Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141.

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.This study was financially supported by grant 101-2917-I-564-035 from the Taiwan National Science Council to JLH; by a Wellcome Trust Fellowship (093966/Z/10/Z) to MPW; an MRC Project Grant and Wellcome Trust Programme Grant (G1000236, WT090323MA) to GWW and PT, European Regional Development Fund and the State Budget of Czech Republic (RECAMO, CZ.1.05/ 2.1.00/03.0101) to ER; a Wellcome Trust Principal Research Fellowship (084957/Z/08/Z) to PJL; and a Medical Research Council (MRC) grant (MC_UU_12014/3) to GSW and AJD. This study was additionally supported by the Cambridge Biomedical Research Centre, UK.This is the final published version. It first appeared at http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004811

Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors

Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors

High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms.

Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.This work was supported by a Wellcome Trust Senior Clinical Research Fellowship (108070) to MPW, a strategic award to Cambridge Institute for Medical Research from the Wellcome Trust (100140), MRC Project Grants to GWGW, PT, ECYW and RJS (MR/L018373/1, MR/P001602/1), a Wellcome Trust Programme Grant (WT090323MA) to GWGW, ECYW and PT, and an MRC Programme Grant (MC_UU_12014/3) to AJD. This study was additionally supported by the Cambridge Biomedical Research Centre, UK

Human cytomegalovirus glycoprotein UL141 targets the TRAIL death receptors to thwart host innate antiviral defenses.

Death receptors (DRs) of the TNFR superfamily contribute to antiviral immunity by promoting apoptosis and regulating immune homeostasis during infection, and viral inhibition of DR signaling can alter immune defenses. Here we identify the human cytomegalovirus (HCMV) UL141 glycoprotein as necessary and sufficient to restrict TRAIL DR function. Despite showing no primary sequence homology to TNF family cytokines, UL141 binds the ectodomains of both human TRAIL DRs with affinities comparable to the natural ligand TRAIL. UL141 binding promotes intracellular retention of the DRs, thus protecting virus infected cells from TRAIL and TRAIL-dependent NK cell-mediated killing. The identification of UL141 as a herpesvirus modulator of the TRAIL DRs strongly implicates this pathway as a regulator of host defense to HCMV and highlights UL141 as a pleiotropic inhibitor of NK cell effector function

PMP comparing wild-type and US2-deficient HCMV shows a requirement for US2 in the downregulation of cell surface targets.

<p>(A) Scatter plots of proteins identified in PMP and quantified by 2 or more unique peptides. Fold change (HFF infected with wild-type HCMV versus HFF infected with ΔUS2 HCMV) is shown as log₂ ratio on the x axis and the summed peptide intensity on the y-axis as log₁₀. Proteins unaltered by HCMV US2 gene deletion locate to the center of the plots (0-fold change), whereas proteins left or right of center represent respectively proteins down- or up-regulated by HCMV in a US2-dependent manner. Significance B was used to estimate p values. (<b>B</b>) US2 is required for integrin and thrombomodulin downregulation during HCMV infection. HFF cells infected with GFP tagged HCMV ΔUL16-18 or ΔUL16-18ΔUS1-11 (moi 0.5) were analyzed by cytofluorometric analysis of the indicated proteins at 72 hours post-infection. (<b>C</b>) HFFs infected with HCMV wild-type or ΔUS2 (moi 0.5) were analyzed by flow cytometry at 72 hours post-infection. Cell surface staining for MHC-I enabled gating for HCMV infected (MHC-I^lo) cells (left panel) and subsequent analysis of the integrin and thrombomodulin expression (right panels). See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004811#ppat.1004811.s007" target="_blank">S3 Table</a></b>.</p

UL141 retains CD112 in the ER and enhances US2 mediated degradation.

<p>ER retention of CD112 is mediated by UL141 and only detected upon depletion of US2 or TRC8. (<b>A</b>) HFF infected with mock, HCMV wild-type, ΔUS2 or ΔUL141 (moi 5) were analyzed for the indicated proteins at 72 hours post infection, in the presence (shTRC8+) or absence (shTRC8-) of TRC8 depletion. (<b>B</b>) UL141 retains CD112 in the ER prior to TRC8-dependent degradation by US2. THP-1 cells stably expressing US2, US11, UL141-Myc or shTRC8 were solubilized in 1% digitonin, immunoprecipitated with anti-Myc antibody and the indicated proteins visualized by immunoblot. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004811#ppat.1004811.s004" target="_blank">S4 Fig</a></b>.</p

US2 induces the proteasomal degradation of integrin α4 and α5 and prevents maturation of the β1 integrin.

<p>(<b>A and B</b>) US2 induces proteasomal degradation of integrins α4 and α5. THP-1 cells stably expressing US2, US11 or depleted of TRC8 (shTRC8) were analyzed by immunoblots for the indicated proteins. Cells were treated with the proteasome inhibitor lactacystin for 18 hours before harvest as indicated. (<b>C-D</b>) Integrin 4 is proteasomally degraded in US2-expressing cells. THP-1 cells expressing integrin α4-HA were labeled with [³⁵S]methionine-cysteine and pulse-chased for the indicated times in the presence or absence of MG132, followed by integrin α4 immunoprecipitation using the HA-tag <b>(E)</b> β1 integrin accumulates in its precursor form in the presence of US2. THP-1 cells were pulse-chase labelled as above and endogenous β1 integrin was immune precipitated using anti-integrin β1 antibody. (<b>F and G</b>) US2 associates with integrin α4 and recruits the TRC8 E3 ligase via its cytosolic tail. Cells stably expressing full-length US2 (US2) or US2 with Δ186–199 residues (US2ΔC') in combination with TRC8-HA or integrin α4-HA were solubilised in 1% digitonin, immunoprecipitated with anti-HA antibody and visualized by immunoblot. (<b>H</b>) US2 induces integrin α4 ubiquitination. THP-1 cells expressing integrin α4-HA in combination with US2 or empty vector were pulse-chase labeled for the indicated times. Integrin α4 was immune precipitated using the HA-epitope tag, eluted from beads, fully denatured to dissociated non-covalently linked proteins and re-precipitated using α-HA or α-ubiquitin antibodies.</p

High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms.

Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.This work was supported by a Wellcome Trust Senior Clinical Research Fellowship (108070) to MPW, a strategic award to Cambridge Institute for Medical Research from the Wellcome Trust (100140), MRC Project Grants to GWGW, PT, ECYW and RJS (MR/L018373/1, MR/P001602/1), a Wellcome Trust Programme Grant (WT090323MA) to GWGW, ECYW and PT, and an MRC Programme Grant (MC_UU_12014/3) to AJD. This study was additionally supported by the Cambridge Biomedical Research Centre, UK