Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase

Abstract

Six affinity reagents containing chemically reactive groups, either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside phosphate linkages of the oligothymidylate primers, were used to covalently modify the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). After covalent binding of these modified primer analogs to the enzyme, the addition of [alpha-32P]dTTP, in the presence of a complementary template, led to elongation of the primer. This reaction was catalyzed by the active site of the enzyme carrying the covalently bound primer. The relative efficiency of labeling of the p66/p51 heterodimer compared to the p66/p66 and p51/p51 homodimers of HIV-1 RT was in agreement with the previously determined affinity of the various enzyme forms toward different primers. The analogues preferentially modified the p66 subunit of the HIV-1 RT heterodimer. The labeling of all RT forms by synthetic primer analogues showed significant and specific competition by the natural primer of HIV-1 RT, tRNA(Lys). In addition, the kinetics of inactivation of RT by primer analogues was studied. The affinity of the enzyme to those derivatives in the presence of poly(A) template was about 5-10 times higher than in the absence of template. Moreover, the maximal rates of HIV-1 RT inactivation by analogues in the absence of template were 3-4 times higher. Our results suggest that the mechanism of oligonucleotide primer binding to HIV-1 RT is different in the presence or absence of template.(ABSTRACT TRUNCATED AT 250 WORDS)

High‐Affinity Interaction of Human Immunodeficiency Virus Type‐1 Reverse Transcriptase with Partially Complementary Primers

“…According to Majumdar et al (1988Majumdar et al ( , 1989) and our results (Andreola et al, 1993), HIV-1 RT is able to bind the primer before the template. We have shown that, in the absence of template, HIV-1 RT could correctly recognize the 3'9erminal nucleotide of primers bearing chemically reactive groups at the 5' end.…”

“…The protection of RT by tRNA from modification by reactive analogs of oligonucleotides (Andreola et al, 1993) supports this suggestion. In fact, RT, unlike DNA polymerases, has a specific site for tRNA.…”

“…We have already described the interaction of HIV-I RT with primers, and unlike DNA polymerases. RT can bind the primer either in the absence or in the presence of template (Andreola et al, 1993). This led us to the hypothesis that RT complex formation with non-complementary short oligonucleotides could occur in the presence of template.…”

“…We have shown that, in the absence of template, HIV-1 RT could correctly recognize the 3'9erminal nucleotide of primers bearing chemically reactive groups at the 5' end. In principle, the interaction of HIV-1 RT with the 5'-terminal part of the reactive derivatives in the absence of the template (Andreola et al, 1993) and with partially complementary primers in the presence of the template (this article), can occur, due to additional contacts of the primer with the enzyme.…”

“…Previously, we have analyzed the interaction of HIV-1 RT with synthetic templates and primers of different length and structure . We have shown that HIV-1 RT can bind the primer either in the presence or in the absence of the template (Andreola et al, 1993). This behaviour is clearly unusual since the interaction of primers with procaryotic, eucaryotic and archaebacterial DNA polymerases is always template dependent.…”