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

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

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

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

Interactions between herpesvirus-encoded TAP-inhibitors and their target.

<p>Upper illustration: model of the TAP transporter, comprising the two subunits TAP1 and TAP2. Each subunit contains a transmembrane domain (TMD), encompassing 10 and 9 transmembrane (TM) helices for TAP1 and TAP2, respectively. The outer N-terminal helices of TAP1 and TAP2 (TMD0) form an autonomous binding platform for tapasin, whereas the core 6 TM helices are necessary for peptide transport. A peptide-binding domain is located within the cytosolic extensions of the TM helices. In addition, TAP1 and TAP2 contain a nucleotide-binding domain (NBD) in the cytosol, which harbors two ATP-binding sites. Lower illustrations: schematic representations of the interaction between the viral proteins and TAP. The sites where TAP is affected are indicated. HSV-1 ICP47 prevents peptide transport by physically obstructing the peptide-binding site. PRV, BoHV-1 and EHV (EHV-1 and EHV-4) UL49.5 leave the transporter in a transformation-incompetent conformation, thereby preventing the structural changes that are needed to translocate peptides over the ER membrane. BoHV-1 UL49.5 is known to interact with a region within the core domain of TAP, comprising the C-terminal 6 TM domains of both TAP1 and TAP2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref132" target="_blank">132</a>]. BoHV-1 UL49.5 induces the degradation of both TAP subunits, and EHV UL49.5 prevents ATP binding to TAP. HCMV US6 blocks TAP by inducing conformational changes that result in diminished ATP binding to TAP1. The protein interacts with TM domains 7–10 of TAP 1 and TM 1–4 of TAP2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref095" target="_blank">95</a>]. EBV BNLF2a inhibits peptide transport by interfering with both peptide and ATP binding to TAP.</p

Phylogenetic tree for selected members of the family Herpesviridae.

<p>The Bayesian tree is based on amino acid sequence alignments for six large, well-conserved genes, namely the orthologs of HSV-1 genes UL15, UL19, UL27, UL28, UL29, and UL30, and is derived from McGeoch and Davison [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref081" target="_blank">81</a>]. Assignments to genera and subfamilies are shown on the right. Abbreviations not mentioned in the text are: MDV-1, Marek's disease virus type 1; MDV-2, Marek's disease virus type 2; HVT, herpesvirus of turkey; ILTV, infectious laryngotracheitis virus; PsHV-1, psitticid herpesvirus 1; GTHV, green turtle herpesvirus; THV, tupaia herpesvirus; GPCMV, guinea pig cytomegalovirus; CalHV-3, callitrichine herpesvirus 3; AHV-1, alcelaphine herpesvirus 1; OHV-2, ovine herpesvirus 2; PLHV-1, porcine lymphotropic herpesvirus 1; HVS, herpesvirus saimiri; HVA, herpesvirus ateles; and RRV, rhesus rhadinovirus. Red, blue, orange, and green shading indicate viruses that encode the ICP47, UL49.5, US6, or BNLF2a TAP inhibitor genes, respectively, and corresponding coloring of virus abbreviations indicate viruses in which these genes have been shown to be functional TAP inhibitors. Light orange shading identifies all members of the <i>Cytomegalovirus</i> genus that have a US6 gene. Light blue shading indicates all members of the herpesvirus family that code for a UL49.5 gene that might be involved in chaperoning maturation of glycoprotein M.</p

The characteristics of a conditionally ORF63/ORF70-expressing mutant virus.

<p>(A) The SVV genome consists of unique long (UL) and a unique short (US) segments and each of them being bound by inverted repeat sequences, TRL, IRL and IRS and TRS respectively. The left end of the SVV genome contains an additional invert repeat sequence. Using a recombinant SVV BAC we generated an SVV mutant in which we fused the destabilizing dihydrofolate reductase (DHFR) to the C-terminus of both ORF63 and ORF70 (63-DHFR). (B) Mono layers of Vero cells were infected with wild type or ORF63/70-DHFR SVV in the presence of 10 μM TMP and harvested at 3, 24, 48, 72, 96, 120, 144 and 168 hours p.i. The titer of each virus was determined at each time point by plaque assay. (C) Vero cells infected with ORF63/70-DHFR SVV were cultured in the presence of 10 μM TMP (first panel), after which the virus was passaged two times on Vero cells in the absence of TMP. In the third panel, TMP was added again to the culture. Viral plaques were identified by staining with crystal violet. (D) TRFs were infected with wild type and ORF63/70-DHFR SVV for 48 hours and stimulated with 5000 U/ml uIFN for 20 minutes. Cells were lysed and expression of IRF9, STAT2 and phosphorylated STAT2 (pSTAT2) was confirmed by SDS-PAGE and western blot using specific antibodies. Lysates were specifically stained for ORF31 and ORF63 to confirm infection. GAPDH was used a loading control. (E) TRFs were infected with ORF63/70-DHFR SVV for 48 hours in the presence of the indicated concentrations of TMP. Expression of IRF9, ORF63 and ORF31 were detected SDS-PAGE and western blot using specific antibodies. GAPDH was used as a loading control. One of three independent experiments is shown.</p