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, 105 (31), 10943-8

HIV-1 Reverse Transcriptase Connection Subdomain Mutations Reduce Template RNA Degradation and Enhance AZT Excision

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HIV-1 Reverse Transcriptase Connection Subdomain Mutations Reduce Template RNA Degradation and Enhance AZT Excision

Krista A Delviks-Frankenberry et al. Proc Natl Acad Sci U S A.

Abstract

We previously proposed that mutations in the connection subdomain (cn) of HIV-1 reverse transcriptase increase AZT resistance by altering the balance between nucleotide excision and template RNA degradation. To test the predictions of this model, we analyzed the effects of previously identified cn mutations in combination with thymidine analog mutations (D67N, K70R, T215Y, and K219Q) on in vitro RNase H activity and AZT monophosphate (AZTMP) excision. We found that cn mutations G335C/D, N348I, A360I/V, V365I, and A376S decreased primary and secondary RNase H cleavages. The patient-derived cns increased ATP- and PPi-mediated AZTMP excision on an RNA template compared with a DNA template. One of 5 cns caused an increase in ATP-mediated AZTMP excision on a DNA template, whereas three cns showed a higher ratio of ATP- to PPi-mediated excision, indicating that some cn mutations also affect excision on a DNA substrate. Overall, the results strongly support the model that cn mutations increase AZT resistance by reducing template RNA degradation, thereby providing additional time for RT to excise AZTMP.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic representation of HIV-1-based constructs and their replicative capacity. (A) Structure of pHL[WT] and pHL[TAMs]. Three vectors were constructed with viruses from each patient: the A vectors contain the entire cn (gray box); the B vectors contain the previously identified AZT resistance mutations in cn reverted back to WT amino acids (gray box with white stripes); the C vectors contain the previously identified AZT resistance mutations from the cn added back into a WT cn (white box with gray stripes). (B) cn sequences for vectors A– C from viruses from each treatment-experienced patient (T-3, T-4, T-6, T-8, and T-10). Vector A lists all patient-derived cn mutations that were different from pNL43. Vector B lists the mutations remaining after reversion of the AZT resistance mutations back to WT amino acids. Vector C lists the cn mutations obtained from patient-derived viruses that were added back into a WT cn. Note: Only the A360I substitution in T-4 was reverted in the B vector; when both A360I and A371V substitutions were reverted in the T-4 A vector, RNase H activity was reduced in a similar manner (data not shown). (C) Replicative capacities relative to the TAMs control (dotted line) are shown for each virus. Error bars represent SEM from at least two independent experiments. LTR, long terminal repeat; fpt, fingers, palm, and thumb region (amino acids 1–287); PR, protease; rh, RNase H domain. Note: Replicative capacity for the A vectors was previously described (14).
Fig. 2.
Fig. 2.
Association of AZT-R with RNase H primary cleavage for cn mutations. (A) AZT IC50s were measured for each virus and compared with the TAMs control (dotted line). Fold changes in AZT-R compared with the TAMs control are shown above each bar. Error bars represent the SEM from two to five independent experiments. Note: AZT IC50s for vectors A and B were previously reported (14). (B) Primary RNase H cleavages were assayed on a substrate containing a 32P-labeled (star) 18-nt RNA (thin black line) annealed to a complementary 18-nt DNA (thick black line). The percentages of substrate cleaved (14- and 15-nt bands) are listed below each lane in the autoradiogram and represent the average of two independent experiments. I, input control; RH−, RNase H mutant D498N.
Fig. 3.
Fig. 3.
RNase H secondary cleavages. (A) Secondary (−8 cut) RNase H cleavages were assayed on a substrate containing a 32P-labeled (star) 41-nt RNA primer (thin black line) annealed to a 77-nt DNA template (thick black line). The percentages of secondary cleavages over time are graphically shown below each autoradiogram; error bars represent the SEM from at least two independent experiments. (B) The percentage of secondary cleavages for T-3, T-4, T-6, and T-8. RH−, RNase H mutant D498N.
Fig. 4.
Fig. 4.
Purified RT RNase H primary and secondary cleavages. (A) Primary RNase H cleavages from purified WT, TAMs, and T-3C RTs were assayed on a substrate containing a 32P-labeled (star) 18-nt RNA (thin black line) annealed to a complementary 18-nt DNA (thick black line). The percentage of substrate cleaved (14- and 15-nt bands) with serial dilutions of the purified RTs (16× to 0.5×) is listed below each lane in the autoradiogram and represents the average of two independent experiments. (B) Secondary (−8 cut) RNase H cleavages for purified WT, TAMs, and T-3C RTs were assayed over time on a substrate containing a 32P-labeled (star) 41-nt RNA primer (thin black line) annealed to a 77-nt DNA template (thick black line). The percentages of secondary cleavages are graphically shown below each autoradiogram; error bars represent the SEM from two independent experiments. I, input control.
Fig. 5.
Fig. 5.
AZTMP excision–extension assays. (A) Representative autoradiogram of the kinetics of DNA synthesis catalyzed by patient-derived cn virion-derived RTs (A vectors) evaluated by using a blocked primer (19mer) on an RNA template. The 32P-labeled (star) blocked DNA primer (thick black line) bound to the 42-nt RNA template is shown above the autoradiogram, with black dots representing sites of AZTTP incorporation. I, input control. (B and C) Efficiency of ATP-mediated excision–extension on an RNA template (B) or a DNA template (C) in the presence of AZTTP and 3.3 mM ATP. (D and E) Efficiency of PPi-mediated excision–extension on an RNA template (D) or a DNA template (E) in the presence of AZTTP and 100 μM PPi. (F and G) Comparison of ATP-mediated (F) and PPi-mediated AZTMP excision–extension (G) on RNA and DNA templates. The percentages of full-length products for the TAMs RT were set to 1.0 for 20-, 30-, 45-, and 60-min time points, and the fold change in the percentage full-length products obtained for each A vector are represented as an average value relative to the TAMs control. (H and I) Polymerization rates on the RNA (H) and DNA (I) templates were normalized to TAMs control (TAMs full-length product at 30 min was 100%). (J and K) Comparison of ATP-mediated AZTMP excision–extension (J) and PPi-mediated AZTMP excision–extension (K) on RNA and DNA templates for purified TAMs RT and purified T-3C RT. Analyses were performed as described for F and G. Error bars represent the SEM from at least two independent experiments. Asterisks represent statistically significant differences from the TAMs control (P < 0.006).

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