BST-2 Is Differentially Expressed in K5-Expressing Cells

<p>HeLa cells were transduced with Ad-WT, Ad-K5, Ad-vpu, or Ad-MARCH-VIII. Then 24 h post-infection, cells were harvested and whole cell lysates were analyzed for the abundance of BST-2 and MHC I by Western blotting. Note that BST-2 is highly glycosylated and runs as multiple bands. The high molecular weight band marked with an asterisk is non-specific. Equal protein loading was confirmed by visualizing the ER resident chaperone Bap31 as well as general protein staining with Ponceau red ([A], left). The intensity of each band was quantified using densitometry ([A], right). The specificity of BST-2 downregulation was confirmed by transfecting K5 or a catalytically inactive K5-RING mutant, which showed no effect on either MHC I or BST-2 (B). The protein bands corresponding to BST-2 are more intense in (B) than in (A) because of longer exposure of the autoradiograph as well as variation in electrophoretic separation. Note that the same lysates were analyzed in lanes 1–3 of the blots in both (A) and (B).</p

Ubiquitination and Lysosomal Degradation of ALCAM in the Presence of K5

<div><p>(A) Ubiquitination of ALCAM was examined by co-transfection of HeLa cells with ALCAM-HA as well as the indicated E3 enzymes. Then 24 h post-transfection, cells were lysed in 1% CHAPS, and ALCAM-HA was immunoprecipitated using anti-HA antibody. Samples were resolved on an 8% SDS-PAGE gel, transferred to PVDF, and immunoblotted (WB) with the anti-ubiquitin (Ubi) antibody P4D1 (top) or anti-HA (bottom). Ubiquitinated ALCAM was visible upon co-transfection of K5 and MARCH-VIII, but not with the inactive K5DE12 mutant and the unrelated HIV immune modulator vpu.</p><p>(B) To determine whether ubiquitinated ALCAM was degraded by proteasomes or in lysosomes, the fate of newly synthesized ALCAM-HA was determined by metabolic labeling for 10 min with S³⁵ Met/Cys and chasing the label for the indicated times (hours) in the presence of the indicated inhibitors. Following lysis, ALCAM-HA was immunoprecipitated using the HA antibody and samples were treated overnight with endoglycosidase H followed by electrophoretic separation. Note the increased recovery of ALCAM at 8 h in the presence of the endosomal/lysosomal proton pump inhibitor concanamycin A (ConA), but not in the presence of the proteasomal inhibitor MG132 (50 μmol).</p><p>(C) Surface expression of ALCAM can be restored by overexpressing a dominant negative version of the AAA-ATPase Vps4, which is essential for targeting proteins to MVBs. HeLa cells were transfected as indicated together with GFP to identify transfected cells. Then 24 h post-transfection, cells were harvested and the surface expression of either MHC I or ALCAM was analyzed using flow cytometry. The graph shows the ratio of mean fluorescence intensity from K5-transfected cells to that of control cells after gating for GFP. Data are averaged from three separate experiments.</p></div

Schematic Representation of the SILAC Labeling and Purification Protocol

<p>HeLa cells were grown for 5 d in labeling medium to ensure complete labeling. They were then infected with either adenovirus vector (light sample) or adenovirus-expressing K5 (heavy sample). Then 24 h post-infection, cells were harvested and counted, and equal numbers of cells combined. Samples were lysed in a Dounce homogenizer and unlysed cells removed by centrifugation. Membrane and soluble proteins were separated by centrifugation. The membrane pellet was resuspended, and different membrane fractions were separated over a discontinuous sucrose gradient. The bands corresponding to the plasma membrane (PM), Golgi, and ER fractions were removed and the proteins pelleted. The resulting pellet was then washed with sodium carbonate to remove non-integral membrane proteins, followed by ammonium bicarbonate to remove salts and other impurities. The final pellet was resuspended in 8 M urea and digested with trypsin for MS/MS analysis.</p

Protein Identification and Differential Peptide Quantification

<div><p>(A) Approximately 500–700 proteins were identified by at least one peptide in each fraction; however, fewer than 150 proteins per fraction were identified in all three replicates. Of these, only five proteins changed more than 1.5-fold in the plasma membrane (PM) fraction, while only one protein changed more than 1.5-fold in all three replicates of either the Golgi or ER fractions.</p><p>(B) Proteins present in all three replicates of each fraction were analyzed for their predicted subcellular distribution using annotations in the Swiss-Prot proteomics database.</p><p>(C) Comparison of signal intensities obtained for selected peptides from the indicated proteins. The red line indicates the actual raw data recovered from the mass spectrometer; the blue line is a hypothetical best fit line drawn to help analyze elution time of the light and heavy peptides. The orange arrows indicate the time at which the initial MS/MS scan was initiated. Peptides derived from unaffected proteins, such as CD44, display a similar intensity for both the light and heavy isotopes. Peptides derived from differentially expressed proteins, such as BST-2, show clearly different intensity peaks for the light and heavy peptides. Note the slightly different scale for intensity of each peptide.</p></div

K5 Downregulates ALCAM

<div><p>(A) C-terminally HA-tagged ALCAM was co-transfected with K5 or control plasmid. Then 24 h post-transfection, cells were harvested and the abundance of ALCAM-HA in each lysate was measured by Western blotting with anti-HA antibody.</p><p>(B) Cell surface expression of endogenous ALCAM in the presence or absence of K5 was determined by flow cytometry. HeLa cells were co-transfected with K5 and a green fluorescent protein (GFP)–expressing plasmid to identify transfectants. Then 24 h post-transfection, cells were harvested and stained with antibodies against MHC I, ALCAM, CD9, and CD29. Both vector (solid black) and K5-transfected (white) cells were gated for GFP-expressing cells.</p><p>(C) ALCAM downregulation by K5-related proteins was determined by transfecting HeLa cells with viral K3 family members (K3 and M153R) and the indicated human MARCH proteins. Also examined were mutant K5 proteins with enzymatically inactive RING-CH domains (K5-RING) or lacking acidic residues implicated in subcellular targeting (K5DE12). Neither K5 mutant reduced ALCAM levels. KSHV K3 was unable to downregulate ALCAM, whereas the myxomavirus M153R protein significantly reduced ALCAM surface expression. Two of the MARCH proteins, MARCH-IV and MARCH-IX, strongly downregulated ALCAM, while MARCH-VIII showed a minimal effect.</p><p>(D) To determine whether ALCAM expression was affected by KSHV, latently infected immortalized DMVECs were treated with PMA to induce expression of lytic genes including K5. Surface levels of either MHC I or ALCAM were measured by flow cytometry 24 and 48 h post-induction. CD81, measured at 24 h, was used as a control. The ratio between the mean fluorescence intensity of infected and uninfected samples from these experiments is shown as fold change on the right.</p></div

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

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