Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases

<div><p>The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.</p></div

PLP2 is required for the export of a K3•MHC-I complex from the ER.

<p>(<b>A</b>) Validation of the cell lines used for the experiment. FLAG-K3 and PLP2 were immunoprecipitated from the indicated [³⁵S]-methionine pulse-labelled cell types, resolved by SDS-PAGE and analysed by autoradiography. (<b>B</b>) K3-bound MHC-I remains EndoH-sensitive in the absence of PLP2. The indicated cell types were [³⁵S]-methionine pulse-labelled for 12 min, chased for the indicated times and solubilised in 1% digitonin. FLAG-K3 was immunoprecipitated using the anti-FLAG M2 mAb, dissociated in 1% SDS, MHC-I molecules re-precipitated with the anti-MHC-I heavy chain mAb HC10 and then either EndoH (+) or mock digested (−) before the samples were resolved by SDS-PAGE and analysed by autoradiography. (<b>C</b>) Loss of PLP2 does not affect the normal MHC-I synthetic pathway. Free [³⁵S]-methionine labelled MHC-I molecules were immunoprecipitated from the post-M2 supernatants (from B) using the W6/32 mAb, EndoH (+) or mock digested (−), and analysed by SDS-PAGE and autoradiography. (<b>D</b>) PLP2 co-immunoprecipitates with K3 and K5. FLAG-K3 and K5 were immunoprecipitated from digitonin lysates of HeLa cells using the anti-FLAG M2 mAb, and co-immunoprecipitated PLP2 detected by immunoblot.</p

An ER-retained PLP2 mutant can still bind K5 but cannot support the K5-mediated downregulation of B7-2.

<p>(<b>A</b>) Addition of a di-lysine ER retention motif onto the C-terminal tail of PLP2 relocalises the protein to the ER. HeLa cells were transduced with lentiviral vectors expressing mCherry-PLP2 or mCherry-PLP2_KKAA, fixed, and immunostained for calnexin to mark the ER. (<b>B</b>) K5 can still bind the PLP2_KKAA mutant. K5 was immunoprecipitated from K5-PLP2^GT cells expressing the PLP2_KKAA mutant, resolved by SDS-PAGE, and co-immunoprecipitating PLP2 detected by immunoblot. (<b>C</b>) The ER-retained PLP2_KKAA cannot support the K5-mediated downregulation of B7-2. K5-PLP2^GT cells were transduced with a lentiviral vector encoding the indicated PLP2 mutant proteins and analysed for cell surface B7-2 expression by flow cytometry.</p

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96

PLP2 is required for the function of K3.

<p>(<b>A</b>) Schematic representation of the dre-mediated excision of K5 to generate a ‘clean’ PLP2 knockout cell. (<b>B</b>) Excision of K5 from K5-PLP2^GT cells. Immunoblot analysis of FLAG-tagged K3, FLAG-tagged K5 and PLP2 from the indicated cell lines. The resulting PLP2^GT cells no longer express K5. (<b>C</b>) K3 requires PLP2 to downregulate MHC-I. Cell surface expression of MHC-I was analysed by flow cytometry in the indicated cells. Due to incomplete transduction with PLP2, there is a population of K3-PLP2^rescue cells that fail to downregulate MHC-I (right plot). (<b>D</b>) K3 function in HepG2 is rescued following expression of exogenous PLP2. Cell surface expression of MHC-I was analysed in the indicated cells. As K3 and GFP are expressed from different promoters in the expression construct, it is likely that GFP levels do not accurately reflect K3 levels in the cell, and hence even some of the GFP-negative cells have sufficient K3 to downregulate MHC-I. (<b>E</b>) Knockdown of PLP2 in BC-3 cells. BC-3 cells were transduced with lentiviral vectors encoding either a non-targeting shRNA (shControl), or shRNAs against PLP2 (sh1-PLP2 and sh4-PLP2), and transduced cells selected using puromycin. PLP2 levels in the resulting lines were analysed by immunoblot. (<b>F</b>) Knockdown of PLP2 does not affect cell surface MHC-I levels in BC-3 cells. Cell surface levels of MHC-I in the indicated cell lines were analysed by flow cytometry. (<b>G</b>) Knockdown of PLP2 prevents the downregulation of MHC-I upon reactivation of KSHV in BC-3 cells. The indicated cell lines were treated with sodium butyrate, and 24 hours later cell surface MHC-I levels were analysed by flow cytometry.</p

PLP2 is required for the downregulation of novel K5 targets.

<p>(<b>A</b>) Schematic view of the plasma membrane profiling (PMP) experiment. (<b>B</b>) Comparsion of the plasma membrane proteome of WT KBM7 cells versus K5-KBM7 cells. A scatterplot of proteins identified by PMP and quantified by >2 unique peptides is shown. The summed ion intensity (y-axis) is shown as log₁₀. Proteins downregulated more than three-fold from the plasma membrane in the presence of K5 are highlighted in orange. A fully annotated plot is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003772#ppat.1003772.s005" target="_blank">Figure S5</a>. (<b>C–D</b>) Validation of novel PLP2-dependent K5 targets. The expression of the indicated K5 targets proteins in WT KBM7 (black), K5-KBM7 (red) and K5-PLP2^GT (blue) were analysed by (<b>C</b>) flow cytometry and (<b>D</b>) immunoblot.</p

PLP2 is required for the function of K5.

<p>(<b>A</b>) PLP2 protein is not expressed in K5-PLP2^GT cells. K5 and PLP2 were analysed by immunoblot from cell lysates of the indicated cell lines. (<b>B</b>) PLP2 is required for the K5-mediated downregulation of B7-2 in KBM7 cells. The K5-PLP2^GT clone was transduced with a lentiviral vector expressing PLP2. Untransduced cells were removed by puromycin selection, and the surface expression of B7-2 in the indicated cell types was analysed by flow cytometry. Re-expression of PLP2 in K5-PLP2^GT cells restores the K5-mediated downregulation of B7-2. Incomplete puromycin selection means that a small population of cells remain that are not transduced with PLP2, which therefore do not recover the K5-mediated downregulation of B7-2. (<b>C</b>) K5 requires PLP2 to downregulate a range of its targets. Cell surface expression of MHC-I, PECAM and ICAM-1 were analysed in the indicated cell types by flow cytometry. (<b>D</b>) HepG2 cells do not express detectable PLP2 protein. Immunoblot analysis of PLP2 from cell lysates of the indicated cell lines. (<b>E</b>) K5 cannot function in HepG2 cells owing to the absence of PLP2. Lentiviral expression of K5 alone in WT HepG2 cells does not affect cell surface MHC-I expression as measured by flow cytometry, but co-expression of K5 and PLP2 leads to downregulation of cell surface MHC-I.</p

PLP2 is required for K3 and K5 to ubiquitinate their substrate MHC-I.

<p>(<b>A</b>) Schematic representation of PLP2 topology. (<b>B–D</b>) K3 and K5 are unable to ubiquitinate MHC-I in the absence of PLP2. In (<b>B</b>), the indicated cell lines were pulse-labelled for 10 minutes with [³⁵S]-methionine, chased for 45 minutes, and MHC-I immunoprecipitated with the conformationally sensitive W6/32 mAb, and after dissociation and denaturation in 1% SDS, MHC-I molecules re-precipitated with the anti-MHC-I heavy chain mAb HC10, resolved by SDS-PAGE and analysed by autoradiography. In (<b>C</b>) and (<b>D</b>), MHC-I molecules were immunoprecipitated with the W6/32 mAb from the indicated cell types, and ubiquitinated MHC-I species were detected by immunoblot using the anti-ubiquitin antibody VU-1 (right panels) and the input cell lysates blotted for the indicated proteins (left panels).</p

A haploid genetic screen identifies a requirement for the PLP2 gene in the K5-mediated downregulation of B7-2.

<p>(<b>A</b>) Schematic view of the genetic screen, designed such that knockout of a gene essential for K5 function in K5-KBM7 cells would rescue cell surface expression of K5 targets. (<b>B</b>) K5 is active in KBM7 cells. Wild-type (WT) KBM7 cells (black line) and K5-KBM7 (red line) were labelled for the indicated K5 target proteins and analysed by flow cytometry. (<b>C</b>) Selecting mutant B7-2^high cells by FACS. Near-haploid K5-KBM7 cells were mutagenised with gene-trap retrovirus and rare B7-2^high cells selected by two sequential rounds of FACS. Approximate sorting gates are indicated. (<b>D</b>) Sequencing of retroviral insertion sites in the selected B7-2^high population revealed 14 independent insertions in the PLP2 gene on the X chromosome.</p

Verification of expression, secretion, activity and localization of TsUBE2L3.

<p><b>A</b>. <i>T</i>.<i>sp</i> SP, HEK 293T cell lysate and <i>T</i>. <i>spiralis</i> muscle larvae lysate were separated by SDS-PAGE and immuno-blotted with anti-<i>Hs</i>UBE2L3 and anti-tubulin antibodies. <b>B.</b> Auto-ubiquitination of parkin with Ub-biotin was probed by streptavidin-HRP blot. Reactions from left to right: lane 1: human E1 (UBE1A), E3 (parkin) and Ub-biotin only (no E2). Reactions in lanes 2–4 included human E1, parkin and Ub-biotin, with the following E2 substitution: lane 2: <i>T</i>.<i>sp</i> SP after resin-bound BSA depletion (BSA), lane 3: <i>T</i>.<i>sp</i> SP after resin-bound anti-tubulin depletion (α-tubulin), lane 4: <i>T</i>.<i>sp</i> SP after resin-bound α-<i>Hs</i>UBE2L3 depletion (α-<i>Hs</i>UBE2L3). In lanes 5–7 the elution fractions from the depletions shown in lanes 2–4 were used to substitute E2. <b>C</b>. The pixel intensity of lanes 2–7 of the depletion assay (B) was analysed using an ImageJ gel analysis plugin. For each of the three samples tested in the assay (BSA, α-tubulin and α-<i>Hs</i>UBE2L3) the sum of the pixel intensity of the depletion plus elution lanes was taken as 100% (intensity of lane 2 + 5 for BSA and so on). The relative percentages of the depletion and elution lanes were then calculated (intensity of lane 2/(2+5) x 100) and plotted. <b>D</b>. A <i>Ts</i>UBE2L3-specific antibody was made and its specificity was assessed by immuno-blot against <i>T</i>.<i>sp</i> L1 lysate, <i>T</i>.<i>sp</i> SP and rat (<i>R</i>.<i>n</i>) skeletal muscle tissue (SMT) lysate. The arrow indicates the expected size of <i>Ts</i>UBE2L3. <b>E.</b> <i>T</i>. <i>spiralis</i> infected rat skeletal muscle tissue (R.n SMT) was sectioned and analyzed by immuno-histofluorescence. A single <i>T</i>.<i>sp</i> L1 inside the nurse cell (n), surrounded by a collagen capsule (c) is displayed. Tissue was probed with anti-<i>Ts</i>UBE2L3 antibodies (Alexa-568, red) and nuclei were stained using DAPI (blue). Brightfield (BF)/DAPI/anti-<i>Ts</i>UBE2L3 and DAPI/anti-<i>Ts</i>UBE2L3 merged images are displayed. As a control, infected <i>R</i>.<i>n</i>SMT was probed with PBS and DAPI only. Arrows indicate stacks of stichocyte cells in the stichosome. <b>F.</b> Annotated diagram of <i>T</i>. <i>spiralis</i> L1 morphology. Image adapted and reprinted from Trichinosis in Man and Animals by S.E. Gould under a CC BY license, with permission from Charles C. Thomas Publisher LTD, original copyright 1970.</p