SUPPLEMENTARY INFORMATION

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1 SPPLEMENTY INFOMTION 2 =.75 SHPE + 1 (eplicate 2) SHPE + 1 (eplicate 1) Figure S1. eproducibility of SHPE measurements between biological replicates. eactivities corresponding to extension reactions performed with primer 9 are shown plotted on a logarithmic scale. eplicates were performed on independent HIV-1 N genome preparations, by different individuals (J.M.W. and.w.l.), roughly one year apart. 1

2 SPPLEMENTY INFOMTION ount (rel. units) a SHPE reactivity b 2.69 ±.61 (48).6 ±.9 (41) SHPE reactivity SHPE reactivity 1 unpaired internal pairs Sovent accessibility (Å 2 ) Figure S2. SHPE reactivities are strongly sensitive to N secondary structure, but not to solvent accessibility. These SHPE data are from the Nase P specificity domain N 1,11, which has a compact structure with significant, tightly packed, tertiary interactions 12. Solvent accessibility was calculated using a 1.4 Å radius probe for the ribose 2'-oxygen atom. (a) SHPE reactivities do not correlate with solvent accessibility ( 2 =.4). Small panels at top give SHPE reactivity distributions for nucleotides whose solvent accessibility are low, med or high (in red, orange and green, respectively). Distributions are similar and all regions contain nucleotides with both high and low reactivities. (b) SHPE reactivities strongly discriminate between unpaired and base paired nucleotides. Box plot representations are shown. Numerical values give the mean ± standard deviation; the number of measurements is in parentheses. Internal base pairs are defined as positions that are paired as visualized crystallographically and are adjacent to other canonically paired nucleotides 11. The strong predictive relationship between SHPE reactivity and secondary structure is consistent with benchmarks showing SHPE measures local disorder in N

3 SPPLEMENTY INFOMTION ccessibility of splice acceptor sites consensus SHPE reactivity N Splice acceptor S2 S3a S4 S4c S4a S4b S5 S7a S7b S7 S8 Position in genome Mean SHPE reactivity arely used Figure S3. SHPE reactivities at splice acceptor sites in the NL4-3 genome. Histograms show SHPE reactivities at each splice acceptor site across the five nucleotide consensus motif. The mean SHPE reactivity at each acceptor is colored according to the scale used throughout the manuscript. The mean SHPE reactivity across all five nucleotide windows in the HIV genome is.34; thus, all splice acceptors have high SHPE reactivities, except the three (underlined) characterized as rarely used sites

4 SPPLEMENTY INFOMTION a inter-protein linkers mean reactivity Number of bootstrapped data sets (out of 1,) b 2 p = protein domain junctions mean reactivity p = Mean SHPE eactivity Figure S4. Histograms comparing mean SHPE reactivities for inter-protein linker regions and protein domain junctions with distributions of equivalent-length sequences obtained from random regions in the HIV genome by a bootstrap statistical analysis. p-values give the probability that the low SHPE reactivities in the collection of genome elements occurred by chance. 4

5 SPPLEMENTY INFOMTION.6 M- yp loop -N N-p6 SHPE reactivity NL4-3 position Toeprint intnesity (au) ~ ~ ~ Figure S5. Distribution of ribosome pause sites in ag at the M- and -N junctions. Pause sites were identified by inhibition of reverse transcriptase-mediated primer extension, after first inhibiting ribosome processivity with cycloheximide. (Top panel) Median SHPE reactivities in ag over a 75 nt widow, reproduced from Fig. 1 in the main text, are shown in blue. ed line shows median SHPE reactivity over entire genome. Domain junctions and the cyclophilin loop are indicated explicitly. ray bars indicate regions scanned in the toeprinting experiment; the strongest pause sites are indicated with vertical bars. (Lower panels) Toeprinting intensity as a function of genome position. 5

6 SPPLEMENTY INFOMTION a P3 slippery 1625 sequence P2 159 frameshift stimulatory stem 158 P NL4-3 b M Y P3 M Y Y N Y Y Y Y P2 M Y Y M Y M Y P1 Y Y Y M K N group M consensus or or or or ny Base pair conservation 1% >9% <7% Figure S6. Structure of the HIV-1 gag-pol frameshift element. (a) SHPE-constrained secondary structure. Nucleotides are colored according to their SHPE reactivities using the scale shown in Fig. 3b. (b) Sequence and structural conservation for the 3-helix junction model across 37 HIV-1 group M reference sequences. 6

7 ' T 5' 5' poly signal PBS DIS SL2 PSI gag-pol frameshift PPT B D E F H I J K L M N O P Q S T V W X Structure of the NL4-3 HIV-1 N enome (5' Half) pol (IN) end vpr start gag () start gag (M) end gag (M) start gag (p2) start B D gag () end E gag (p2) end gag (N) start F gag (N) end gag (p1) start TF peptide start H I J gag (p1) end gag (p6) start K L TF peptide end pol (P) start M N gag (p6) end O pol (P) end pol (T) start P Q pol (T) end pol (Nase) start S pol (Nase) end pol (IN) start T vif start V W X Protein oding egions to 3' half m1 5' 3' tn 3 Lys 5' 3' SHPE eactivity not analyzed (53 nts) Figure S7. Detailed secondary structure for the NL4-3 HIV-1 genome, including nucleotide identities. Divided into two panels. doi: 1.138/nature8237 SPPLEMENTY INFOMTION 7

8 Structure of the NL4-3 HIV-1 N enome (3' Half) () n E I V IV III IIc IIb IIa ev B.S. 3' T 3' poly signal -3' PPT Ee Ff g Hh Ii Jj Kk Ll Mm Nn Oo Y Z a Bb c Dd 76 vif end tat exon1 start vpr end nef end Y Z a tat/rev exon 1 end rev exon 1 start vpu start Bb c Dd env signal peptide start env signal peptide end env (gp12) start env (gp41) start env (gp12) end Ee Ff g Hh Ii tat/rev exon 2 start rev end env end tat end nef start Jj Kk Ll Mm Nn Oo Protein oding egions to 5' half doi: 1.138/nature8237 SPPLEMENTY INFOMTION doi: 1.138/nature8237 SPPLEMENTY INFOMTION 8

9 SPPLEMENTY INFOMTION Table S1: NL4-3 SHPE primer sequences Number Primer Sequence Binding Site in the NL4-3 enome 1 TTTTT TTTTTTT TTTTTTTTTT TTTTTTTT TTTTTT TTTTTTTTT TTTTTTT TTTT TTTTTTTTTTTT TTTTTTTTT TTTT TTT TTTTTTTTTT TTTTTT TTTTTTT TTTT TTTTTTT TTTTTTTT TTTTT TTTTTTTT T TTTTTTTTTT TTTTTTT TTTTTT TTTTTT TTTTT TTTTTTT TTTTT TTTTTTTT TT TTTTTTTTTTTTTTTTTTTTTT / poly() 9

10 SPPLEMENTY INFOMTION Table S2: Protein Domains in Figure 1d Protein PDB (ref.) Protein esidues olor M/ 2OL Blue reen ed Yellow Pink Yellow (-terminal) ray P 3PHV Blue T/Nase H (p66) 1HMV ed Yellow Pink Yellow ray reen Magenta IN 1WJ ed Yellow 1BIS Pink Yellow 1QM Magenta gp Blue 18-2 Yellow Blue gp41 1SZT , ed 1

11 SPPLEMENTY INFOMTION Supplementary eferences 1. Kelly, B.N. et al. Implications for viral capsid assembly from crystal structures of HIV-1 ag(1-278) and (N)( ). Biochemistry 45, (26). 2. Worthylake, D.K., Wang, H., Yoo, S., Sundquist, W.I. & Hill,.P. Structures of the HIV-1 capsid protein dimerization domain at 2.6 Å resolution. cta rystallogr. D Biol. rystallogr. 55, (1999). 3. Lapatto,., Blundell, T., Hemmings,., Overington, J., Wilderspin,., Wood, S., Merson, J.., Whittle, P.J., Danley, D.E., eoghegan, K.F., et al. X-ray analysis of HIV- 1 proteinase at 2.7 Å resolution confirms structural homology among retroviral enzymes. Nature 342, (1989). 4. odgers, D.W. et al. The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1. Proc. Natl. cad. Sci. S 92, (1995). 5. ai, M. et al. Solution structure of the N-terminal zinc binding domain of HIV-1 integrase. Nat. Struct. Biol. 4, (1997). 6. oldgur, Y. et al. Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium. Proc. Natl. cad. Sci. S 95, (1998). 7. Eijkelenboom,.P. et al. The DN-binding domain of HIV-1 integrase has an SH3-like fold. Nat. Struct. Biol. 2, (1995). 8. Kwong, P.D. et al. Structure of an HIV gp12 envelope glycoprotein in complex with the D4 receptor and a neutralizing human antibody. Nature 393, (1998). 9. Tan, K., Liu, J., Wang, J., Shen, S. & Lu, M. tomic structure of a thermostable subdomain of HIV-1 gp41. Proc. Natl. cad. Sci. S 94, (1997). 1. Mortimer, S.. & Weeks, K.M. fast-acting reagent for accurate analysis of N secondary and tertiary structure by SHPE chemistry. J. m. hem. Soc. 129, (27). 11. Wilkinson, K.. et al. Influence of nucleotide identity on ribose 2'-hydroxyl reactivity in N. N 15, in press (29). 12. Krasilnikov,.S., Yang, X., Pan, T. & Mondragon,. rystal structure of the specificity domain of ribonuclease P. Nature 421, (23). 13. herghe,.m., Shajani, Z., Wilkinson, K.., Varani,. & Weeks, K.M. Strong correlation between SHPE chemistry and the generalized NM order parameter (S 2 ) in N. J. m. hem. Soc. 13, (28). 14. Purcell, D.F. & Martin, M.. lternative splicing of human immunodeficiency virus type 1 mn modulates viral protein expression, replication, and infectivity. J. Virol. 67, (1993). 11

Architecture and secondary structure of an entire HIV-1 RNA genome

Architecture and secondary structure of an entire HIV-1 RNA genome Vol 46 6 ugust 29 doi:1.138/nature8237 RTILES rchitecture and secondary structure of an entire HIV-1 RN genome Joseph M. Watts 1, Kristen K. Dang 2, Robert J. orelick 5, hristopher W. Leonard 1, Julian