Mechanisms involved in the selection of HIV-1 reverse transcriptase thumb subdomain polymorphisms associated with nucleoside analogue therapy failure.
Previous studies showed an increased prevalence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) thumb subdomain polymorphisms Pro272, Arg277, and Thr286 in patients failing therapy with nucleoside analogue combinations. Interestingly, wild-type HIV-1(BH10) RT contains Pro272, Arg277, and Thr286. Here, we demonstrate that in the presence of zidovudine, HIV-1(BH10) RT mutations P272A/R277K/T286A produce a significant reduction of the viral replication capacity in peripheral blood mononuclear cells in both the absence and presence of M41L/T215Y. In studies carried out with recombinant enzymes, we show that RT thumb subdomain mutations decrease primer-unblocking activity on RNA/DNA complexes, but not on DNA/DNA template-primers. These effects were observed with primers terminated with thymidine analogues (i.e., zidovudine and stavudine) and carbovir (the relevant derivative of abacavir) and were more pronounced when mutations were introduced in the wild-type HIV-1(BH10) RT sequence context. RT thumb subdomain mutations increased by 2-fold the apparent dissociation equilibrium constant (K(d)) for RNA/DNA without affecting the K(d) for DNA/DNA substrates. RNase H assays carried out with RNA/DNA complexes did not reveal an increase in the reaction rate or in secondary cleavage events that could account for the decreased excision activity. The interaction of Arg277 with the phosphate backbone of the RNA template in HIV-1 RT bound to RNA/DNA and the location of Thr286 close to the RNA strand are consistent with thumb polymorphisms playing a role in decreasing nucleoside RT inhibitor excision activity on RNA/DNA template-primers by affecting interactions with the template-primer duplex without involvement of the RNase H activity of the enzyme.
Betancor
Gilberto
G
Centro de Biología Molecular Severo Ochoa, c/Nicolás Cabrera 1, Campus de Cantoblanco, Madrid, Spain.
Puertas
Maria C
MC
Nevot
María
M
Garriga
César
C
Martínez
Miguel A
MA
Martinez-Picado
Javier
J
Menéndez-Arias
Luis
L
D002460:Cell Line; D002478:Cells, Cultured; D015224:Dideoxynucleosides / Q000627:therapeutic use*; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D011110:Polymorphism, Genetic / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use*; D018119:Stavudine / Q000627:therapeutic use*; D014779:Virus Replication / Q000187:drug effects; D015215:Zidovudine / Q000627:therapeutic use*
D015224:Dideoxynucleosides; D018894:Reverse Transcriptase Inhibitors; C057503:carbovir; D015215:Zidovudine; D018119:Stavudine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase