean gene intensity from each of the four biological replicates is plotted on the y axis, and RCMV gene number on the x axis.
FIG. 6.
FIG. 6.
RCMV gene expression is tissue specific. RT-PCR TaqMan analysis was used to confirm and quantify viral-gene expression in RNA samples from in vivo-infected native heart, allograft heart, lung, liver, kidney, spleen, and SMG. All tissues were harvested at 7 days p.i., except SMG (21 days p.i.). RCMV-infected REFs harvested at 24 h p.i. were used for in vitro-in vivo comparisons. Primers specific for the RCMV genes R78, r119.1, r119.2, r151, and r152.4 were used to quantify viral-gene expression. RNA samples were normalized to expression of L32 (cellular ribosomal protein), and the relative copy numbers were determined using plasmids containing each viral gene. The limit of detection for this assay was 100 copies.
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