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. 2004 Nov;78(22):12529-36.
doi: 10.1128/JVI.78.22.12529-12536.2004.

A cyclooxygenase-2 homologue encoded by rhesus cytomegalovirus is a determinant for endothelial cell tropism

Cary A Rue  1 , Michael A Jarvis, Amber J Knoche, Heather L Meyers, Victor R DeFilippis, Scott G Hansen, Markus Wagner, Klaus Früh, David G Anders, Scott W Wong, Peter A Barry, Jay A Nelson
Affiliations

A cyclooxygenase-2 homologue encoded by rhesus cytomegalovirus is a determinant for endothelial cell tropism

Cary A Rue et al. J Virol. 2004 Nov.

Abstract

Cyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates the synthesis of prostaglandins from arachidonic acid. The eicosanoids function as critical regulators of a number of cellular processes, including the acute and chronic inflammatory response, hemostasis, and the innate immune response. Human cytomegalovirus (HCMV), which does not encode a viral COX-2 isoform, has been shown to induce cellular COX-2 expression. Importantly, although the precise role of COX-2 in CMV replication is unknown, COX-2 induction was shown to be critical for normal HCMV replication. In an earlier study, we identified an open reading frame (Rh10) within the rhesus cytomegalovirus (RhCMV) genome that encoded a putative protein (designated vCOX-2) with high homology to cellular COX-2. In the current study, we show that vCOX-2 is expressed with early-gene kinetics during RhCMV infection, resulting in production of a 70-kDa protein. Consistent with the expression of a viral COX-2 isoform, cellular COX-2 expression was not induced during RhCMV infection. Finally, analysis of growth of recombinant RhCMV with vCOX-2 deleted identified vCOX-2 as a critical determinant for replication in endothelial cells.

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Figures

FIG. 1.
FIG. 1.
Genomic localization and structure of the RhCMV vCOX-2 gene. (A) Schematic showing position of the vCOX-2 gene within the RhCMV genome. The vCOX-2 exons are shown below, with the predicted translational start (+1) at nt 10933 and stop (UGA) site at nt 8521 to 8523. (B) Alignment of the amino acid sequences of vCOX-2 cloned from cDNA with the human cCOX-2 protein. Identical residues are shown in grey. Active-site residues of human cCOX-2 are marked by asterisks.
FIG. 2.
FIG. 2.
Northern analysis of vCOX-2 gene expression during RhCMVWT infection of Telo-RFs. Telo-RFs were infected with RhCMVWT at an MOI of 3, and total cellular RNA was isolated at the indicated times p.i. (in hours). Foscarnet (FosC) was added to cells as indicated. RNA (10 μg/lane) was separated by electrophoresis and transferred prior to Northern analysis using 32P-labeled DNA probes corresponding to vCOX-2 (exon 1) and GAPDH.
FIG. 3.
FIG. 3.
Construction and characterization of a RhCMVvCOX-2HA recombinant expressing an HA-tagged endogenous vCOX-2 protein. (A) Schematic showing construction of RhCMVvCOX-2HA/BAC-Cre. To generate RhCMVvCOX-2HA/BAC-Cre, an HA epitope was fused in frame with the 3′ end of the vCOX-2 ORF by E/T recombination. Following EcoRI digestion, insertion of the HA tag results in loss of a 9.4-kb vCOX-2-containing fragment and production of new 7.8- and 3.1-kb fragments. The FRT-flanked Kanr marker was then removed by Flp recombinase-mediated excision, resulting in a 7.8- to 6.3-kb band shift. Also shown is DNA electrophoresis of EcoRI-digested RhCMVvCOX-2HA/BAC-Cre, showing predicted DNA band shifts. Southern blotting using a Kanr-specific probe shows insertion of a single Kanr marker in the appropriate 7.8-kb EcoRI fragment and Kanr removal following Flp recombinase induction. (B) Western analysis of RhCMVvCOX-2HA-infected Telo-RFs. Rhesus macaque Telo-RFs were infected with either RhCMVWT or RhCMVvCOX-2HA at an MOI of 10, and protein was harvested at the times indicated p.i. Viral DNA polymerase was inhibited by the addition of phosphonoacetic acid (PAA) as indicated. Proteins were separated by SDS-PAGE, transferred, and assayed by Western immunoblotting for the presence of vCOX-2HA (using anti-HA antibody) or actin (loading control).
FIG. 4.
FIG. 4.
Quantitative RT-PCR showing absence of rhesus cCOX-2 gene induction during RhCMV infection of Telo-RFs. Telo-RFs were infected with RhCMVWT (black bars) and RhCMVΔvCOX-2 (hatched bars) at an MOI of 3, and the level of cCOX-2 expression was measured by quantitative RT-PCR at increasing times p.i. Primers specific to endogenous cCOX-2 were used, and amplified products were detected with SYBR green. The results shown are representative of two independent experiments.
FIG. 5.
FIG. 5.
Construction and characterization of an RhCMVΔvCOX-2 recombinant. (A) Schematic showing construction of RhCMVΔvCOX-2/BAC-Cre. RhCMVΔvCOX-2/BAC-Cre was constructed by using E/T recombination to replace the entire vCOX-2 ORF with an Ampr-lacZ cassette. Following BamHI digestion, deletion of the vCOX-2 ORF results in the shift of a 7.7- to 7.1-kb fragment that is reactive with an Ampr-lacZ probe in Southern blotting. (B) Northern analysis showing absence of vCOX-2- reactive transcripts from RhCMVΔvCOX-2-infected Telo-RFs.
FIG. 6.
FIG. 6.
Growth analysis of RhCMVΔvCOX-2, RhCMVvCOX-2Revt, and RhCMVWT. Telo-RFs (A) or ECs (B) were infected at an MOI of 0.01 with either RhCMVΔvCOX-2, RhCMVvCOX-2Revt, or RhCMVWT. Samples were collected at the indicated days p.i., and titers were determined by plaque assay on Telo-RFs. Results are the averages of three experiments, and standard errors are shown.
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