Supplemental Materials and Methods

1 1Schmidt et al.

2HIV-1 Vpu Blocks Recycling and Biosynthetic Transport of the Intrinsic Immunity Factor

3CD317/Tetherin to Overcome the Virion Release Restriction

5SUPPLEMENTAL MATERIAL

6SUPPLEMENTAL MATERIALS AND METHODS

7Cells and transfections. Human cell lines TZM-bl, HT1080, and HEK 293T were obtained

8from the American Type Culture Collection and cultivated under standard condition in

9Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FCS), 1%

10penicillin-streptomycin, and 1% L-glutamine (all from GIBCO). Jurkat T cells, which express

11high levels of CD317, were kept in RPMI medium supplemented with 10% FCS, 1%

12penicillin-streptomycin, and 1% L-glutamine. TZM-bl cells were transfected by

13Lipofectamine 2000 (Invitrogen) according to manufactures protocol typically in a cell

14culture dish (Φ 10-cm) using a total DNA amount of 10 μg. 293T cells were transfected by

15calcium phosphate-DNA precipitation. Primary monocyte-derived macrophage cultures were

16derived by 10 day differentiation of peripheral blood monocytes from Ficoll-gradient purified

17PBMCs by adherence on cover slips and cultivation in RPMI1640 complete medium

18supplemented with human AB serum, in principle as reported (1).

19 Drugs. Cycloheximide, Brefeldin A, and Primaquine were from Sigma Aldrich.

20ALLN was from Calbiochem.

21 Confocal immunofluorescence microscopy. Cells grown on coverglasses were fixed

22in 4% paraformaldehyde/PBS, followed by permeabilization for 2 min with 0.1% Triton X-

23100 in PBS. Cells were blocked for 1 hr with 1% bovine serum albumin in PBS and stained

24for CD317-HAint using an anti-HA mAb (Santa Cruz) and Alexa 568-conjugated secondary

25antibodies. Anti-TGN46 (AbD Serotec) and anti-TfR (Invitrogen) mAbs were used as primary

2 - 1 - 1 Schmidt et al. 2 1antibodies together with appropriate secondary antibodies (Invitrogen). Nuclei were

2counterstained with Hoechst (Invitrogen). Coverslips were mounted in Mowiol (Sigma

3Aldrich) and analyzed with a Zeiss LSM 510 confocal microscope with a 100x PLAN-APO

4objective lens. Images were recorded with the Zeiss proprietary software LSM 5 and

5processed with Adobe Photoshop 11.0.

6 For the analysis of intracellular transport of CD317, transfected cells were incubated

7with unconjugated anti-HM1.24/CD317 mAb (gift from Chugai Pharmaceuticals) at 4°C

8followed by various cultivation periods at 37°C to allow uptake of surface-bound CD317-

9antibody complexes. Following fixation at the indicated time points, cells were stained

10without or following permeabilization with 0.1% Triton X-100/PBS to reveal surface or total

11cellular pools of CD317-antibody complexes, respectively. Following staining for anti-

12HM1.24-CD317 complexes using Alexa 568-conjugated secondary antibodies, alone or in

13combination with the anti-TGN46 mAb, cells were mounted and analyzed as described above.

14 Viruses. Proviral plasmids pHIV-1NL4-3WT (BH10 Env) and pHIV-1NL4-3∆vpu (BH10

15Env) were from Valerie Bosch. pBR HIV-1NL4-3WT IRES.GFP and pBR HIV-1NL4-3∆vpu

16IRES.GFP were provided by Frank Kirchoff (2). These replication-competent viruses carry an

17internal ribosom entry site (IRES) as well as the GFP gene behind the nef ORF. To prepare

18virus stocks, supernatants from provirus-transfected 293T cells were harvested on day 3 post-

19transfection and concentrated using Amicon centrifugal filters (Millipore). Virus titers were

20determined on TZM-bl cells in a luminometric infectivity assay (3) or by flow cytometry for

21the GFP-encoding viruses (4). HIV-1 release was quantified either by determination of the

22virus titer or as the ratio of the p24CA concentration in the supernatant divided by the total

23p24CA concentration (cell-associated + in the supernatant), in principle as reported (5).

24 CD317 endocytosis assay. The CD317 endocytosis assay was performed in principle

25as depicted in Fig. S1A and as reported previously (6). Briefly, TZM-bl cells were transfected

26with vectors encoding Vpu.GFP or GFP. Twenty-four hours post-transfection, cells were

3 - 2 - 1 Schmidt et al. 2 1incubated with saturating concentrations of the unconjugated anti-HM1.24/CD317 mAb (1 µg

2mAb per 1.5x106 cells) diluted in ice-cold binding medium (Dulbecco’s modified Eagle

3medium supplemented with 2% fetal bovine serum and 20 mM Hepes, pH 7.5). Alternatively,

4rabbit polyclonal anti-BST-2 antiserum (1:200) from Klaus Strebel was used. Washed cells

5were then shifted to 37°C for various time periods (t = 0 to 40 min) and finally stained with

6APC-conjugated goat-anti-mouse IgG antibodies (Jackson ImmunoResearch) (or goat-anti-

7rabbit Alexa 660, Invitrogen) at 4°C prior to FACS analysis. CD317 endocytosis was

8quantified by analysis of the APC-mean fluorescence intensity (MFI) at different time points

9detecting the remaining anti-HM1.24 mAb-labelled CD317 population on the surface of

10viable cells with identical GFP intensity. The percentages shown are based on the MFIs at

11different time points and expressed relative to the MFI at t = 0, which was set to 100%. Flow

12cytometric analyses were carried out on a FACS Calibur with BD CellQuest Pro 4.0.2

13 siRNA-mediated β-TrCP depletion. Seeded cells were transfected twice on

14consecutive days by jetPRIME (PEQLAB Biotechnology) with siRNAs specific for β-TrCP1

15mRNA (5'- GAAUUCACUUAGAC AGACA-3') and β-TrCP2 mRNA (5'-

16AGAUUAUCCAGGAUAUAGA-3'), or nonspecific control siRNA (5'-AGGUAGUGUAA

17UCGCCUUG-3') (each 50 pmol), in principle as reported (7). During the second transfection

18expression plasmids for Vpu.GFP or GFP were added and cells analyzed in the indicated

19assays one day later.

20 β-TrCP knockdown quantification. Total RNA was extracted by a standard Trizol-

21chloroform protocol and precipitated with isopropyl alcohol. RNA pellets were washed with

2275% ethanol, dissolved in water, and stored at -80°C until use. After treatment with DNA-free

23DNase (Ambion) and cDNA synthesis (NEB), relative quantitative PCR analyses were

24performed on the ABI Prism 7500 sequence detection system (Applied Biosystems). β-TrCP

25mRNA levels were quantified with primers specific for β-TrCP1 and β-TrCP2 (8), and

26TaqMan-specific probes for β-TrCP1 and β-TrCP2 (7). β-TrCP mRNA levels were quantified

3 - 3 - 1 Schmidt et al. 2 1by using the 2-∆∆Ct method with the human RNaseP gene as endogenous reference control.

2Pooled triplicates were analyzed for each condition. Data analysis was conducted using the

37500 System Software (Applied Biosystems).

4 Western blotting. Cell pellets were directly resuspended in SDS-lysis buffer. Proteins

5were run on a 12.5% SDS-PAGE and transferred onto nitrocellulose membrane. Blocked

6membranes were probed with the following primary antisera/antibodies: rabbit polyclonal

7antiserum to Vpu (provided by Klaus Strebel), anti-HIV-1 p24CA (provided by Hans-Georg

8Kräusslich), anti-MAPK (Santa Cruz). Horseradish peroxidase-coupled secondary antibodies

9were used for ECL-based detection.

10 Statistical evaluation. Statistical significance was determined using the paired

11Student’s t-test.

14References

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. Nolkemper, I. Baumann, P. Plinkert, J. 22 Reichling, and O. T. Keppler. 2008. Aqueous extracts from peppermint, sage and 23 lemon balm leaves display potent anti-HIV-1 activity by increasing the virion density. 24 Retrovirology 5:27. 254. Venzke, S., N. Michel, I. Allespach, O. T. Fackler, and O. T. Keppler. 2006. 26 Expression of Nef downregulates CXCR4, the major coreceptor of human 27 immunodeficiency virus, from the surfaces of target cells and thereby enhances 28 resistance to superinfection. J. Virol. 80:11141-11152. 295. Goffinet, C., I. Allespach, S. Homann, H. M. Tervo, A. Habermann, D. Rupp, L. 30 Oberbremer, C. Kern, N. Tibroni, S. Welsch, J. Krijnse-Locker, G. Banting, H. 31 G. Krausslich, O. T. Fackler, and O. T. Keppler. 2009. HIV-1 antagonism of 32 CD317 is species specific and involves Vpu-mediated proteasomal degradation of the 33 restriction factor. Cell Host Microbe 5:285-297. 346. Michel, N., I. Allespach, S. Venzke, O. T. Fackler, and O. T. Keppler. 2005. The 35 Nef protein of human immunodeficiency virus establishes superinfection immunity by 36 a dual strategy to downregulate cell-surface CCR5 and CD4. Curr. Biol. 15:714-723.

3 - 4 - 1 Schmidt et al. 2 17. Tervo, H. M., S. Homann, I. Ambiel, J. V. Fritz, O. T. Fackler, and O. T. 2 Keppler. 2011. beta-TrCP is dispensable for Vpu's ability to overcome the 3 CD317/Tetherin-imposed restriction to HIV-1 release. Retrovirology 8:9. 48. Butticaz, C., O. Michielin, J. Wyniger, A. Telenti, and S. Rothenberger. 2007. 5 Silencing of both beta-TrCP1 and HOS (beta-TrCP2) is required to suppress human 6 immunodeficiency virus type 1 Vpu-mediated CD4 down-modulation. J. Virol. 7 81:1502-1505. 8 9

3 - 5 -