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. 2009 Oct;83(20):10797-807.
doi: 10.1128/JVI.01093-09. Epub 2009 Jul 29.

Human cytomegalovirus exploits ESCRT machinery in the process of virion maturation

Ritesh Tandon  1 , David P AuCoin, Edward S Mocarski
Affiliations

Human cytomegalovirus exploits ESCRT machinery in the process of virion maturation

Ritesh Tandon et al. J Virol. 2009 Oct.

Abstract

The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4DN) in HCMV replication. Vps4DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4DN or CHMP1DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.

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Figures

FIG. 1.
FIG. 1.
DN ESCRT proteins inhibit CMV replication. HCMV IE1 protein was coexpressed with WT or DN proteins of cellular ESCRT machinery in HFs, and cells were infected with IE1-deficient HCMV (CR208). (A) At the endpoint (6 dpi), ppUL44+ foci were quantified. (B) ESCRT inhibition allowed expression of delayed early viral antigen (ppUL44). The native cytoplasmic fluorescence of GFP-tagged Tsg101DN, eYFP tagged-CHMP1ADN, and nuclear fluorescence of eGFP-tagged IE1 is shown in the left panels. Vps4A was detected by anti-FLAG primary antibody and Alexa-488 secondary antibody. ppUL44 concentrated in the nuclear replication compartments in the primary transfected cell (arrowheads) in all transfections suggesting progression to a late stage of viral replication. (C and D) Giemsa-stained viral plaques were quantified in a similar CR208 complementation assay at 10 dpi.
FIG. 2.
FIG. 2.
UL32-PTAP mutant complements UL32 defect similar to WT protein in a secondary spread assay. WT (Towne) or ΔUL32-BAC strains were transfected in HFs, along with indicated expression constructs, and the eGFP+ foci were counted at 10 days posttransfection.
FIG. 3.
FIG. 3.
Cellular rearrangement of ESCRT proteins during HCMV infection. HFs transfected with plasmids expressing Vps4AWT, Vps4ADN, CHMP1A WT, or CHMP1A DN proteins were infected with HCMV Towne virus at 24 hpt. Cells were fixed and photographed for immunofluorescence microscopy at day 5 postinfection. Arrowheads point to the AC in infected cells. The native fluorescence of eYFP-tagged CHMP1ADN protein (M, N, O, and P) and immunofluorescence localization of CHMP1AWT protein using anti-myc primary antibody and Alexa-488 secondary antibody (I, J, K, and L) is shown. Vps4A was detected by anti-FLAG primary antibody and Alexa-488 secondary antibody (A, B, C, D, E, F, G, and H). Hoechst 33342 staining (blue) marks the nucleus in merged images (B, D, F, H, J, L, N, and P).
FIG. 4.
FIG. 4.
HCMV-infected HFs express gM-gN glycoproteins in the presence of DN or WT proteins of the ESCRT machinery. Epifluorescence micrograph of cells coexpressing control eGFPC1 protein (A to C), myc-tagged CHMP1AWT protein (D to F), or eYFP-tagged CHMP1ADN protein (G to I) and DsRed-tagged viral proteins. CHMP1AWT protein was detected using anti-myc primary antibody and Alexa 488-conjugated secondary antibody (E and F). Transfected cells were infected at 24 hpt and were fixed and photographed at day 5 postinfection. Hoechst 33342 (blue) stains for the nucleus (C, F, and I). Arrowheads point to the AC in infected cells (C, F, and I).
FIG. 5.
FIG. 5.
Viral plaque assays to analyze ability of cytoplasmic viral particles in lysates from ESCRT inhibited or uninhibited cells to form plaques on IE1-complementing cells. CR208 complementation assays were set up as described previously (A) and at day 3 (B) or day 6 (C) postinfection, and the cells were harvested, sonicated, and titered on ihfie1.3 cells. Plaques were counted at day 10 postinoculation.
FIG. 6.
FIG. 6.
Proposed model for HCMV exploitation of host-cell ESCRT machinery incorporating the results obtained in the present study. Mutation of PTAP domain in pUL32 (pp150) (circled 1) and DN inhibition of Tsg101 (circled 2), ALIX (circled 3), or WWP1 (HECT Ub E3) (circled 4) did not affect HCMV replication but inhibition of CHMP1A (ESCRT-III component) (boxed 5) or Vps4A (ATPase) (boxed 6) impaired HCMV replication. HCMV is proposed to either utilize novel late domains and novel ESCRT recruiting proteins for engaging ESCRT, or it depends on ESCRT machinery for the biogenesis of MVBs for viral protein processing.
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