<p> 1 1Schmidt et al.</p><p>2HIV-1 Vpu Blocks Recycling and Biosynthetic Transport of the Intrinsic Immunity Factor </p><p>3CD317/Tetherin to Overcome the Virion Release Restriction</p><p>4</p><p>5SUPPLEMENTAL MATERIAL</p><p>6SUPPLEMENTAL MATERIALS AND METHODS</p><p>7Cells and transfections. Human cell lines TZM-bl, HT1080, and HEK 293T were obtained </p><p>8from the American Type Culture Collection and cultivated under standard condition in </p><p>9Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FCS), 1% </p><p>10penicillin-streptomycin, and 1% L-glutamine (all from GIBCO). Jurkat T cells, which express </p><p>11high levels of CD317, were kept in RPMI medium supplemented with 10% FCS, 1% </p><p>12penicillin-streptomycin, and 1% L-glutamine. TZM-bl cells were transfected by </p><p>13Lipofectamine 2000 (Invitrogen) according to manufactures protocol typically in a cell </p><p>14culture dish (Φ 10-cm) using a total DNA amount of 10 μg. 293T cells were transfected by </p><p>15calcium phosphate-DNA precipitation. Primary monocyte-derived macrophage cultures were </p><p>16derived by 10 day differentiation of peripheral blood monocytes from Ficoll-gradient purified </p><p>17PBMCs by adherence on cover slips and cultivation in RPMI1640 complete medium </p><p>18supplemented with human AB serum, in principle as reported (1).</p><p>19 Drugs. Cycloheximide, Brefeldin A, and Primaquine were from Sigma Aldrich. </p><p>20ALLN was from Calbiochem. </p><p>21 Confocal immunofluorescence microscopy. Cells grown on coverglasses were fixed </p><p>22in 4% paraformaldehyde/PBS, followed by permeabilization for 2 min with 0.1% Triton X-</p><p>23100 in PBS. Cells were blocked for 1 hr with 1% bovine serum albumin in PBS and stained </p><p>24for CD317-HAint using an anti-HA mAb (Santa Cruz) and Alexa 568-conjugated secondary </p><p>25antibodies. Anti-TGN46 (AbD Serotec) and anti-TfR (Invitrogen) mAbs were used as primary</p><p>2 - 1 - 1 Schmidt et al. 2 1antibodies together with appropriate secondary antibodies (Invitrogen). Nuclei were </p><p>2counterstained with Hoechst (Invitrogen). Coverslips were mounted in Mowiol (Sigma </p><p>3Aldrich) and analyzed with a Zeiss LSM 510 confocal microscope with a 100x PLAN-APO </p><p>4objective lens. Images were recorded with the Zeiss proprietary software LSM 5 and </p><p>5processed with Adobe Photoshop 11.0.</p><p>6 For the analysis of intracellular transport of CD317, transfected cells were incubated </p><p>7with unconjugated anti-HM1.24/CD317 mAb (gift from Chugai Pharmaceuticals) at 4°C </p><p>8followed by various cultivation periods at 37°C to allow uptake of surface-bound CD317-</p><p>9antibody complexes. Following fixation at the indicated time points, cells were stained </p><p>10without or following permeabilization with 0.1% Triton X-100/PBS to reveal surface or total </p><p>11cellular pools of CD317-antibody complexes, respectively. Following staining for anti-</p><p>12HM1.24-CD317 complexes using Alexa 568-conjugated secondary antibodies, alone or in </p><p>13combination with the anti-TGN46 mAb, cells were mounted and analyzed as described above.</p><p>14 Viruses. Proviral plasmids pHIV-1NL4-3WT (BH10 Env) and pHIV-1NL4-3∆vpu (BH10 </p><p>15Env) were from Valerie Bosch. pBR HIV-1NL4-3WT IRES.GFP and pBR HIV-1NL4-3∆vpu </p><p>16IRES.GFP were provided by Frank Kirchoff (2). These replication-competent viruses carry an</p><p>17internal ribosom entry site (IRES) as well as the GFP gene behind the nef ORF. To prepare </p><p>18virus stocks, supernatants from provirus-transfected 293T cells were harvested on day 3 post-</p><p>19transfection and concentrated using Amicon centrifugal filters (Millipore). Virus titers were </p><p>20determined on TZM-bl cells in a luminometric infectivity assay (3) or by flow cytometry for </p><p>21the GFP-encoding viruses (4). HIV-1 release was quantified either by determination of the </p><p>22virus titer or as the ratio of the p24CA concentration in the supernatant divided by the total </p><p>23p24CA concentration (cell-associated + in the supernatant), in principle as reported (5).</p><p>24 CD317 endocytosis assay. The CD317 endocytosis assay was performed in principle </p><p>25as depicted in Fig. S1A and as reported previously (6). Briefly, TZM-bl cells were transfected</p><p>26with vectors encoding Vpu.GFP or GFP. Twenty-four hours post-transfection, cells were </p><p>3 - 2 - 1 Schmidt et al. 2 1incubated with saturating concentrations of the unconjugated anti-HM1.24/CD317 mAb (1 µg</p><p>2mAb per 1.5x106 cells) diluted in ice-cold binding medium (Dulbecco’s modified Eagle </p><p>3medium supplemented with 2% fetal bovine serum and 20 mM Hepes, pH 7.5). Alternatively,</p><p>4rabbit polyclonal anti-BST-2 antiserum (1:200) from Klaus Strebel was used. Washed cells </p><p>5were then shifted to 37°C for various time periods (t = 0 to 40 min) and finally stained with </p><p>6APC-conjugated goat-anti-mouse IgG antibodies (Jackson ImmunoResearch) (or goat-anti-</p><p>7rabbit Alexa 660, Invitrogen) at 4°C prior to FACS analysis. CD317 endocytosis was </p><p>8quantified by analysis of the APC-mean fluorescence intensity (MFI) at different time points </p><p>9detecting the remaining anti-HM1.24 mAb-labelled CD317 population on the surface of </p><p>10viable cells with identical GFP intensity. The percentages shown are based on the MFIs at </p><p>11different time points and expressed relative to the MFI at t = 0, which was set to 100%. Flow </p><p>12cytometric analyses were carried out on a FACS Calibur with BD CellQuest Pro 4.0.2 </p><p>13 siRNA-mediated β-TrCP depletion. Seeded cells were transfected twice on </p><p>14consecutive days by jetPRIME (PEQLAB Biotechnology) with siRNAs specific for β-TrCP1 </p><p>15mRNA (5'- GAAUUCACUUAGAC AGACA-3') and β-TrCP2 mRNA (5'-</p><p>16AGAUUAUCCAGGAUAUAGA-3'), or nonspecific control siRNA (5'-AGGUAGUGUAA </p><p>17UCGCCUUG-3') (each 50 pmol), in principle as reported (7). During the second transfection </p><p>18expression plasmids for Vpu.GFP or GFP were added and cells analyzed in the indicated </p><p>19assays one day later. </p><p>20 β-TrCP knockdown quantification. Total RNA was extracted by a standard Trizol-</p><p>21chloroform protocol and precipitated with isopropyl alcohol. RNA pellets were washed with </p><p>2275% ethanol, dissolved in water, and stored at -80°C until use. After treatment with DNA-free</p><p>23DNase (Ambion) and cDNA synthesis (NEB), relative quantitative PCR analyses were </p><p>24performed on the ABI Prism 7500 sequence detection system (Applied Biosystems). β-TrCP </p><p>25mRNA levels were quantified with primers specific for β-TrCP1 and β-TrCP2 (8), and </p><p>26TaqMan-specific probes for β-TrCP1 and β-TrCP2 (7). β-TrCP mRNA levels were quantified</p><p>3 - 3 - 1 Schmidt et al. 2 1by using the 2-∆∆Ct method with the human RNaseP gene as endogenous reference control. </p><p>2Pooled triplicates were analyzed for each condition. Data analysis was conducted using the </p><p>37500 System Software (Applied Biosystems).</p><p>4 Western blotting. Cell pellets were directly resuspended in SDS-lysis buffer. Proteins</p><p>5were run on a 12.5% SDS-PAGE and transferred onto nitrocellulose membrane. Blocked </p><p>6membranes were probed with the following primary antisera/antibodies: rabbit polyclonal </p><p>7antiserum to Vpu (provided by Klaus Strebel), anti-HIV-1 p24CA (provided by Hans-Georg </p><p>8Kräusslich), anti-MAPK (Santa Cruz). Horseradish peroxidase-coupled secondary antibodies </p><p>9were used for ECL-based detection.</p><p>10 Statistical evaluation. Statistical significance was determined using the paired </p><p>11Student’s t-test.</p><p>12</p><p>13</p><p>14References</p><p>151. Welsch, S., O. T. Keppler, A. Habermann, I. Allespach, J. Krijnse-Locker, and 16 H. G. Krausslich. 2007. HIV-1 buds predominantly at the plasma membrane of 17 primary human macrophages. PLoS Pathog. 3:e36. 182. Wildum, S., M. Schindler, J. Munch, and F. Kirchhoff. 2006. Contribution of Vpu, 19 Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency 20 virus type 1-infected T cells to superinfection. J. Virol. 80:8047-8059. 213. Geuenich, S., C. Goffinet, S. Venzke, S. 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