172R 172K TAM-2/172R TAM-2/172K. AZT concentration [nm] AZT concentration [nm] MgCl 2 2.5K 2.5K 5K 2.5K 5K 2.5K K 5K 2.5K 5K 2.5K 50 2.

Transcription

1 A MgCl 2 172R 172K TAM-2/172R TAM-2/172K AZT concentration [nm] B 172R 172K TAM-2/172R TAM-2/172K AZT concentration [nm] ATP + ATP - Supplemental Figure 1. Primer extension of HIV-1 RT polymorphisms of 172R and K in the presence of AZT with or without ATP. Primer extension activities of HIV-1 RT TAM-2 with 172R or 172K were determined by drug inhibition assay in the absence or presence of AZT and ATP. The presence of an arginine rather than a lysine at codon 172 resulted in significantly higher primer extension only when the assays were carried out in the presence of ATP. A nucleic acid template/primer comprising a nt DNA template annealed to a Cy3-5 -labeled 18 nt DNA primer was used. The reactions were run for 4 min, and fractionated on a 6% polyacrylamide gel.

2 Time [min] - MgCl 2 172R 172K TAM-2/ 172R TAM-2/ 172K RNA template Supplemental Figure 2. RNase H cleavage activity of HIV-1 RT with polymorphisms at codon 172. RNase H activity of RTs 172R, 172K, TAM-2/172R and TAM- 2/172K were evaluated using 5 nm T RNA /P DNA and 2 nm each HIV-1 RT in a buffer containing 5 mm Tris-HCl, ph 7.8 and 5 mm NaCl. Reactions were initiated by the addition of 6 mm MgCl 2. The reaction times were 2, 5 and 1 min.

3 Supplemental Table 1. X-ray data collection and refinement statistics Data collection Wavelength (Å) 1. Resolution (Å) 2.15 ( ) a Space group C2 Cell dimensions a (Å) b (Å) 72.2 c (Å) 19.3 β ( ) 97.6 Observed reflections 2916 Unique reflections Redundancy 3. (2.9) Completeness (%) 98.8 (99.7) R sym b.51 (.424) Avg I/σ 8.8 (1.4) Refinement statistics Resolution (Å) No. of reflections (working) No. of reflections (test) 3315 c R work d R free Overall B value (Å 2 ) 58. Wilson B value (Å 2 ) 49.1 RMSD bond length (Å).1 RMSD Angle ( ) 1.4 Ramachandran plot (%) e Favored 91.6 Allowed 96.5 Disallowed 3.5 a Values given in parentheses are for the highest resolution shell. b R sym = Σ hkl I <I> / Σ hkl I. c R cryst = Σ hkl F obs F calc / Σ hkl F obs. d R free = R cryst, except 5% of the data excluded from the refinement. e Evaluated by MolProbity. (Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J., Wang, X., Murray, L. W., Arendall, W. B., 3rd, Snoeyink, J., Richardson, J. S., and Richardson, D. C. (27) Nucleic Acids Res 35, W PMCID: PMC )

4 Supplemental Table 2. Antiviral activities of NRTIs against HIV-1s with NRTI resistance mutations in the background of 172K or 172R Excision Mutation(s) AZT EC 5 (fold increase) a 172R 172K 172R 172K none.33 ±.1.54 ±.2 (1.6) 2.2 ±.2 (1) 2.5 ±.9 (1.1) N348I.22 ±.3 (6.7).6 ±.1 (1.8) c 9.2 ± 2.4 (4.2) 5.6 ±.4 (2.5) TAM-1 b.28 ±.6 (8.5).16 ±.5 (4.8) 4.5 ± 1.6 (2) 5.2 ±.9 (2.4) 69ins complex b 2.5 ±.17 (76).22 ±.4 (6.7) c 37.3 ± 3.1 (17) 29.3 ± 2.3 (13) N348I/TAM ±.4 (42).3 ±.3 (9.1) c 9.9 ± 1. (4.5) 6.4 ± 2.3 (2.9) Discrimination TAM-2 b.55 ±.7 (17).3 ±.7 (.9) c 17.4 ± 2.6 (7.9) 5.9 ±.8 (2.7) c K65R.2 ±. (.6).2 ±. (.6) 12. ± 2.7 (5.5) 13.3 ± 4. (6) L74V.6 ±.3 (1.8).5 ±.1 (1.5) 11.7 ± 1.5 (5.3) 1.7 ± 1.4 (4.9) M184V.2 ±.1 (.6).3 ±.1 (.9) 7.4 ± 1.6 (3.4) 6.2 ±.4 (2.8) Q151M complex b 3.4 ±.5 (13) 3.33 ±.4 (11) > (>45) > (>45) Combination L74V/N348I.15 ±.1 (4.5).6 ±.1 (1.8) c 15.3 ± 1.5 (7.) 12.7 ± 1.2 (5.8) L74V/TAM-1.29 ±.12 (8.8).8 ±.2 (2.4) c 11.7 ±.6 (5.3) 7.1 ±.1 (3.2) c L74V /N348I/TAM-1.81 ±.17 (25).22 ±.7 (6.7) c 18. ± 2. (8.2) 5.5 ±.6 (2.5) c M184V/N348I.15 ±.4 (4.5).5 ±.1 (1.5) c 11.7 ±.6 (5.3) 1.7 ± 1.1 (.8) c M184V/TAM-1.31 ±.8 (9.4).1 ±.4 (3.) c 1.1 ± 2.5 (4.6) 7.3 ± 1.8 (3.3) M184V/ N348I/TAM-1.34 ±.12 (1).2 ±.9 (6.1) 11.3 ± 2.9 (5.1) 8.2 ± 1.8 (3.7) a Mean values ± standard deviations from at least three independent experiments. The relative increase in EC 5 for recombinant viruses compared with none/172r are shown in parentheses. b TAM-1 and TAM-2 carry the M41L and T215Y, and D67N, K7R, T215F and K219Q mutations, respectively. 69ins complex carries 69 insertion and TAM1. Q151M complex carries Q151M, A62V, V75I, F77L and F116V. ddi

5 c EC 5 value of NRTI resistant mutations with 172K is significantly different from that with 172R (P<.5 by t-test).

6 Supplemental Table 3. Antiviral activities of NNRTIs against HIV-1s with NNRTI resistance mutations in the background of 172K or 172R Mutation NVP EC 5 (fold increase) a EFV 172R 172K 172R 172K None.5 ±.1.3 ±.1 (.6).2 ±.6.2 ±.4 (1) K13N 5.4 ±.53 (18) 3.2 ± 1.4 (64) b.56 ±.12 (28).36 ±.6 (18) V16M 4.1 ±.76 (82).35 ±.19 (7) b.29 ±.5 (15).14 ±.3 (7) b V18I.8 ±.7 (16).14 ±.6 (2.8) b.4 ±.1 (2).2 ±.1 (1) b Y181C >1 ± (>2) >1 ± (>2).4 ±.1 (2).3 ±.4 (1.5) b Y188L >1 ± (>2) >1 ± (>2).31 ±.14 (155).143 ±.45 (72) N348I 1.1 ±.6 (22).18 ±.7 (3.6) b.5 ±.1 (2.5).3 ±.1 (1.5) a Mean values ± standard deviations from at least three independent experiments (triplicates in each experiment). The relative increase in EC 5 for recombinant viruses compared with none/172r are shown in parentheses. b EC 5 value of NNRTI resistant mutation with 172K is significantly different from that with172r (P<.5 by t-test).

7 Supplemental Table 4. Frequency of 172K in clinical samples treatment group / mutation Total (n) a 172K n (%) P-value b No RTIs (untreated) (.7) NRTI-treated (.7).914 NNRTI-treated (1.2).85 NRTI-associated resistance mutation M41L (.8).744 K65R (4.8).4 D67N (1) ins 34 1 (2.9).2178 K7R (.6).8429 L74V (1.4).1698 Q151M (9.9) <.1 M184V (.4).2121 L21W (.8).698 T215Y (.5).3936 NNRTI associated resistant mutation K13N (.9).538 V16M 44 () >.9999 V18I (.9).5 Y181C (2.3).46 Y188L 72 3 (4.2).159 G19A (1.5).176 a The Stanford HIV Drug Resistance database was accessed on 23 May 28. b The P-value was determined by the Fisher's exact test. The P-values for isolates deposited in the Stanford HIV Drug Resistance Database were determined based on a comparison with the no RTIs (untreated) group. Numbers in bold letters indicate statistically significant values (<.5).