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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.
Supplementary Figure 7: Pr55
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.
Supplementary Figure 8: Pr55
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’
5’- PvuII-AATACCGACGCTCTCGCA-3’
NPsiSL1syIL pNPsiSL1syIL
(PCR)
pN1-
600SL1syIL
(this study)
5’-T7-GGACTCGGCTTGCTGA-3’
5’- PvuII-AATACCGACGCTCTCGCA-3’
NPsiSL1srIL pNPsiSL1srIL
(PCR)
pN1-
600SL1srIL
(this study)
5’-T7-GGACTCGGCTTGCTGA-3’
5’- PvuII-AATACCGACGCTCTCGCA-3’
NPsiSL1∆IL pNPsiSL1∆IL
(PCR)
pN1-
600SL1∆IL
(this study)
5’-T7-GGACTCGGCTTGCTGA-3’
5’- PvuII-AATACCGACGCTCTCGCA-3’
NPsiSL2∆B pNPsiSL2∆B
(PCR)
pN1-600SL2∆B
(this study)
5’-T7-GGACTCGGCTTGCTGA-3’
5’- PvuII-AATACCGACGCTCTCGCA-3’
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’
5’- PvuII-AATACCGACGCTCTCGCA-3’
MPsi∆SL3 pMPsi∆SL3
(PCR)
pDLS∆326-339
(2)
5’-T7-GGACTCGGCTTGCTGA-3’
5’- PvuII-AATACCGACGCTCTCGCA-3’
MPsi∆SL4 pMPsi∆SL4
(PCR)
pDLS∆351-366
(2)
5’-T7-GGACTCGGCTTGCTGA-3’
5’- PvuII-CTAGCCTCCGCTAGTCAA-3’
MPsi∆SL3/4 pMPsi∆SL3/4
(PCR)
pDLS∆351-366
(2)
5’-T7-GGACTCGGCTTGCTGA-3’
5’- PvuII-GCGTACTCACCAGTCGC-3’
MPsiSL1sAL pMPsiSL1sAL
(PCR)
pDISs274-276
(2)
5’-T7-GGACTCGGCTTGCTGA-3’ 5’- PvuII-AATACTGACGCTCTCGCA-3’
MPsiSL3sAL pMPsiSL3sAL
(PCR)
pDLSs331-334
(2)
5’-T7-GGACTCGGCTTGCTGA-3’
5’- PvuII-AATACTGACGCTCTCGCA-3’
MPsiSL4sAL pMPsiSL4sAL
(PCR)
pDLSs358-361
(2)
5’-T7-GGACTCGGCTTGCTGA-3’
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’
5’- PvuII-TCTTTTACATCTATC -3’
N1-600SL1sAL pN1-600 SL1sAL
(PCR)
pNL4.3 S257-
259 (3)
5’-T7- GGTCTCTCTGGTTAG-3’
5’- PvuII-TCTTTTACATCTATC -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).
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