Quantitative membrane proteomics reveals a role for tetraspanin enriched microdomains during entry of human cytomegalovirus

<div><p>Human cytomegalovirus (HCMV) depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC), membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM) cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-β-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells.</p></div

Tetraspanins CD81, CD9 and CD151 play a redundant role in HCMV-entry.

<p>HFFs were transfected with the indicated siRNAs for 64h. Control cells were left untransfected. (A) HCMV attachment to these cells was studied directly after incubating the cells for 1h with AD169 at an MOI of 3 on ice. The cells were lysed and samples were analyzed for pp65 levels of attached virions using SDS-PAGE and Western blot. The graph shows relative pp65 levels in every sample compared to untransfected HCMV-infected cells that were set to 100%. (B) Transfected HFFs were incubated with AD169 at an MOI of 3 for 1h at ice and subsequently moved to 37°C for 1h. The cells were washed with citric acid wash buffer to inactivate HCMV virions at the cell surface and infection proceeded for another 8h after which the cells were harvested and lysed. The samples were analyzed for IE1 expression using SDS-PAGE and Western blot. The graph shows relative IE1 expression in every sample compared to untransfected HCMV-infected cells that were set to 100%. Shown are the means ± standard error of the mean of three independent experiments. Experiments were repeated three times, one representative experiment is shown.</p

HCMV downregulates tetraspanins.

<p>(A) HFFs were infected with the HCMV strains AD169, Toledo, or Powers at an MOI of 3. At 24 hpi the cells were stained for cell surface CD81 expression, or fixed and permeabilized and stained for total CD81 protein expression using flow cytometry. (B) HFFs were infected with AD169 for 24h at an MOI of 3 and the cell surface and total protein expression of CD9 and CD151 was measured as described above. (C) HFFs were left uninfected or infected with HCMV TB40 (MOI of 5), HSV-1 (MOI of 10), VSV (MOI of 10), VACV (MOI of 5) and CPXV (MOI of 5). At 24 hpi the cells were fixed and stained for surface expression of CD81, CD151, CD9 or EGFR using flow cytometry. Before analysis HCMV-, VACV- and CPXV-infected HFFs were gated for GFP. Experiments were repeated two to three times and one representative experiment is shown.</p

MβCD alters the levels of tetraspanin-enriched membrane microdomains-associated proteins and blocks HCMV infection.

<p>(A) HFFs were treated with 8.0 mM MβCD for 1h, then the cells were fixed and cell surface expression of the indicated proteins was determined by flow cytometry. (B) HFFs were incubated with increasing concentrations of MβCD for 1h and subsequently infected with AD169 at an MOI of 3. The cells were fixed at 8 hpi and stained for IE1 and DAPI using IFA. (C) The IE1 positive cells shown in (B) were counted and the percentage of IE1 positive cells was calculated in relation to the DAPI-positive cells. *Indicates cells that were treated with 8 mM MβCD and with cholesterol. Experiments were repeated three times and the result of one representative experiment is shown.</p

Multiple host cell surface proteins show altered expression levels after HCMV infection.

<p>(A) Schematic overview of the proteomic analysis. HFFs were infected with AD169 at an MOI of 3. 24h later, IFA was used to verify the infection levels by intracellular IE1 staining. (B), by SDS-PAGE and Western blot analysis of whole cell lysates for pp28 and IE1 (C) and by monitoring MHC class I downregulation from the cell surface of infected cells using flow cytometry (D). (E) The results of three independent SILAC experiments are summarized. (F) Shown is the area measurement of the eluted peaks corresponding to the peptides of four of the selected proteins. (G) The selected seven proteins for follow-up studies are listed with their respective fold changes.</p

CD81 downregulation occurs in the absence of viral gene expression.

<p>(A) HFFs were left uninfected, or infected with UV-inactivated AD169 or AD169 at an MOI of 3. At 24, 48 and 72 hpi surface expression of CD81 and EGFR was determined by flow cytometry. Upregulation of MHC I by UV-inactivated HCMV and downregulation of MHC I by HCMV were confirmed by flow cytometry at 24 hpi. (B) HFFs were infected with HCMV TB40-GFP in the presence or absence of foscarnet. At 24 hpi the cell surface expression of CD81 and EGFR was assessed using flow cytometry. (C) HFFs were treated with IFNß or Poly I:C for 8h and CD81 cell surface expression was examined by flow cytrometry. (D) HFFs were infected with adenoviruses expressing the specified HCMV proteins or with a control adenovirus expressing the tetracyclin transactivator for 24h, after which CD81 cell surface expression was monitored by flow cytometry. All experiments were repeated two times and the result of one representative experiment is shown. (E) HFFs were infected with AD169 at an MOI of 3 and the cell surface expression of CD81 was examined at 2, 4, 8 and 24 hpi by flow cytometry. Results shown are an average of two independent experiments.</p

Validation of membrane protein downregulation by HCMV.

<p>HFFs were left uninfected or infected with the indicated HCMV strains at an MOI of 3 for 24h. (A) Cell surface proteins were biotinylated and isolated using NeutrAvidin agarose beads. Bound proteins were analyzed by SDS-PAGE and Western blot using the indicated antibodies. (B) Total changes in expression levels of the specified proteins were analyzed in whole cell lysates by SDS-PAGE and Western blot using specific antibodies. Cell surface downregulation of a selection of the proteins was also studied using flow cytometry (C) and IFA (D). Experiments were repeated two times and the result of one representative experiment is shown. For caveolin-1 staining cells were permeabilized with 0.5% Saponin for flow cytometry and 0.2% Triton X-100 for IFA. HCMV IE1 co-staining was performed to identify HCMV infected cells. (E) HFF cells were infected with increasing MOI of HCMV AD169 and at 24 hpi mRNA levels of CD44, CD81 and GRP78 were assessed using by qPCR. The results displayed are an average of three independent experiments that each included three replicates.</p