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. 2017 Nov;7(11):160298.
doi: 10.1098/rsob.160298.

Cellular v-ATPase is required for virion assembly compartment formation in human cytomegalovirus infection

Jonathan Pavelin  1 , Dominique McCormick  1 , Stephen Chiweshe  1 , Saranya Ramachandran  1 , Yao-Tang Lin  1 , Finn Grey  2
Affiliations

Cellular v-ATPase is required for virion assembly compartment formation in human cytomegalovirus infection

Jonathan Pavelin et al. Open Biol. 2017 Nov.

Abstract

Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral therapy.

Keywords: assembly and egress; herpesvirus; host virus interaction; human cytomegalovirus; v-ATPase.

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Conflict of interest statement

The authors have no competing interests.

Figures

Figure 1.
Figure 1.
Knockdown of ATP6V0C has greater effect on supernatant virus than cell associated virus. Fibroblast cells were transfected with siRNA against ATP6V0C or a control siRNA and infected with TB40/E-GFP at an MOI of 1 and cells and supernatant collected 7 days post-infection. (a) Infectious cell associated and supernatant virus levels were determined by dilution plaque assay following ATP6V0C knockdown. (b) Effects of ATP6V0C knockdown on infectious supernatant virus levels are greater than effects on cell associated virus. (c) Cell associated viral genome levels were equivalent between control cells and ATP6V0C knockdown cells at 7 days post-infection (d) Supernatant virion genome levels were determined by qPCR. Supernatant was collected from cells 7 days post infection. Virions were isolated from supernatant by ultracentrifugation then treated with DNase to degrade non-virion associated viral DNA. Primers against HCMV gB were used to determine viral genome levels and primers to GAPDH (e) were used to confirm successful degradation of non-protected DNA (n = 2).
Figure 2.
Figure 2.
Knockdown of ATP6V0C has no effect on levels of representative viral immediate early or late transcripts. Fibroblast cells were transfected with ATP6V0C siRNA or a control siRNA and infected 48 hpi. Total RNA was harvested at indicated time points. Levels (a) of IE86, (b) UL83 and (c) UL75 were determine by qRT-PCR using specific primer probe assays (n = 2). (d) Cell-associated viral protein levels were determined by western blot analysis at the indicated time points post-infection from control cells (siNeg) or cells knocked down for ATP6V0C (siA6).
Figure 3.
Figure 3.
ATP6V0C is not required for generation of viral capsids in the nucleus. To directly analyse the effects of ATP6V0C knockdown on intracellular viral particle formation, infected cells were imaged by transmission electron microscopy at 120 hpi. Fibroblast cells were transfected with (a) ATP6V0C siRNA or (b) negative control siRNA and infected with HCMV (AD169). (c) Enlarged image from (b) showing three capsid types: A—empty capsid, B—capsid with scaffold, C—DNA containing capsid. (d) Manual counting of images revealed no significant difference in nuclear capsid formation following ATP6V0C knockdown. Nine individual frames from three independent cells were counted for each condition. A total of 508 capsid particles were counted for control cells and 516 particles counted for ATP6V0C knockdown cells.
Figure 4.
Figure 4.
ATP6V0C is required for HCMV virion assembly compartment biogenesis. Fibroblast cells were transfected with ATP6V0C siRNA or negative control siRNA and at 72 h post-transfection were infected with AD169. At 144 hpi cells were fixed, permeabilised and stained for early endosomes or trans-Golgi vacuoles, (EEA1:green or TGN46:green), viral tegument protein (pp28:red) and nuclei (DAPI:blue). Images were acquired on a Zeiss LSM710 confocal microscope. Images presented here are maximum-intensity projections compiled from multiple 0.33 µm slices through the z-axis.
Figure 5.
Figure 5.
Loss of VAC due to ATP6V0C1 knockdown is quantifiable and significant. (a) Fibroblast cells were transfected with ATP6V0C siRNA or negative control siRNA and at 72 h post-transfection were infected with AD169. At 144 hpi cells were fixed, permeabilised and stained for early endosomes or trans-Golgi vacuoles, (EEA1:green), viral tegument protein (pp28:red) and nuclei (DAPI:blue). Images represent single slices through the Z-axis. (b) Representative scatter-plot showing average pixel signal intensity in red (pp28) and green (EEA1) channels from multi-cell images (n = 16 for ATP6V0C and n = 9 for negative siRNA). Individual images in the Z-field were analysed using Fiji image analysis software. (c) Pearson's R-value for colocalization of TGN46 or EEA1 and pp28 in ATP6V0C or negative control siRNA transfected fibroblast cells (n = 20). *p-value < 0.05; **p-value < 0.01.
Figure 6.
Figure 6.
Failure of VAC formation following AT6V0C knockdown is not due to gross defects in cellular membrane organization prior to infection. (a) Fibroblast cells were transfected with ATP6V0C siRNA or negative control siRNA. At 72 h post-transfection cells were fixed, permeabilised, and stained for early endosomes or trans-Golgi vacuoles. Images are maximum-intensity projections compiled from multiple 0.33 µM slices through the z-axis. (b) Wide field view of fibroblast cells from (a). Image is a single optical slice. (c) Western blot analyses of ATP6V0C siRNA and negative control and transfected fibroblast cells against markers of trans-Golgi vacuoles (TGN46), early endosomes (EEA1). Infected cells were harvested at 72 hpi. (d) Graph shows quantification of representative western shown in C.+ = ATP6V0C siRNA transfected fibroblast, − = negative control siRNA.
Figure 7.
Figure 7.
ATP6V0C knockdown disrupts changes in nuclear size. (a) Representative image showing nuclei in ATP6V0C and negative control siRNA transfected fibroblast cells infected with HCMV (AD169). Using Fiji imaging software, masks were drawn around nuclei and nuclear area was calculated. (b) Nuclear areas in ATP6V0C and negative control siRNA transfected fibroblast cells infected with HCMV (AD169) and mock-infected fibroblast cells. Quantification based on minimum 12 cells per condition per time point. Difference in cross-sectional nuclear area between control cells and ATP6V0C knockdown cells was significant based on two-way ANOVA analysis with repetitions p ≤ 0.01.
Figure 8.
Figure 8.
Disruption of V-ATPase complex results in loss of VAC formation. (a) Immunofluorescence microscopy in AD169 infected fibroblast cells transfected with ATP6V1H siRNA and stained for early endosomes (EEA1/green), viral tegument protein (pp28/red) and nuclei (DAPI/blue). Images represent single slices through the z-axis. (b) Pearson's R-value for colocalization of TGN46 or EEA1 and pp28 in ATP6V1H or negative control siRNA transfected fibroblast cells (n = 20). **p-value < 0.01.
Figure 9.
Figure 9.
Chloroquine inhibits HCMV virus production and VAC formation. (a) Fibroblast cell were pre-treated 24 h before infection with the indicated concentrations of chloroquine. Following infection with TB40/E-GFP supernatant was harvested 7 days post-infection and virus levels determined by serial dilution plaque analysis. (n = 4, error bars indicate standard deviation, *p-value < 0.05). (b) Total RNA was extracted from control cells and cells treated as described above with 25 mM chloroquine (CQ), to determine viral transcript levels by qRT-PCR. Transcript levels were normalized to GAPDH and compared to untreated sample (UN) (n = 2, error bars indicate standard deviation). (c) Fibroblast cells were treated with 25 mM of chloroquine then infected with AD169 at an MOI of 1. Cells were fixed 96 hpi and analysed by immunofluorescence for viral pp28 (red), cellular GM130 (green) and nuclear stain DAPI (blue).
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