Nonenvelopment Role for the ESCRT-III Complex during Human Cytomegalovirus Infection

Abstract

Secondary envelopment of human cytomegalovirus (HCMV) occurs through a mechanism that is poorly understood. Many enveloped viruses utilize the endosomal sorting complexes required for transport (ESCRTs) for viral budding and envelopment. Although there are conflicting reports on the role of the ESCRT AAA ATPase protein VPS4 in HCMV infection, VPS4 may act in an envelopment role similar to its function during other viral infections. Because VPS4 is normally recruited by the ESCRT-III complex, we hypothesized that ESCRT-III subunits would also be required for HCMV infection. We investigated the role of ESCRT-III, the core ESCRT scission complex, during the late stages of infection. We show that inducible expression of dominant negative ESCRT-III subunits during infection blocks endogenous ESCRT function but does not inhibit virus production. We also show that HCMV forms enveloped intracellular and extracellular virions in the presence of dominant negative ESCRT-III subunits, suggesting that ESCRT-III is not involved in the envelopment of HCMV. We also found that as with ESCRT-III, inducible expression of a dominant negative form of VPS4A did not inhibit the envelopment of virions or reduce virus titers. Thus, HCMV does not require the ESCRTs for secondary envelopment. However, we found that ESCRT-III subunits are required for efficient virus spread. This suggests a role for ESCRT-III during the spread of HCMV that is independent of viral envelopment.IMPORTANCE Human cytomegalovirus (HCMV) is a prevalent opportunistic pathogen in the human population. For neonatal and immunocompromised patients, HCMV infection can cause severe and possibly life-threatening complications. It is important to define the mechanisms of the viral replication cycle in order to identify potential targets for new therapies. Secondary envelopment, or acquisition of the membrane envelope, of HCMV is a mechanism that needs further study. Using an inducible fibroblast system to carefully control for the toxicity associated with blocking ESCRT-III function, this study determines that the ESCRT proteins are not required for viral envelopment. However, the study does discover a nonenvelopment role for the ESCRT-III complex in the efficient spread of the virus. Thus, this study advances our understanding of an important process essential for the replication of HCMV.

Keywords: CHMP; ESCRT; HCMV; VPS4; assembly; envelopment; spread.

FIG 1

High levels of dominant negative ESCRT-III subunits prevent the initiation of HCMV infection. (A) Model of ESCRT recruitment during ILV formation. (B) Titers of viruses expressing a parental control, GFP, or CHMP6-GFP at 96 hpi (MOI, 3) in the presence or absence of 100 ng/ml doxycycline (Dox). Viral titers are from three independent replicates. (C) Immunofluorescence staining of immediate early proteins (red) 9 dpi (MOI, 0.05) in the presence of Dox. (D) Immunofluorescence staining of pp28 (red) at 96 hpi in cells infected with a virus expressing CHMP6-GFP or a control virus at 96 hpi. Nuclei were labeled with DAPI (blue). Bars, 10 μm. (E) Western blot analysis of viral proteins pUL44, pp150, and pp28 at 96 hpi (MOI, 3) from fibroblasts infected with viruses expressing GFP or CHMP6-GFP. (F) Titers of viruses expressing CHMP4C-GFP or CHMP2A-GFP. Viral titers are from three independent experiments.

FIG 2

ESCRT function is inhibited in inducible ESCRT-III fibroblasts. (A) GFP expression of inducible fibroblasts expressing GFP, CHMP6-GFP, CHMP4B-GFP, or CHMP4C-GFP 24 h post-Dox addition. Nuclei were labeled with DAPI (blue). Bars, 10 μm. (B) Western blot analysis of HCMV-infected cells expressing CHMP6-GFP from the HCMV genome (HCMV) or of fibroblasts following lentiviral transduction (Lenti) and selection for cells containing the inducible construct. Dox was present throughout the infection (96 h). (C) Western blot analysis of endogenous and GFP-tagged CHMP6, CHMP4B, and CHMP4C at 96 hpi after Dox induction for 0, 48, 72, or 96 h. NS, nonspecific band detected by the CHMP4B antibody; *, unknown band detected by the CHMP6 antibody only in Dox-induced samples. (D) EGF degradation in ESCRT-III dominant negative (green) fibroblasts. EGF (red) was imaged 3 h after a 30-min pulse in the presence or absence of Dox. Nuclei are labeled with DAPI. Bars, 10 μm. (E) HSV-1 infection of fibroblasts expressing GFP, CHMP6-GFP, or CHMP4-GFP at 24 hpi (MOI, 5). Dox was added 24 h prior to infection. Significant differences (P < 0.05) between samples are marked with asterisks. Infections were carried out in three independent experiments.

FIG 3

HCMV viral protein expression and cVAC formation are unaffected by dominant negative ESCRT-III subunits. (A) Western blot analysis of viral proteins 96 hpi (MOI, 3) in fibroblasts expressing CHMP6-GFP, CHMP4B-GFP, or CHMP4C-GFP. Hours (above the blot) indicate the duration of Dox induction. (B) Immunofluorescence staining for pp28 (red) and gB (green) 96 hpi. Fibroblasts were treated with Dox for 48 h prior to fixation. Nuclei were labeled with DAPI. Bars, 10 μm. (C to E) Cell viability assays on fibroblasts expressing GFP, CHMP6-GFP, CHMP4B-GFP, or CHMP4C-GFP 96 hpi (MOI, 3) as measured by a trypan blue (C), XTT (D), or CellTiter-Glo (E) assay. For each cell type, viability is shown relative to no Dox treatment. Significant differences (P < 0.05) between samples are marked with asterisks. All viability assay results are from at least three independent experiments.

FIG 4

ESCRT-III is not required for productive HCMV infection. (A to C) Virus titers at 96 hpi (MOI, 3) of fibroblasts expressing GFP or CHMP6-GFP (A), CHMP4B-GFP or CHMP4C-GFP (B), or CHMP3-GFP or CHMP2A-GFP (C) with HCMV strain AD169. (D) Western blot analysis of endogenous and GFP-tagged CHMP3 and CHMP2A at 96 hpi after Dox induction for 0, 48, 72, or 96 h. NS, nonspecific band detected by the CHMP3 antibody. (Inset) Mock-treated and 96-hpi lysates probed with the CHMP2A antibody. (E) Intracellular and extracellular virus titers (96 hpi) on fibroblasts selected for constructs expressing GFP, CHMP6-GFP, or CHMP4B-GFP, infected with HCMV AD169 in the presence or absence of Dox (48-h induction). (F) Virus titers at 96 hpi on fibroblasts with HCMV strain TB40/E. No significant differences were found between samples (P > 0.05). Titers are from at least three independent experiments. (G) Micrographs depicting the localization of CHMP-GFP proteins in relation to pp28 (red). Bars, 10 μm.

FIG 5

Dominant negative ESCRT-III does not inhibit HCMV secondary envelopment. (A) Electron micrographs of enveloped intracellular and extracellular virions in fibroblasts expressing GFP, CHMP6-GFP, or CHMP4B-GFP at 96 h after infection with AD169 (MOI, 3). Fibroblasts were treated with Dox for 48 h prior to fixation. (B) Graph showing percentages of membrane-associated capsids that have not completed envelopment compared to percentages of fully enveloped capsids. Total numbers of capsids were 182 (GFP), 190 (CHMP6), and 181 (CHMP4B).

FIG 6

VPS4A is not required for HCMV envelopment. (A) Fibroblasts were transduced with inducible WT GFP-VPS4A or VPS4A^E228Q and were imaged 24 h post-Dox treatment. Nuclei were labeled with DAPI (blue). Bars, 10 μm. (B) Western blot analysis of cellular protein EphA2 and viral proteins pUL44, pp150, and pp28 at 96 hpi after AD169 infection (MOI, 3) of fibroblasts expressing WT VPS4A (WT) or dominant negative VPS4A^E228Q (DN). (C) Virus titers on fibroblasts expressing WT VPS4A or VPS4A^E228Q 24 h after HSV-1 infection. Dox was added to cells 24 h prior to infection. The asterisk indicates a significant difference (P < 0.05) between samples. (D and E) HCMV AD169 (D) and TB40/E (E) titers at 96 h after infection (MOI, 3) of fibroblasts expressing WT VPS4A or VPS4A^E228Q. No significant differences were found between samples (P > 0.05). Viral titers are from three independent experiments. (F) Electron micrographs of enveloped intracellular and extracellular virions from VPS4A^E228Q cells 96 h after infection with HCMV AD169 (MOI, 3). Dox was added to cells for 48 h prior to fixation.

FIG 7

Dominant negative ESCRT-III subunits slow HCMV spread during low-MOI infection. Shown are low-MOI (0.05) infections of fibroblasts expressing inducible GFP, CHMP6-GFP, or CHMP4B-GFP with AD169 Tet activator HCMV. (A) Immunofluorescence staining for immediate early protein (red) 3, 9, and 15 dpi. Dox was added to cells at the beginning of infection, and fresh Dox was added every 3 days thereafter. (B) The percentage of the monolayer infected was calculated by comparing DAPI and immediate early protein staining fluorescence within the monolayer from three independent experiments. (C) Percentage of the monolayer infected in fibroblasts expressing CHMP4C-GFP and CHMP3-GFP during low-MOI infection with Dox treatment as described for panel A.