CONTENIDO

Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis

06/02/2015

Gilberto Betancor, Mar Álvarez, Barbara Marcelli, Cristina Andrés, Miguel A. Martinez and Luis Menéndez-Arias || Nucleic Acids Research, 2015 doi:10.1093/nar/gkv077

Abstract

HIV-1 reverse transcriptase (RT) connection subdomain mutations at positions 348, 369 and 376 have been associated with resistance to non-nucleoside RT inhibitors (NNRTIs). N348I may interfere with the initiation of (+)-strand DNA synthesis by reducing polypurine tract (PPT) removal in the presence of nevirapine. The effect of NNRTIs on the RNase H-mediated cleavage of PPT-containing templateprimers has been studied with wild-type HIV-1 RT and mutants N348I, T369I, T369V, T376S and N348I/T369I.

 

In the presence of NNRTIs, all RTs were able to stimulate PPT cleavage after primer elongation. The enhancing effects of nevirapine and efavirenz were reduced in RTs carrying mutation N348I, and specially N348I/T369I. However, those mutations had no effect on rilpivirine-mediated cleavage. Prior to elongation, the PPT remains resilient to cleavage, although efavirenz and rilpivirine facilitate RNase H-mediated trimming of its 3_-end. The integrity of the 3_-end is essential for the initiation of (+)-strand DNA synthesis.

 

In the presence of dNTPs, rilpivirinewas themost effective inhibitor of (+)-strand DNA synthesis blocking nucleotide incorporation and preventing usage of available PPT primers. The N348I/T369I RT showed reduced ability to generate short RNA products revealing a cleavage window defect. Its lower RNase H activity could be attributed to enhanced rigidity compared to the wild-type enzyme.

 

Read article online in Nucleic Acids Research 

Gilberto Betancor, Mar Álvarez, Barbara Marcelli, Cristina Andrés, Miguel A. Martinez and Luis Menéndez-Arias

Grupos de IrsiCaixa vinculados: Variabilidad genética y fenotípica del VIH y del VHC