p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1--interactions with primer tRNA(Lys3), initiation of cDNA synthesis, and effect of inhibitors.
Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) initiates reverse transcription from tRNA(Lys3). HIV-1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNA(Lys3) was studied. Two recombinant forms of HIV-1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani-Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago-Litvak, L. (1992) FEBS Lett. 301, 23-28]. Here we show that HIV-1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNA(Lys3) to the primer-binding site of the HIV-1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non-nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form. Part of this work was devoted to the study of the complex between the recombinant forms of HIV-1 RT and its primer tRNA. Each enzymatic form was cross-linked to tRNA(Lys3) in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 x tRNA, the label was mainly found to migrate with the p66 subunit, although some cross-linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNA(Lys3). Moreover, cross-linking of tRNA(Lys3) with HIV-1 RT p66/p51 in the presence of a DNA template containing the primer-binding-site sequence yielded an enzymatically active complex.
Dufour
E
E
EP-630, CNRS-Université Victor Segalen Bordeaux, IFR 66 Pathologies Infectieuses, France.
El Dirani-Diab
R
R
Boulmé
F
F
Fournier
M
M
Nevinsky
G
G
Tarrago-Litvak
L
L
Litvak
S
S
Andreola
M L
ML
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis*; D019281:Dimerization; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011728:Pyridones / Q000737:chemistry / Q000494:pharmacology; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013698:Templates, Genetic
C096774:5-ethyl-6-methyl-3-nitro-4-((3',5'-dimethylphenyl)thio)pyridin-2(1H)-one; D003432:Cross-Linking Reagents; D017931:DNA Primers; D018076:DNA, Complementary; D011728:Pyridones; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase