Contribution of the P51 subunit of HIV-1 reverse transcriptase to enzyme processivity

doi:10.1016/0006-291X(92)90688-H

Biochemical and Biophysical Research Communications

Volume 184, Issue 2, 30 April 1992, Pages 986-992

https://doi.org/10.1016/0006-291X(92)90688-H Get rights and content

Abstract

Human immunodeficiency virus Type I reverse transcriptase is active as either the homodimer ( $p66 p66$ ) or the heterodimer ( $p66 p51$ ). Purified recombinant p66 and p51 expressed in yeast were reconstituted in the presence of 60 mM sodium pyrophosphate to enhance dimer formation. Comparison of the processivity of these two active reconstituted forms shows that the heterodimer is more processive than the homodimer with a cycle almost twice as long as judged by assays utilizing poly (U,G) as a challenger to primer-template. Binding assays demonstrated that the heterodimer has a higher affinity for primer-template than the homodimer and that the p51 subunit has an affinity equal to that of the heterodimer. These results suggest that the p51 subunit functions to increase processivity in the heterodimer.

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Cited by (11)

A new generation of peptide-based inhibitors targeting HIV-1 reverse transcriptase conformational flexibility
2009, Journal of Biological Chemistry
The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction “the dimerization step,” followed by conformational changes “the maturation step,” yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide P_AW that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. P_AW only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, P_AW prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.
Functional Characterization of Chimeric Reverse Transcriptases with Polypeptide Subunits of Highly Divergent HIV-1 Group M and O Strains
2001, Journal of Biological Chemistry
Human immunodeficiency virus (HIV)-1 strains have been divided into three groups: main (M), outlier (O), and non-M non-O (N). Biochemical analyses of HIV-1 reverse transcriptase (RT) have been performed predominantly with enzymes derived from HIV-1 group M:subtype B laboratory strains. This study was designed to optimize the expression and to characterize the enzymatic properties of HIV-1 group O RTs as well as chimeric RTs composed of group M and O p66 and p51 subunits. The DNA-dependent DNA polymerase activity on a short heteropolymeric template-primer was similar with all enzymes,i.e. the HIV-1 group O and M and chimeric RTs. Our data revealed that the 51-kDa subunit in the chimeric heterodimer p66^M:B/p51^O confers increased heterodimer stability and partial resistance to non-nucleoside RT inhibitors. Chimeric RTs (p66^M:B/p51^O and p66^O/p51^M:B) were unable to initiate reverse transcription from tRNA $_{3}^{Lys}$ using HIV-1 group O or group M:subtype B RNA templates. In contrast, HIV-1 group O and M RTs supported (−)-strand DNA synthesis from tRNA $_{3}^{Lys}$ hybridized to any of their corresponding HIV-1 RNA templates. HIV-2 RT could not initiate reverse transcription on tRNA $_{3}^{Lys}$ -primed HIV-1 genomic RNA. These findings suggest that the initiation event is conserved between HIV-1 groups, but not HIV types.
Chimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: Critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases
1998, Journal of Molecular Biology
The reverse transcriptase (RT) of HIV-1 and feline immunodeficiency virus (FIV) consist of two subunits of 51 kDa (p51) and 66 kDa (p66). In order to elucidate the role of p51 in the heterodimer, chimeric HIV-1/FIV RT heterodimers were constructed and characterized. The FIV RT p51/HIV-1 RT p66 chimera showed a 2.5-fold higher RNase H activity than the natural HIV-1 RT, a 50% lower strand displacement DNA synthesis activity and resistance to the two RT inhibitors 3′-azido-3′-deoxythymidine triphosphate (AZTTP) and Nevirapine. The HIV-1 RT p51/FIV RT p66 chimera on the other hand had very similar properties to the natural FIV RT. The differences observed upon exchange of the p51 subunits suggest that the three-dimensional structure of the p51 subunit in the RT heterodimers is not completely conserved between the human and the feline lentiviruses. Finally, our data suggest an important role for the p51 subunit in maintaining the optimal structural integrity of the RT heterodimer. The different effects of the small subunits on the sensitivity to known RT inhibitors might be of importance in the development of novel drugs against HIV-1 RT.
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2010, Biological Chemistry
Insertion of a small peptide of six amino acids into the β7-β8loop of the p51 subunit of HIV-1 reverse transcriptase perturbs theheterodimerand affects its activities
2002, BMC Biochemistry

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Contribution of the P51 subunit of HIV-1 reverse transcriptase to enzyme processivity

Abstract

Biochem. Biophys. Res. Comm

Cell

J. Biol. Chem

Biochem. Biophys. Res. Comm

J. Biol. Chem

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Science