Nitric Oxide Inhibits the HIV-1 Reverse Transcriptase Activity Tiziana Persichini,* Marco Colasanti,* Maurizio Fraziano,† Vittorio Colizzi,† Claudio Medana,‡ Fabio Polticelli,* Giorgio Venturini,* and Paolo Ascenzi* ,1 *Department of Biology, University of Rome ‘Tre’, Viale Marconi 446, I-00146 Rome, Italy; †Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica, I-00133 Rome, Italy; and ‡Department of Pharmaceutical Chemistry and Technology, University of Torino, Via Pietro Giuria 9, I-10125 Torino, Italy Received March 18, 1999 Nitric oxide (NO) is a polypotent regulatory molecule involved in a variety of activities, such as the modula- tion of the catalytic activity of cysteine-containing en- zymes. The present study reports the modulation of the HIV-1 reverse transcriptase activity by NO, released by the NO-donors 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1- triazene (NOC-18), ()-(E)-4-ethyl-2-[(E)-hydroxyimino]- 5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnon- imine (SIN-1), 4-(phenylsulfonyl)-3-((2-(dimethylamino) ethyl)thio)furoxan oxalate (SNO-102), and sodium nitro- prusside (SNP). NO inhibits dose-dependently the HIV-1 reverse transcriptase activity, likely due to oxidation of Cys residue(s). Present results, representing a new in- sight into the modulation mechanism of the HIV-1 re- verse transcriptase activity, may be relevant to develop new strategies for inhibition of HIV-1 replication. © 1999 Academic Press Key Words: HIV-1 reverse transcriptase; nitric oxide; NO-donor; enzyme inhibition. The replication of the human immunodeficiency virus-1 (HIV-1) requires the activity of three viral- encoded enzymes, i.e. the reverse transcriptase, the integrase, and the protease. The HIV-1 reverse tran- scriptase carries out several reactions, including re- verse transcription of the viral RNA into cDNA, con- version of the newly synthesized DNA strand into duplex DNA by the polymerase machine and removal of the unused RNA by the intrinsic RNaseH activity. HIV-1 reverse transcriptase lacks an error-correction mechanism, introducing between five and ten mis- matches per genome per round of replication. Thus, the high error-rate of HIV-1 reverse transcriptase may explain the rapid selection of drug-resistant mutants during treatment [1-3]. The HIV-1 reverse transcriptase is processed from the polyprotein gene product of the gag-pol gene com- plex to give a p66-p51 heterodimer. The shape of HIV-1 reverse transcriptase is reminiscent of a ‘right hand’ with DNA threaded between the ‘fingers’ and a ‘thumb’ domains of the p66 subunit. The ‘palm’ subdomain of p66 contains the polymerase active site. The catalytic region for the RNaseH activity is located in the C-terminal portion of the p66 subunit and is joined to the ‘palm’ by a subdomain known as ‘connection’ [4,5]. The catalytic activity of the HIV-1 reverse transcrip- tase can be inhibited by nucleoside analogues and non- nucleoside compounds. Nucleoside analogues act as chain terminators during DNA synthesis, binding to the enzyme active site formed by catalytic residues Asp110, Asp185 and Asp186 and by Gln182 and Met184. On the other hand, a number of non- nucleoside inhibitors (e.g. nevirapine [6]) bind to a hy- drophobic pocket involving residues Tyr181 and Tyr188 and located in the ‘palm’ subdomain of the p66 subunit, some 10 Å from the active site. Upon binding of non-nucleoside inhibitors, conformational changes occur in the catalytic centre of the HIV-1 reverse tran- scriptase. In this respect, Trp229 is displaced from the hydrophobic pocket and the phenolic rings of Tyr181 and Tyr188 flip to allow inhibitor entry. Such struc- tural rearrangements lead to repositioning of a three stranded -sheet containing the catalytic residues Asp110, Asp185 and Asp186. In this way, the non- nucleoside inhibitor-bound enzyme is locked into an inactive conformation [1,2,4,5,7]. 1 To whom all correspondence should be addressed. Fax: 0039+ 06+55176321. E-mail: ascenzi@bio.uniroma3.it. Abbreviations: HIV-1, human immunodeficiency virus-1; AIDS, acquired immunodeficiency syndrome; NO, nitric oxide; NOC-18, 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene; NOR-3, ()-(E)-4- ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide; NOR-3*, NO-de- prived NOR-3; SIN-1, 3-morpholinosydnonimine; SIN-1*, NO- deprived SIN-1; SNO-102, 4-(phenylsulfonyl)-3-((2-(dimethylamino ethyl)thio)furoxan oxalate; SNP, sodium nitroprusside; DTT, dithio- threitol; dNTP, deoxynucleotide triphosphate. Biochemical and Biophysical Research Communications 258, 624 – 627 (1999) Article ID bbrc.1999.0581, available online at http://www.idealibrary.com on 624 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.