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. 2013 Sep;176(1-2):312-20.
doi: 10.1016/j.virusres.2013.07.002. Epub 2013 Jul 11.

The human cytomegalovirus US27 gene product enhances cell proliferation and alters cellular gene expression

Angela P Lares  1 , Carolyn C Tu, Juliet V Spencer
Affiliations

The human cytomegalovirus US27 gene product enhances cell proliferation and alters cellular gene expression

Angela P Lares et al. Virus Res. 2013 Sep.

Abstract

Human cytomegalovirus (HCMV) is a prevalent pathogen worldwide. Although generally harmless in healthy individuals, HCMV can pose a serious threat to immune compromised individuals and developing fetuses in utero. HCMV encodes four genes predicted to give rise to G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. The US28 gene product is a functional chemokine receptor that enhances cell growth in some cell types but induces apoptosis in others. In contrast, the US27 gene product has not been demonstrated to signal either constitutively or in a ligand-induced manner. In this study, US27 was expressed in transfected cells, and both cell proliferation and DNA synthesis were significantly increased compared to control cells. PCR array analysis revealed that expression of US27 led to changes in a limited number of cellular genes, but genes that were up-regulated included the pro-survival factor Bcl-x, AP-1 transcription factor components jun and fos, and the IL-6 family cytokine oncostatin M. These results demonstrate that US27 can impact host cell physiology and may shed light on the function of this orphan viral GPCR.

Keywords: Chemokine receptor; Cytomegalovirus; GPCR; HCMV.

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Figures

Figure 1
Figure 1. Cells expressing HCMV US27 exhibit enhanced cell growth
A) HEK293 cells were seeded into white 96-well plates at a density of 1 × 104 cells per well and then transfected with the indicated p3XFLAG expression vectors using Fugene transfection reagent (Roche Biosciences, Basel, Switzerland) at a ratio of 3:1 (μl Fugene:μg plasmid DNA per manufacturer’s instructions). Cell number was monitored via the addition of CellTiter-Glo reagent (Promega, Madison, WI) at the indicated time points and luminescence evaluated using a Turner Biosystems Veritas Microplate Luminometer. B) Transfection efficiency was determined by flow cytometry after staining cells at the indicated times post-transfection with anti-FLAG antibody and FITC-conjugated secondary in permeabilization buffer. C) HEK293 cells were seeded and transfected with the indicated pEGFP expression vectors as described, then cell growth was monitored using CellTiter-Glo reagent and luminometry. D) Transfection efficiency was determined by flow cytometry for cells expressing EGFP. E) HeLa cells were seeded at 5 × 103 cells per well and transfected with the indicated pEGFP expression vectors as described, then cell growth monitored using CellTiter-Glo reagent and luminometry. F) Cos7 African green monkey cells were seeded at 1 × 104 cells per well and transfected with the indicated pEGFP expression vectors as described, then cell growth monitored via the addition of Cell Titer Glo reagent and luminometry. Error bars represent standard error of three triplicate data points.
Figure 2
Figure 2. Stable cell lines expressing US27 have a shorter doubling time and synthesize DNA at a higher rate than controls
HEK293 cells were transfected with p3XFLAG vectors and stable, clonal cell lines were produced using antibiotic selection as described (Stapleton et al., 2012). Expression of the transgene was evaluated via A) western blot staining of cell lysates using anti-FLAG antibody followed by AP-conjugated secondary or anti-total MAPK as a control and b) immunofluorescence staining of fixed, permeabilized cells with anti-FLAG antibody followed by FITC-conjugated secondary. C) Cells were seeded in 6-well dishes at 2 × 105 cells per well, then harvested and counted using a hemacytometer at the indicated time points. D) Doubling time was calculated based on total number of cell divisions for over the 72 hour growth period for triplicate wells of the standard growth curve. E) Cell growth was measured via the addition of CellTiter-Glo reagent and luminometry. F) The rate of DNA synthesis for HEK293 stable cell lines was measured using bromodeoxyuridine (BrdU) incorporation (Roche Bioscience, South San Francisco, CA) and luminometry at the indicated times. Error bars represent standard error of three triplicate data points. Statistical analysis was performed by student’s t-test and * indicates p<0.05. These results are representative of four independent experiments.
Figure 3
Figure 3. JAK-STAT pathway gene expression levels in cells expressing US27, US28, or CXCR3
RNA from stable cell line was extracted using RNEasy Midi Kit (Qiagen, Valencia, CA), cDNA was prepared using the RT2 First Strand Kit (Qiagen), and then diluted cDNA was mixed with the RT2 SYBR green master mix (Qiagen) according to the manufacturer’s instructions and loaded into the Human Jak-Stat RT2-PCR Profiler Array (SABiosciences, Valencia, CA). Real Time PCR was performed using the CFX96 (BioRad, Hercules, CA) by heating to 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Data was analyzed using the ΔΔCt method according to the SABiosciences web portal (www.SABiosciences.com/pcrarray.dataanalysis.php) and further recalculated manually. The same threshold value was used across all plates in the same data analysis to ensure accurate reading of quality controls. The data were normalized across all plates to the following housekeeping genes: beta-2-microglobulin (B2M), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta actin (ACTB). Controls for genomic DNA contamination RNA quality, and PCR performance were all in the recommended ranges. Data are represented as fold change compared to expression levels in HEK293 cells. The results are the average of three biological replicates.
Figure 4
Figure 4. JAK-STAT pathway gene expression levels in 293-US27 cells
The fold change values from three biological replicates of the PCR array were analyzed in comparison to 293-CXCR3 cells as a baseline control. Error bars indicate standard error; * indicates genes with a 1.8-fold change or higher.
Figure 5
Figure 5. GPCR pathway gene expression levels in cells expressing US27, US28, or CXCR3
RNA from each stable cell line was extracted using RNEasy Midi Kit (Qiagen, Valencia, CA), cDNA was prepared using the RT2 First Strand Kit (Qiagen), and then diluted cDNA was mixed with the RT2 SYBR green master mix (Qiagen) according to the manufacturer’s instructions and loaded into the Human GPCR RT2-PCR Profiler Array (SABiosciences, Valencia, CA). Data analysis was performed as described for the JAK-STAT array in the legend for Figure 3 except that the GPCR results are the average of two biological replicates.
Figure 6
Figure 6. Jun and Fos protein levels are higher in cells expressing either US27 or US28
Stable cell lines expressing either US27, US28 or CXCR3 or control HEK293 cells were lysed, proteins separated by SDS-PAGE, and then immunoblotted with antibodies directed against jun, fos, or total MAPK (Santa Cruz Biotechnology, Santa Cruz, CA) followed by AP-conjugated secondary antibody.
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References

    1. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991;1072(2–3):129–157. - PubMed
    1. Bais C, Santomasso B, Coso O, Arvanitakis L, Raaka EG, Gutkind JS, Asch AS, Cesarman E, Gershengorn MC, Mesri EA. G-protein-coupled receptor of Kaposi’s sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator. Nature. 1998;391(6662):86–89. - PubMed
    1. Beisser PS, Goh CS, Cohen FE, Michelson S. Viral chemokine receptors and chemokines in human cytomegalovirus trafficking and interaction with the immune system. CMV chemokine receptors. Curr Top Microbiol Immunol. 2002;269:203–234. - PubMed
    1. Beisser PS, Grauls G, Bruggeman CA, Vink C. Deletion of the R78 G protein-coupled receptor gene from rat cytomegalovirus results in an attenuated, syncytium-inducing mutant strain. J Virol. 1999;73(9):7218–7230. - PMC - PubMed
    1. Beisser PS, Vink C, Van Dam JG, Grauls G, Vanherle SJ, Bruggeman CA. The R33 G protein-coupled receptor gene of rat cytomegalovirus plays an essential role in the pathogenesis of viral infection. J Virol. 1998;72(3):2352–2363. - PMC - PubMed

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