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. 2015;14(17):2748-63.
doi: 10.1080/15384101.2015.1066535.

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

Nektaria Goulidaki  1 , Saud Alarifi, Saad H Alkahtani, Ahmed Al-Qahtani, Demetrios A Spandidos, Christos Stournaras, George Sourvinos
Affiliations

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

Nektaria Goulidaki et al. Cell Cycle. 2015.

Abstract

Human Cytomegalovirus (HCMV), an ubiquitous β-herpesvirus, is a significant pathogen that causes medically severe diseases in immunocompromised individuals and in congenitally infected neonates. RhoB belongs to the family of Rho GTPases, which regulates diverse cellular processes. Rho proteins are implicated in the entry and egress from the host cell of mainly α- and γ-herpesviruses, whereas β-herpesviruses are the least studied in this regard. Here, we studied the role of RhoB GTPase during HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells showed that RhoB was translocated to the assembly complex/compartment (AC) of HCMV, a cytoplasmic zone in infected cells where many viral structural proteins are known to accumulate and assembly of new virions takes place. Furthermore, RhoB was localized at the AC even when the expression of the late HCMV AC proteins was inhibited. At the very late stages of infection, cellular projections were formed containing RhoB and HCMV virions, potentially contributing to the successful viral spread. Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a significant reduction of the virus titer and could also affect the accumulation of AC viral proteins at this subcellular compartment. RhoB knockdown also affected actin fibers' structure. Actin reorganization was observed at late stages of infection originating from the viral AC and surrounding the cellular projections, implying a potential interplay between RhoB and actin during HCMV assembly and egress. In conclusion, our results demonstrate for the first time that RhoB is a constituent of the viral AC and is required for HCMV productive infection.

Keywords: HCMV; RhoB; actin; assembly compartment; assembly complex; cellular projections; cytoskeleton; pUL32; pUL97; pp65.

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Figures

Figure 1.
Figure 1.
RhoB is upregulated upon HCMV infection and localizes at the AC in HCMV-infected cells. HFFs were either mock infected (A) or infected with HCMV AD169 at MOI = 0.5 pfu/cell. (B and C) RhoB cytoplasmic expression pattern in HCMV infected cells 2 d.p.i. and 3 d.p.i., respectively. HFFs were stained for the viral nuclear protein pUL44 (green) and RhoB (red). Nuclei were stained with DAPI. (D and E) HCMV-infected cells were fixed either 2 d.p.i. or 3 d.p.i. and they were immunostained for the viral AC markers pp65, pUL97, pUL32 and for RhoB, followed by incubation with a mouse-specific Alexa Fluor 488 conjugate (green, viral proteins) or a rabbit-specific Cy3 conjugate (red, RhoB) and DAPI. (F) Kinetics of RhoB expression during the course of HCMV infection determined by western blot. (G) Subcellular localization of RhoB (red) in association with the cytoplasmic (cyt) distribution of the trans-Golgi Network marker TGN-38 (green, upper panel) and Rab5 (green, lower panel) 3 d.p.i. The cells were also co-stained for pUL44 (green) as a nuclear (nuc) marker of infection progress. (bar: 10 μm).
Figure 2.
Figure 2.
Dynamic recruitment of RhoB at the viral AC in live HCMV infected fibroblasts and cytoplasmic translocaion of RhoB and pUL32 at late stages of infection. (A) Cellular distribution of RhoB following transfection with the mRFP-RhoB plasmid in a live non-infected cell (a). Live human fibroblasts transiently expressing mRFP-RhoB were infected with the UL32-EGFP-HCMV-TB40 virus at MOI=5 pfu/cell and the image was captured 5 d.p.i. (b–d). (B) Live HFFs as above were monitored by time-lapse microscopy and images were obtained every 6 hours, initiating at 108 hours post-infection (h.p.i.). Presented are selected double-labeled images that illustrate the dynamic recruitment of RhoB at the pUL32-containing AC. Arrow indicates the position of the HCMV AC. (C) Visualization of RhoB (red) and pUL32 (green) translocation in the cytoplasm of a single live-infected cell similarly transfected and infected as in A and B, above, at 6 d.p.i. (a), 7 d.p.i (b) and 8 d.p.i. (c) Arrows indicate the HCMV AC site (bar: 10 μm).
Figure 3.
Figure 3.
RhoB localization in cellular projections at late stages of HCMV infection. (A) Live HFFs expressing mRFP-RhoB were infected with the UL32-EGFP-HCMV-TB40 virus at MOI=5 pfu/cell. Images were obtained by timelapse microscopy 11 d.p.i. (B) Striking localization of the endogenous RhoB at the edges of cellular projections (arrows) containing pUL32-EGFP labeled virus particles at very late stages of HCMV infection. (C) The endogenous distribution of RhoB was visualized in fixed cells by indirect immunofluorescence. The enlargement illustrates the intimate contacts established between UL32-EGFP-HCMV-TB40 infected cells in non-confluent cell cultures. Nuclei were stained with DAPI. (bar: 10 μm).
Figure 4.
Figure 4.
Cellular projections containing elongated actin stress fibers enclose accumulations of new virions at late stages of HCMV infection. HFFs were either mock infected (a) or infected with the UL32-EGFP-HCMV-TB40 virus at MOI = 5 pfu/cell and fixed 8 d.p.i. (b–d). Actin fibers were stained with rhodamine-labeled phalloidin and the nuclei were stained with DAPI. The enlargements (e–g) show in detail the stress fibers formed, surrounding pUL32-EGFP labeled virions. (bar: 10 μm).
Figure 5.
Figure 5.
In HCMV infected cells, RhoB localizes at the AC in the absence of late viral proteins from the AC. HFFs were either mock infected and treated with ganciclovir (GCV) (a–c and j–l) or were infected with HCMV AD169 at MOI = 0.5 pfu/cell and either treated with the drug solvent (d–f and m–o) or with GCV (g–i and p–r). Cells were fixed 4 d.p.i. and immunostained for the viral AC markers, pp65 or pUL32 (green), as well as for RhoB (red). Nuclei were stained with DAPI. (bar: 25 μm).
Figure 6.
Figure 6.
The accumulation of several viral proteins at the AC is affected in RhoB-depleted cells. HFFs were transduced with the lentiviral vector, TRIPZshRhoB, and the expression of the RhoB shRNA cassette was either induced by treatment with doxycycline (+Dox) or not (−Dox). The scrambled shRNA TRIPZ lentiviral vector (TRIPZshscr) was used as a negative control. Doxycycline-treated TRIPZshRNA-expressing cells exhibited red fluorescence. The lentiviral-transduced and doxycycline-treated cells, as well as lentiviral-transduced untreated control cells, were infected either with the UL32-EGFP-HCMV-TB40 virus at MOI = 5 pfu/cell and images were captured 5 d.p.i. (A) or with HCMV AD169 at MOI = 0.5 pfu/cell and stained for pp65 (B) or pUL97 (C) 5 d.p.i. The arrows indicate the localization of the AC. Nuclei in fixed cells were stained with DAPI. (bar: 10 μm).
Figure 7.
Figure 7.
Depletion of RhoB results in the reduction of HCMV progeny virion production. (A) HFFs were first transduced with either the doxycycline-inducible lentiviral vector, TRIPZshRhoB, or control vectors [empty vector (EV) or shRNA scramble vector (shscr)], in the presence or absence of doxycycline. The transduced cells were subsequently infected with HCMV AD169 at MOI = 0.5 pfu/cell. Supernatants from all lentiviral transduced HCMV infected cells were harvested 5 days after infection, fresh HFFs were subsequently superinfected with these supernatants, and fixed and stained for viral IE1 expression 24 hours later. Cell nuclei were counterstained using DAPI. (bar: 1 μm). (B) IE1 gene expression of fresh superinfected HFFs described in (A) was analyzed by counting representative microscopy fields. A minimum of 500 cells were counted for each vector from each of 3 independent experiments. The percentage of IE1-positive cells was quantified and statistical analysis was performed by One-Way Anova. Erros bars, SD. Genomic and viral DNA and total protein extracts were examined by qPCR (C) and western blotting using the indicated antibodies (D) in similarly transduced and HCMV infected cells as in (A)
Figure 8.
Figure 8.
Actin reorganization at the AC in HCMV infected cells. Human fibroblasts were infected with HCMV AD169 at MOI = 0.5 pfu/cell. Cells were fixed at the indicated time points after infection and stained for pUL44 (nuclear) and RhoB (cytoplasmic) proteins. Actin fibers were visualized by direct immunofluorescence using rhodamine-labeled phalloidin. Nuclei were stained with DAPI (bar: 10 μm).
Figure 9.
Figure 9.
Disassembly of actin fibers does not dramatically disperse the viral AC but reduces the viral yield. (A) HCMV AD169-infected fibroblasts, 5 d.p.i., were treated either with Cytochalasin B (CytoB, b, c, e) or with nocodazol (Noc, f) or the corresponding organic solvent (a, d) for 1 hour. The cells were then fixed and stained for RhoB (a, b, d–f), pp65 (c) and α-tubulin (d–f). Actin filaments were stained with FITC-labeled phalloidin (a–c) while nuclei were stained with DAPI. (bar: 10 μm). (B) Supernatants harvested from the above Cytochalasin B-treated and control cells were quantified on fresh HFFs by IE1 fluoresence, counting representative microscopy fields, examining more than 500 cells from each of 3 independent experiments. The percentage of cells expressing IE1 was quantified and statistical analysis was carried out by One-Way Anova. Error bars, SD.
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