supplementary figure 1: characterization of pr55gag a ... · tris-hcl [ph 7.5], 300 mm nacl, 5 mm...

Supplementary Figure 1: Characterization of Pr55Gag

. (a) Characterization of the Pr55Gag

protein used in this study by 10% SDS PAGE. The Gag∆p6 protein was loaded on the same

gel for comparison. (b) Dynamic light scattering of Pr55Gag

in the RNA binding buffer.

Supplementary Figure 2: Pr55

Gag binding to individual SL motifs in the absence of

competitor RNA.

The Pr55Gag

concentration in lanes 1 to 6 was 0, 50, 100, 200, 400, and 600 nM, respectively.

M and D correspond to the monomeric and dimeric species, respectively, of NflSL1 and

NapSL1.

Supplementary Figure 3: Effects of mutations in SL1 on Pr55

Gag binding to the MAL Psi

domain

Binding curves of Pr55Gag

to mutants in SL1 in the MAL isolate Psi context. Data are

represented as mean ± SEM (n = 3).

Supplementary Figure 4: SHAPE analysis of N1-600WT RNA and key mutants. SHAPE

reactivities superimposed on a structural representation of (a) N1-600WT, (b) N1-295WT, (c)

N1-600∆flSL1, and (d) N1-600SL1srIL RNAs. Data are an average of two independent

experiments.

Supplementary Figure 5: Denaturing PAGE and in vitro dimerization of terminal

deleted and SL1 mutant RNAs. Eight % denaturing-PAGE and in vitro dimerization gel

electrophoresis assays were run for terminal deleted and SL1 mutant RNAs. For in vitro

dimerization assays, RNA fragments were incubated in 1X Pr55Gag

binding buffer (30 mM

Tris-HCl [pH 7.5], 300 mM NaCl, 5 mM MgCl2) and run on a native 0.8% agarose gel (TB

0.5×, 0.1 mM MgCl2). For dimerization assays, each (a) NL4-3 and (b) MAL terminal deleted

dimer RNA is run alongside a heat denatured control RNA. (c) NL4-3 (d) MAL SL1 mutant

dimer RNA is run alongside a single heat denatured PsiWT control.

Supplementary Figure 6: Pr55Gag

footprinting on HIV-1 genomic RNA using RNase V1.

RNA N1- 600WT was modified in the absence and in the presence of increasing Pr55Gag

concentrations as indicated above each panel. Sequencing reactions performed to identify the

position of the modifications are shown on the left. Strong/weak protections induced by

Pr55Gag

are indicated by dark/light green dots; RNase V1 cuts that were not affected by

Pr55Gag

are indicated by black dots.


Gag footprinting on HIV-1 genomic RNA using BzCN:

RNA N1-600WT was modified in the absence and in the presence of increasing Pr55Gag

concentrations as indicated above each panel. Sequencing reactions performed in parallel are

shown on the left. Strong/weak protections induced by Pr55Gag

are indicated by dark/light

green dots; Strong/weak Pr55Gag

-induced reactivity increases are marked by red/orange dots;

unaffected modifications are indicated by black dots.


Gag footprinting on HIV-1 genomic RNA using Kethoxal

(a) or DMS (b). RNA N1-600WT was modified in the absence and in the presence of

increasing Pr55Gag

concentrations as indicated above each panel. Sequencing reactions

performed in parallel are shown on the left. Strong/weak protections induced by Pr55Gag

are

indicated by dark/light green dots; modifications that were not affected by Pr55Gag

are

indicated by black dots.

Supplementary Figure 9: Comparison of Pr55Gag

binding to M1-615WT and MSL1-

615WT RNA: Schematic drawing of gRNA and relative binding affinity of Pr55Gag

to M1-

615WT and MSL1-615WT RNAs. Binding of Pr55Gag

was evaluated by filter binding and

data are represented as mean ± SEM (n = 3).

SUPPLEMENARY TABLES

Supplementary Table 1: RNAs produced by in vitro transcription of plasmids described

in previous publications

RNA Plasmid Restriction Enzyme Reference

N1-600WT pNL4.3–615 PvuII 1

N1-600ENV pT-env/vpu EcoRI 1

N1-600VPR pT-vpr1/3 EcoRI 1

N1-600TAT pT-tat EcoRI 1

N1-600REV pT-rev EcoRI 1

N1-600NEF pT-nef EcoRI 1

N1-400WT pNL4.3–615 HaeIII 1

N1-295WT pNL4.3–615 RsaI 1

M1-1615WT pJCB PvuII 2

M1-415WT pJCB HaeIII 2

M1-311WT pJCB RsaI 2

M305-615WT pJCA SmaI 2

M1-615∆apSL1 pJCB∆265-287 PvuII 2

M1-615∆SL3 pJCBDLS∆326-339 PvuII 2

M1-615∆SL4 pJCBDLS∆351-366 PvuII 2

M1-615SL1sAL pJCBDISC275 PvuII 2

M1-615SL3sAL pJCBDLSS331-334 PvuII 2

M1-615SL4sAL pJCBDLSS358-361 PvuII 2

The name of the restriction enzyme used to linearize the plasmid prior to in vitro transcription

is indicated in the third column.

Supplementary Table 2: RNAs produced from plasmids constructed for this study and

plasmid construction

RNA Plasmid

(PCR or Site

Directed

Mutagenesis)

Starting Plasmid

(Ref.)

Oligos

NPsiWT pNPsiWT

(PCR)

pNL4.3–615 (1) 5’-T7-GGACTCGGCTTGCTGA-3’

5’- PvuII-AATACCGACGCTCTCGCA-3’

NPsi∆SL2 pNPsi∆SL2

(PCR)

pN1-600∆SL2

(this study)

5’-T7-GGACTCGGCTTGCTGA-3’ 5’- PvuII-AATACCGACGCTCTCGCA-3’

NPsiSL2sAL pNPsiSL2sAL

(PCR)

pN1-

600SL2sAL

(this study)

5’-T7-GGACTCGGCTTGCTGA-3’


NPsiSL1syIL pNPsiSL1syIL

(PCR)

pN1-

600SL1syIL

(this study)



NPsiSL1srIL pNPsiSL1srIL

(PCR)

pN1-

600SL1srIL

(this study)



NPsiSL1∆IL pNPsiSL1∆IL

(PCR)

pN1-

600SL1∆IL

(this study)



NPsiSL2∆B pNPsiSL2∆B

(PCR)

pN1-600SL2∆B

(this study)



NPsiSL2sLS pNPsiSL2sLS

(PCR)

pN1-

600SL2sLS

(this study)

5’-T7-GGACTCGGCTTGCTGA-3’ 5’- PvuII-AATACCGACGCTCTCGCA-3’

MPsiWT pMPsiWT

(PCR)

pJCB (2) 5’-T7-GGACTCGGCTTGCTGA-3’ 5’- PvuII-AATACCGACGCTCTCGCA-3’

MPsi∆SL1 pMPsi∆SL1

(PCR)

pDIS∆265-287

(2)

5’-T7-GGCGACTGGTGAGTACGCCAA-3’



(PCR)

pDLS∆326-339

(2)




(PCR)

pDLS∆351-366

(2)


5’- PvuII-CTAGCCTCCGCTAGTCAA-3’

MPsi∆SL3/4 pMPsi∆SL3/4

(PCR)

pDLS∆351-366

(2)


5’- PvuII-GCGTACTCACCAGTCGC-3’

MPsiSL1sAL pMPsiSL1sAL

(PCR)

pDISs274-276

(2)

5’-T7-GGACTCGGCTTGCTGA-3’ 5’- PvuII-AATACTGACGCTCTCGCA-3’


(PCR)

pDLSs331-334

(2)


5’- PvuII-AATACTGACGCTCTCGCA-3’


(PCR)

pDLSs358-361

(2)


5’- PvuII-AATACTGACGCTCTCGCA-3’

MPsiSL1∆IL pMPsiSL1∆IL

pJCB (2) 5’-CTGAGGTGCACACAGCAAGCCGAGAGCGGCGACTGGTGAG-3’

5’-CTCACCAGTCGCCGCTCTCGGCTTGCTGTGTGCACCTCAG-3’

MPsiSL1srIL pMPsiSL1srIL pJCB (2) 5’-CTGAGGTGCACACAGCAAGTTTCGAGAGCGGCGACTGGTGAG-3’

5’-CTCACCAGTCGCCGCTCTCGAAACTTGCTGTGTGCACCTCAG-3’

MPsiSL1syIL pMPsiSL1syIL

pJCB (2) 5’-CTGAGGTGCACACAGCAAGGAACGAGAGCGGCGACTGGTGAG-3’

5-‘CTCACCAGTCGCCGCTCTCGTTCCTTGCTGTGTGCACCTCAG-3’

N1-600∆flSL1 pN1-600 ∆flSL1

(PCR)

pNL4.3∆243-

277 (3)

5’-T7-GGTCTCTCTGGTTAG-3’

5’- PvuII-TCTTTTACATCTATC -3’

N1-600∆SL2 pN1-600∆SL2 pNL4.3–615 (1) 5’-CAAGAGGCGAGGGGCAAAAATTTTGACTAG-3’

5’-CTAGTCAAAATTTTTGCCCCTCGCCTCTTG-3’

N1-600∆SL3 pN1-600∆SL3

(PCR)

pNL4.3 ∆SL3

(4)

5’-T7-GGTCTCTCTGGTTAG-3’


N1-600SL1sAL pN1-600 SL1sAL

(PCR)

pNL4.3 S257-

259 (3)

5’-T7- GGTCTCTCTGGTTAG-3’


N1-600SL2sAL pN1-600SL2sAL pNL4.3–615 (1) 5’-GAGGGGCGGCGACTGCAAAGTACGCCAAAAAT -3’ 5’-ATTTTTGGCGTACTTTGCAGTCGCCGCCCCTC-3’

N1-600SL1∆IL pN1-600SL1∆IL pNL4.3–615 (1) 5’-AGCGCGCACGGCAAGCCGAGGGGCGGCGAC-3’

5’-GTCGCCGCCCCTCGGCTTGCCGTGCGCGCT-3’

N1-600SL1syIL pN1-600SL1syIL pNL4.3–615 (1) 5’-GCGCGCACGGCAAGTTTCGAGGGGCGGCGAC-3’

5’-GTCGCCGCCCCTCGAAACTTGCCGTGCGCGC-3’

N1-600SL1srIL pN1-600SL1srIL pNL4.3–615 (1) 5’-GCGCGCACGGCAAGGAACGAGGGGCGGCGAC-3’

5’-GTCGCCGCCCCTCGTTCCTTGCCGTGCGCGC-3’

N1-600SL2∆B pN1-600SL2∆B pNL4.3–615 (1) 5’-CGGCGACTGGTGAGTCGCCAAAAATTTTGA-3’

5’-TCAAAATTTTTGGCGACTCACCAGTCGCCG-3’

N1-600SL2sLS pN1-600SL2sLS pNL4.3–615 (1) 5’- AGAGGCGAGGGGCAATGACTGGTGAGTACATTAAAAATTTTGACT-3’

5’- AGTCAAAATTTTTAATGTACTCACCAGTCATTGCCCCTCGCCTCT-3’

N1-SL2WT pN1-SL2WT

(PCR)

pNL4.3–615 (1) 5’-T7-GGTCTCTCTGGTTAG-3’

5’- PvuII-GCGTACTCACCAGTCGC -3’

N1-SL3WT pN1-SL3WT

(PCR)

pNL4.3–615 (1) 5’-T7-GGTCTCTCTGGTTAG-3’ 5’- PvuII-CTAGCCTCCGCTAGTCAA -3’

N1-SL4WT pN1-SL4WT

(PCR)

pNL4.3–615 (1) 5’-T7-GGTCTCTCTGGTTAG-3’

5’- PvuII-AATACTGACGCTCTCGCA -3’

NSL1-600WT pNSL1-600WT

(PCR)

pNL4.3–615 (1) 5’-T7-GGACTCGGCTTGCTGA-3’ 5’- PvuII-GCACACAATAGAGGACTGCT -3’

MSL1-615WT pMSL1-615WT

(PCR)

pJCB (2) 5’-T7-GGACTCGGCTTGCTGA-3’

5’- PvuII-TGTACACAATAGAGGGTTGC -3’

T7: 5’-TAATACGACTCACTATAG-3’

The second column indicates the name of the plamids and, into parenthesis, whether it has

been obtained by PCR of a plasmid already containing the desired mutation (subcloning) or

by site-directed mutagenesis. The third column indicates the starting plasmid (with reference)

and the last column lists the oligos used for plasmid construction

SUPPLEMENTARY REFERENCES

1. Sinck L, et al. In vitro dimerization of human immunodeficiency virus type 1 (HIV-1)

spliced RNAs. Rna 13, 2141-2150 (2007).

2. Paillart JC, Marquet R, Skripkin E, Ehresmann B, Ehresmann C. Mutational analysis of the

bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA. The

Journal of biological chemistry 269, 27486-27493 (1994).

3. Paillart JC, et al. A dual role of the putative RNA dimerization initiation site of human

immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis.

Journal of virology 70, 8348-8354 (1996).

4. Houzet L, et al. HIV controls the selective packaging of genomic, spliced viral and cellular

RNAs into virions through different mechanisms. Nucleic Acids Res 35, 2695-2704 (2007).

supplementary figure 1: characterization of pr55gag a ... · tris-hcl [ph 7.5], 300 mm nacl, 5 mm...

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