Clinical, virological and biochemical evidence supporting the association of HIV-1 reverse transcriptase polymorphism R284K and thymidine analogue resistance mutations M41L, L210W and T215Y in patients failing tenofovir/emtricitabine therapy.
BACKGROUND
Thymidine analogue resistance mutations (TAMs) selected under treatment with nucleoside analogues generate two distinct genotypic profiles in the HIV-1 reverse transcriptase (RT): (i) TAM1: M41L, L210W and T215Y, and (ii) TAM2: D67N, K70R and K219E/Q, and sometimes T215F. Secondary mutations, including thumb subdomain polymorphisms (e.g. R284K) have been identified in association with TAMs. We have identified mutational clusters associated with virological failure during salvage therapy with tenofovir/emtricitabine-based regimens. In this context, we have studied the role of R284K as a secondary mutation associated with mutations of the TAM1 complex.


RESULTS
The cross-sectional study carried out with > 200 HIV-1 genotypes showed that virological failure to tenofovir/emtricitabine was strongly associated with the presence of M184V (P < 10-10) and TAMs (P < 10-3), while K65R was relatively uncommon in previously-treated patients failing antiretroviral therapy. Clusters of mutations were identified, and among them, the TAM1 complex showed the highest correlation coefficients. Covariation of TAM1 mutations and V118I, V179I, M184V and R284K was observed. Virological studies showed that the combination of R284K with TAM1 mutations confers a fitness advantage in the presence of zidovudine or tenofovir. Studies with recombinant HIV-1 RTs showed that when associated with TAM1 mutations, R284K had a minimal impact on zidovudine or tenofovir inhibition, and in their ability to excise the inhibitors from blocked DNA primers. However, the mutant RT M41L/L210W/T215Y/R284K showed an increased catalytic rate for nucleotide incorporation and a higher RNase H activity in comparison with WT and mutant M41L/L210W/T215Y RTs. These effects were consistent with its enhanced chain-terminated primer rescue on DNA/DNA template-primers, but not on RNA/DNA complexes, and can explain the higher fitness of HIV-1 having TAM1/R284K mutations.


CONCLUSIONS
Our study shows the association of R284K and TAM1 mutations in individuals failing therapy with tenofovir/emtricitabine, and unveils a novel mechanism by which secondary mutations are selected in the context of drug-resistance mutations.
Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Madrid, Spain.
Gilberto
G
Betancor
César
C
Garriga
Maria C
MC
Puertas
María
M
Nevot
Lourdes
L
Anta
José L
JL
Blanco
M Jesús
MJ
Pérez-Elías
Carmen
C
de Mendoza
Miguel A
MA
Martínez
Javier
J
Martinez-Picado
Luis
L
Menéndez-Arias
José Antonio
JA
Iribarren
Estrella
E
Caballero
Esteban
E
Ribera
Josep Maria
JM
Llibre
Bonaventura
B
Clotet
Angels
A
Jaén
David
D
Dalmau
José María
JM
Gatel
Joaquín
J
Peraire
Francesc
F
Vidal
Carmen
C
Vidal
Melchor
M
Riera
Juan
J
Córdoba
José
J
López Aldeguer
María José
MJ
Galindo
Félix
F
Gutiérrez
Marta
M
Álvarez
Federico
F
García
Pilar
P
Pérez-Romero
Pompeyo
P
Viciana
Manuel
M
Leal
José Carlos
JC
Palomares
Juan Antonio
JA
Pineda
Isabel
I
Viciana
Jesús
J
Santos
Patricia
P
Rodríguez
Juan Luis
JL
Gómez Sirvent
Carolina
C
Gutiérrez
Santiago
S
Moreno
Mayte
M
Pérez-Olmeda
José
J
Alcamí
Carmen
C
Rodríguez
Jorge
J
del Romero
Angelina
A
Cañizares
José
J
Pedreira
Celia
C
Miralles
Antonio
A
Ocampo
Luis
L
Morano
Antonio
A
Aguilera
Carolina
C
Garrido
Gustavo
G
Manuzza
Eva
E
Poveda
Vicente
V
Soriano
D000225:Adenine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D019380:Anti-HIV Agents / Q000008:administration & dosage / Q000494:pharmacology; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D024882:Drug Resistance, Viral; D000068679:Emtricitabine; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D063065:Organophosphonates / Q000008:administration & dosage* / Q000494:pharmacology; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D000068698:Tenofovir; D017211:Treatment Failure
D019380:Anti-HIV Agents; D063065:Organophosphonates; D003841:Deoxycytidine; D000068698:Tenofovir; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D000068679:Emtricitabine; D000225:Adenine