Rab27a is required for human cytomegalovirus assembly

Abstract

Human cytomegalovirus (HCMV) completes its final envelopment on intracellular membranes before it is released from the cell. The mechanisms underlying these processes are not understood. Here we studied the role of Rab27a, a regulator of lysosome-related organelle transport, in HCMV production. HCMV infection increased Rab27a expression, and recruitment of Rab27a to membranous strutures at the assembly site. Immuno-gold labelling demonstrated association of Rab27a with viral envelopes. CMV production was reduced after knock-down of Rab27a, and in Rab27a-deficient ashen melanocytes. This study shows a requirement for Rab27a in the CMV life cycle and suggests that CMV and LRO biogenesis share common molecular mechanisms.

Figure 1. Expression and localisation of Rab27a in HCMV-infected cells.

(A) Rab27a protein expression during HCMV infection. Equal number of BJ1 cells mock infected or infected with HCMV at a moi of 3 were lysed at the indicated times. Rab27a was analyzed by Western blot where actin served as loading control Autoradiography films were scanned; images were cropped and assembled with Adobe Photoshop. Molecular weights in kDa are indicated. (B) Subcellular localisation of Rab27a in HCMV-infected cells. BJ1 and BJ1-YFP-Rab27a cells were either mock infected (left panels) or infected with HCMV at a moi of 0.5 (right panels). After 5dpi, cells were fixed, permeabilised and stained with anti-Rab27a (green in upper panels) and anti-HCMV glycoprotein gH (red) antibodies. In BJ1-YFP-Rab27a cells, YFP was directly imaged. DNA was stained with DAPI (blue). Dashed lines show the outline of the cells. Scale bars, 20 µm.

Figure 2. Immuno EM localisation of Rab27a in HCMV-infected cells.

(A and B) Cryosections of BJ1 YFP-Rab27a cells HCMV infected for 5 days at a moi of 3 were labelled for YFP with 10 nm gold-conjugated secondary antibodies. Gold particles were seen over the membrane of vacuoles, small vesicles and tubules, as well as the envelope of virions and dense bodies (black arrowhead), and the vacuole containing them (white arrowhead). N, nucleus; VL, vacuole; V, virion; D, dense bodies. Scale bars, 200 nm.

Figure 3. Immuno EM localisation of Rab27a on the viral envelope of isolated HCMV viral particles.

Isolated viral particles from BJ1 YFP-Rab27a cells HCMV infected for 5 days were permeabilised with saponin and labelled with antibodies against YFP (A) and HCMV tegument viral protein pp28 (B), and 10 nm protein-A gold. (C) General morphology of HCMV virions by negative staining with 2% uranyl acetate. When viral particles were partially disrupted, uranyl acetate revealed the nucleocapsids. Scale bar, 50 nm.

Figure 4. HCMV infection after knock-down of Rab27a.

(A) Mewo cells expressing non-target control and Rab27a shRNAs were lysed, and Rab27a expression was determined by Western blot. Actin served as loading control. Molecular weights in kDa are indicated. (B–D) Infectious viruses produced in BJ1 Rab27a shRNAs expressing cells. BJ1 untransduced, non-target control and Rab27a shRNAs expressing cells were infected with RCMV288 at a moi of 0.5. After 3 days a fraction of the cells was analysed by flow cytometry to assess the number of infected cells by GFP expression (B). Some cells were lysed at 4 dpi, and expression of Rab27a, actin and HCMV was analyzed by Western blot (C). At 5 dpi, supernatants and the remainder of the cells were harvested, and the number of extracellular and cell-associated infectious viruses was determine on fresh BJ1 cells as described in Materials and Methods (D). Data are means plus standard deviation (n = 3). ***, P<0.001. MFI: mean fluorescence intensity. IP: infectious particles.

Figure 5. MCMV infection in Rab27a-deficient melanocytes.

Melan-a and ashen-3 melanocytes were infected with MCMV, and after 24 h some cells were fixed and MCMV IE1 protein expression analysed by immunofluorescence microscopy (magenta in upper panels). At 48 hpi, supernatants and cells were harvested, and the number of extracellular and cell-associated infectious viruses were determined by plaque assay (histograms). Data are means plus standard deviations (n = 2). ***, P<0.001. PFU: plaque forming units.