Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis
Betancor, Gilberto (Centro de Biología Molecular Severo Ochoa)
Álvarez García, Mar (Centro de Biología Molecular Severo Ochoa)
Marcelli, Bárbara (Centro de Biología Molecular Severo Ochoa)
Andrés, Cristina (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Martínez, Miguel Ángel (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Menéndez Arias, Luis (Centro de Biología Molecular Severo Ochoa)

Fecha:	2015
Resumen:	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 template-primers 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, rilpivirine was the most 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.
Ayudas:	Ministerio de Economía y Competitividad BIO2010/1554 Ministerio de Economía y Competitividad BIO2013-48788-C2-1-R Ministerio de Economía y Competitividad SAF2013-41421-R
Nota:	Altres ajuts: MSSSI/EC11-025
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Publicado en:	Nucleic acids research, Vol. 43 Núm. 4 (february 2015) , p. 2259, ISSN 1362-4962

DOI: 10.1093/nar/gkv077
PMID: 25662223

12 p, 4.6 MB

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