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. 2020 Oct;22(4):3066-3072.
doi: 10.3892/mmr.2020.11434. Epub 2020 Aug 19.

Efficient proliferation and mitosis of glioblastoma cells infected with human cytomegalovirus is mediated by RhoA GTPase

Ahmed A Al-Qahtani  1 , Saud Alarifi  2 , Saad Alkahtani  2 , Christos Stournaras  3 , George Sourvinos  4
Affiliations

Efficient proliferation and mitosis of glioblastoma cells infected with human cytomegalovirus is mediated by RhoA GTPase

Ahmed A Al-Qahtani et al. Mol Med Rep. 2020 Oct.

Abstract

Human cytomegalovirus (HCMV) is a prevalent viral pathogen, which can cause severe clinical consequences in neonates, immunocompromised individuals, patients with AIDS, and organ and stem cell transplant recipients. HCMV inhibits the host cell cycle progress while the immediate‑early protein 1 (IE1) tethers to condensed chromatin in mitotic cells. The present study investigated the effect of HCMV on the cell cycle in human glioblastoma cells, as well as the role of RhoA GTPase during mitosis in the same context. Live cell microscopy showed that despite the apparent cell cycle arrest at late stages of mitosis in normal fibroblasts, HCMV‑infected U373MG cells successfully went through all stages of cell division. HCMV IE1 protein exhibited a remarkably tight association with mitotic chromosomes from early mitosis to late cytokinesis. Depletion of RhoA significantly impaired the proliferation rate of HCMV‑infected U373MG cells; consistent with this observation, the number of cells entering mitosis was also decreased. These results demonstrated the differential behavior of HCMV during mitosis in a normal and a cancer background. Furthermore, RhoA may be a critical component for the efficient cell division of HCMV‑infected glioblastoma cells, which subsequently ensures the maintenance of viral genomes.

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Figures

Figure 1.
Figure 1.
HCMV causes defects in mitotic phases during cell division normal fibroblasts. HFF cells were transfected with pHcRed1-H2A and super-infected with HCMV CR401 (MOI=3 pfu/cell) expressing EGFP-IE1. (A) A single cell co-expressing IE1 (green) and H2A (red) was monitored by timelapse microscopy and still images were obtained every 3 h showing the defective mitotic progression from (a and b) prophase and (c and d) prometaphase to (e) metaphase and (f-h) defective anaphase, with evident lagging chromosomes. Scale bar, 10 µm. (B) Distribution of the HCMV-infected mitotic cells at the different phases of cell division. HCMV, human cytomegalovirus; IE1, immediate-early protein 1.
Figure 2.
Figure 2.
Glioblastoma cells infected with HCMV successfully complete all phases of cell division. U373MG cells were infected with CR401 IE1-EGFP virus (MOI=3 pfu/cell) and transfected with pHcRed1-H2A. Still images were obtained from live cells by timelapse microscopy. Scale bar, 10 µm. HCMV, human cytomegalovirus; IE1, immediate-early protein 1.
Figure 3.
Figure 3.
Rho silencing results in defects in cell proliferation rate and mitosis efficiency in glioblastoma HCMV infected cells. (A) Specific knockdown of RhoA in U373 cells was confirmed by western blot from whole protein extracts obtained after transfection with the indicated siRNAs (siRhoA or siRNA scramble) of U373MG cells. Actin served as loading control. (B and C) Relative proliferation rate of parental U373MG, siscr and siRhoA either (B) mock infected or (C) infected with HCMV AD169 (MOI=3 pfu/cell). The proliferation rate of cells was determined by seeding 5×103 cells/well and counting the cells in triplicate for up to 3 days by MTT assay. Each experiment was replicated three times and data represent the mean and SE of the three separate experiments. *P<0.05 vs. parental cells; #P<0.05 vs. siscr cells. (D) Proportion of mock-infected mitotic parental U373MG cells, siscr and siRhoA cells compared to each other after counting 10 random microscopy fields. (E) siscr or (F) siRhoA U373MG cells successfully enter mitosis. (G) Proportion of HCMV AD169-infected mitotic parental U373MG cells, siscr and siRhoA cells compared to each other after counting 10 randomly selected microscopy fields. (H) The efficiency of HCMV shedding in siRNA scramble and siRhoA cells was determined by standard plaque assay after titrating the supernatants of the cells on HFF cells. Error bars represent a standard deviation based on results from three experiments. *P<0.05; **P<0.01. siscr, siRNA scramble; siRhoA, siRNA RhoA; siRNA, small interfering RNA; DAPI, 4′,6-diamidino-2-phenylindole; HCMV, human cytomegalovirus.
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