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. 2014 Nov 19;9(11):e113427.
doi: 10.1371/journal.pone.0113427. eCollection 2014.

The DRY box and C-terminal domain of the human cytomegalovirus US27 gene product play a role in promoting cell growth and survival

Carolyn C Tu  1 , Juliet V Spencer  1
Affiliations

The DRY box and C-terminal domain of the human cytomegalovirus US27 gene product play a role in promoting cell growth and survival

Carolyn C Tu et al. PLoS One. .

Abstract

Human cytomegalovirus (HCMV) is a widespread pathogen that can lay dormant in healthy individuals and establish lifelong latent infection. This successful co-existence is facilitated by a number of viral gene products that manipulate host cellular functions and immune responses. Among these immunomodulatory genes are four G-protein coupled receptors (GPCRs) encoded by HCMV, designated US27, US28, UL33, and UL78. Studies have shown the US28 gene product to be a functional chemokine receptor that signals both constitutively and in a ligand-dependent manner, resulting in a wide range of cellular effects. In previous work, we have found that US27 expression results in at least two biological effects: enhanced CXCR4 signaling and increased in cellular proliferation in HEK293 cells. Here, we examined the involvement of two protein domains, the DRY box and the C-terminal intracellular domain (CTD) of US27, in mediating both cell proliferation and survival. While both domains were required for a proliferative effect, loss of either domain only moderately impacted cell survival, suggesting that US27 may interact with cell survival pathways through protein regions other than the DRY box and CTD. Quantitative RT-PCR arrays were used to profile changes in cellular gene expression in the HEK293-US27 cell line, and down-regulation of cell cycle regulators CDKN1A/p21/CIP1 (cyclin dependent kinase inhibitor 1A) and SESN (Sestrin2 or Hi95) was observed. These results indicate that increased cell proliferation due to US27 may be linked to suppression of negative growth regulators, and that these interactions require the DRY box and CTD.

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Conflict of interest statement

Competing Interests: Juliet V. Spencer serves as an Academic Editor for PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Stable cell lines express HCMV US27 or US27 mutants.
A) Cell lysates were separated by SDS-PAGE and then immunoblotted using anti-FLAG M2 antibody or anti-MAPK as a control. (B) Immunofluorescence staining of fixed, permeabilized cells with anti-FLAG M2 antibody followed by Alexa Fluor 514-conjugated mouse secondary. Optical sectioning was performed and one slice of the Z-stack is shown. (C) Cell surface staining with anti-FLAG M2 antibody on non-permeabilized cells, followed by Alexa Fluor 514 secondary antibody.
Figure 2
Figure 2. Expression of US27 conveys a proliferative advantage.
A) Cells were seeded into 96-well plates at a density of 5×103 cells per well and cell number was monitored via the addition of CellTiter-Glo reagent at the indicated time points. B) The rate of DNA synthesis for each cell line was measured using bromodeoxyuridine (BrdU) incorporation and luminometry. C) Cells were seeded in 6-well dishes at 2×105 cells per well, then harvested with trypsin and counted via flow cytometry at the indicated time points. (D) HEK293 and (E) HeLa cells were seeded into 96-well plates at a density of 1×104 cells per well and transfected 24 hours later with the indicated pEGFP expression vector at a ratio of 3∶1 (µl Fugene: µg plasmid DNA), cell number was monitored via the addition of CellTiter-Glo reagent at the indicated time points. Error bars represent standard error of three triplicate data points within one experiment. These results are representative of three independent experiments. ** indicates p<0.001, * indicates p<0.05 by Student t-test.
Figure 3
Figure 3. HEK293 cells expressing wild type US27 are more resistant to apoptosis.
Cells were treated with 10 µM etoposide to induce apoptosis. A) After 48 hours, cells were stained with propidium iodide and Annexin V then analyzed by flow cytometry. B) The average percent positive cells for Annexin V, propidium iodide (PI) or both (double positive, DP) from three independent experiments, as indicated in A above. * indicates p<0.01 by Student t-test compared to US27-cells. Overall cell viability was measured at the indicated time points via the addition of CellTiter-Glo reagent and total luminescence quantified in C) stable HEK293 cell lines, and in transiently transfected D) HEK293 cells and E) HeLa cells following etoposide treatment. Error bars represent standard error of three triplicate data points within one experiment. These results are representative of three independent experiments. ** indicates p<0.001, * indicates p<0.05 by Student t-test. In C, pairwise comparisons were performed between US27/XR3CT and US27, US28, and HEK293 and between US27-DAY and US27 and HEK293.
Figure 4
Figure 4. HCMV US27 correlates with decreased expression of p21 and Sestrin-2.
A) Fold changes in gene expression compared to levels in cells expressing CXCR3. B) RNA was harvested from each stable cell line, reverse transcribed, then gene specific primers were used to amplify either Sestrin-2 or β-actin. C) Cell lysates were separated by SDS-PAGE and immunoblotted with antibodies directed against p21, sestrin-2, or MAPK as indicated.
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