Additional files 1
Supplementary Methods 2
PAK2 binding by SIVmac239 mutated in residues of the putative PAK2 activating 3
structural motif 4
SIVmac239 Nef residues I117, H121, T218 and Y221 were each mutated to three
5
different substitutions by overlapping PCR. The resulting constructs were cloned into
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pCDNA3.1 and transfected into 293T cells as previously described [1]. Western blot of
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lysates was revealed with anti-SIVmac Nef rabbit antiserum [2] followed by horseradish
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peroxidase (HRP)-conjugated anti-rabbit antibodies. HRP conjugates were visualized by
9
using enhanced chemiluminescence. In vitro kinase assay was essentially performed as
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previously described [1]. Immunoprecipitations were performed using the rabbit
anti-11
SIVmac Nef antiserum. Nef/PAK-2 complexes were immunoadsorbed and assayed for
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autophosphorylation activity. Phosphorylated proteins were resolved by sodium dodecyl
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sulfate-polyacrylamide gel electrophoresis, and dried gels were then exposed to a
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phosphorimager screen.
15 16
Assessment of cell surface marker modulation by SIVmac239 Nef mutated in 17
H121R and/or Y221R Nef 18
Cloned single and double mutant SIVmac 239 H121R/Y221R nef sequences were
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cloned into the bicistronic cytomegalovirus-based pCG expression vector coexpressing
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the GFP and Nef and in replication competent HIV-1 NL4-3 based proviral contructs in
the Nef reading frame [3, 4]. pCG plasmids were used to transfect cell lines (Jurkat and
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THP-I)[5]. HIV/SIV Nef/eGFP viral stocks were generated by transfection in 293T cells
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and used to infect stimulated normal donor peripheral blood mononuclear cells (PBMC)
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as described before [4]. After cell culture, transfected or infected cells were stained and
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analysed by flow cytometry as described before [3].
Supplementary references 28
29 30
1. O'Neill E, Kuo LS, Krisko JF, Tomchick DR, Garcia JV, Foster JL: Dynamic
31
evolution of the human immunodeficiency virus type 1 pathogenic factor, 32
Nef. J Virol 2006, 80:1311-1320. 33
2. Garcia JV, Foster JL: Structural and functional correlates between HIV-1 and
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SIV Nef isolates. Virol 1996, 226:161-166. 35
3. Schindler M, Munch J, Brenner M, Stahl-Hennig C, Skowronski J, Kirchhoff F:
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Comprehensive analysis of Nef functions selected in simian 37
immunodeficiency virus-infected macaques. J Virol 2004, 78:10588-10597. 38
4. Schindler M, Munch J, Kutsch O, Li H, Santiago ML, Bibollet-Ruche F,
Muller-39
Trutwin MC, Novembre FJ, Peeters M, Courgnaud V, et al: Nef-mediated
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suppression of T cell activation was lost in a lentiviral lineage that gave 41
rise to HIV-1. Cell 2006, 125:1055-1067. 42
5. Schindler M, Wildum S, Casartelli N, Doria M, Kirchhoff F: Nef alleles from
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children with non-progressive HIV-1 infection modulate MHC-II expression 44
more efficiently than those from rapid progressors. AIDS 2007, 21:1103-45
1107.
46 47 48
Supplementary Table 1: primers used to generate mutant and tagged Nef protein expression constructs
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Forward Reverse
SIVmac239 H121R 5’-ccgtctggagatctgcgacagagac-3’ 5’-cttttataaatctagacatgtctattgccaatttg-3’ SIVmac239 Y221R 5’-ccgtctggagatctgcgacagagac-3’ 5’-catatgcctcataagtcctggccagagttgg-3’
HIV1 SF2-AU1 tag 5’-gctctagaatatgggtggcaag-3’ 5’-tttacgcgttttactagttcatatatagcgataggtgtcgcagtctttgtagtactccggat-3’ SIVmac293-AU1 tag 5’-cgtctagaatatgggtggagctatttcc-3’ 5’-tcccttacgcgttatatatagcgataggtgtcgcgagtttccttcttgtc-3’
Supplementary Figure legends
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Figure S1. SIVmac239 Nef displays a PAK2-activating structural domain surface. 52
Autophosphorylation activity of Nef/PAK-2 complexes using the SIVmac239 Nef
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mutants in residues I117, H121, T218 and Y221 as indicated. Substituting amino acids
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are indicated with their letter code (top). Controls (left side of upper gel) were empty
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vector and wild-type SIVmac239 Nef, respectively. Arrow head indicates position of the
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complex. Lower gel shows Western blot to detect Nef proteins in lysate, arrow head
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indicates protein bands specifically detected.
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Figure S2. Cell surface marker modulation by SIVmac239 mutated in PAK2-59
activating structural domain surface . 60
(A) Flow cytometric analysis of HIV/SIV Nef/eGFP infected PBMC stained for CD4,
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CD3, CD28 and MHC-I as indicated, and of transfected THP-I cells (bottom row),
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stained for MHC class II invariant chain Ii (CD74). Top line indicates construct used,
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resp. none (mock), Nef- construct (Nef-), wild-type SIVmac239 Nef (239) or the three
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different mutants hereof (239 H121R, 239 Y221R and 239 H121R Y221R respectively).
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Dot plots show GFP marker expression as a measure of Nef expression vs. the stained
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marker. (B) Fold downregulation of the mean fluorescence intensity of surface markers
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stained on PBMC, normalized against Nef- (=1) and measured by flow cytometry as
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shown in (A). X-axis indicates construct used, resp. none (mock), Nef- construct (nef-),
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wild-type SIVmac239 Nef (239nef) or the three different mutants hereof (239H121R,
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239Y221R and 239H121RY221R respectively. Bar charts show average ± standard
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deviation of experiments in 2 donors. (C) Fold downregulation of the mean fluorescence
intensity of surface markers stained on Jurkat cells, normalized against Nef- (=1) and
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measured by flow cytometry. X-axis indicates construct used, resp. none (mock), Nef-
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construct (nef-), Nef sequence unable to express due to multiple stop codons (nef*),
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wild-type SIVmac239 Nef (239nef) or the three different mutants hereof (239H121R,
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239Y221R and 239H121RY221R respectively. Bar charts show average ± standard
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deviation of 2 experiments. (D) Fold upregulation of Ii and fold downregulation of MHC
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class II (mean fluorescence intensity) stained on THP-I cells, normalized against Nef-
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(=1) and measured by flow cytometry. X-axis indicates construct used, resp. none
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(mock), Nef- construct (nef-), Nef sequence unable to express due to multiple stop
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codons (nef*), wild-type SIVmac239 Nef (239nef) or the three different mutants hereof
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(239H121R, 239Y221R and 239H121RY221R respectively. Bar charts show average ±
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standard deviation of 2 experiments.
Figure S1 86 87 I117 H121 T218 Y221 88 Empty Wild F R W F R W A E K I F R 89 vector type 90 91 92 93 94 32 P-PAK2 Nef
Figure S2A
Figure S2B 97 98 0 1 2 3 4 5 6 mo ck n ef -239nef 239H1 21R 239Y 221R 239H1 21Y 221R X -fo ld d o w n re g u la ti o n (n e f-) CD3 down, PBMC, n=2 CD28 down, PBMC, n=2 0 1 2 3 4 5 6 7 m o c k n e f-2 3 9 n e f 2 3 9 H 1 2 1 R 2 3 9 Y2 2 1 R 2 3 9 H 1 2 1 Y2 2 1 R X-fo ld d o w n re g u la ti o n (n e f-) 0,0 0,5 1,0 1,5 2,0 2,5 mo ck n ef -239nef 239H1 21R 239Y 221R 239H1 21Y 221R X -f o ld d o w n reg u la tio n ( n ef -) CD4 down, PBMC, n=2 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 mo ck n ef -239nef 239H1 21R 239Y 221R 239H1 21Y 221R X -fo ld d o w n re g u la ti o n (n e f-) MHCI down, PBMC, n=2
Figure S2C 99 100 101 102 0 2 4 6 8 10 12 14 16 18 20 m ock nef -nef * 23 9ne f 23 9H 12 1R 23 9Y221 R 23 9H 12 1Y221 R X -f o ld d o w n reg u la tio n ( n ef -) CD3 down, Jurkat, n=2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 m ock nef -nef * 23 9ne f 23 9H 12 1R 23 9Y221 R 23 9H 12 1Y221 X -f o ld d o w n reg u la tio n ( n ef -) CD4 down, Jurkat, n=2 0 5 10 15 20 25 30 m ock nef -nef * 23 9ne f 23 9H 12 1R 23 9Y221 R 23 9H 12 1Y221 R X -f o ld d o w n reg u la tio n ( n ef -) CD28 down, Jurkat, n=2 0 2 4 6 8 10 12 14 m ock nef -nef * 23 9ne f 23 9H 12 1R 23 9Y221 R 23 9H 12 1Y221 X -f old do w nreg ulat ion ( ne f-)
Figure S2D 103 104 105 106 0,0 0,5 1,0 1,5 2,0 2,5 m ock nef -nef * 23 9ne f 23 9H 12 1R 23 9Y221 R 23 9H 12 1Y221 R X -f old do w nreg ulat ion ( ne f-)
MHC-II down, THP-I, n=2
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 m ock nef -nef * 23 9ne f 23 9H 12 1R 23 9Y221 R 23 9H 12 1Y221 R X -f o ld u p reg u la tio n ( n ef -) Ii up, THP-I, n=2
