Borano-nucleotides: new analogues to circumvent HIV-1 RT-mediated nucleoside drug-resistance.
Journal - Nucleosides, nucleotides & nucleic acids (United States )
Alpha-boranophosphates suppress RT-mediated resistance when the catalytic rate of incorporation (kpol) of the analogue 5'-triphosphate is responsable for drug resistance, such as in the case of K65R mutant and ddNTPs, and Q151M toward AZTTP and ddNTPs. This suppression is also observed with BH3-d4T and BH3-3TC toward their clinically relevant mutants Q151M and M184V. Moreover, the presence of the borano (BH3-) group renders the incorporation of the analogue independent from amino-acid substitutions in RT. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analogue.
|ISSN : ||1525-7770|
|Mesh Heading : ||Amino Acids Anti-HIV Agents Boron Boron Compounds Chemistry, Pharmaceutical Drug Design HIV Reverse Transcriptase Kinetics Mutation Nucleic Acid Conformation Oxygen Phosphates chemistry pharmacology methods antagonists & inhibitors chemistry chemistry pharmacology|
|Mesh Heading Relevant : ||Drug Resistance, Viral chemical synthesis chemistry pharmacology|
Coronavirus Nonstructural Protein 16 Is a Cap-0 Binding Enzyme Possessing (Nucleoside-2'O)-Methyltransferase Activity
Journal - Journal of Virology
The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-l-methionine (AdoMet)-dependent RNA (nucleoside-2'O)-methyltransferase (2'O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap (7MeGpppAC3-6) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2'O position of the first transcribed nucleotide, thus converting 7MeGpppAC3-6 into 7MeGpppA2'OMeC3-6. The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2'O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2'O methylation follows an order of events in which 2'O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase.
Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using -Boranophosphate Nucleoside Analogs*
Journal - Journal of Biological Chemistry
A class of amino acid substitutions in drug-resistant HIV-1reverse transcriptase (RT) is responsible for the selectivelyimpaired incorporation of the nucleotide analog inhibitor intoDNA. We have shown previously that -boranophosphate nucleosideanalogs suppress RT-mediated resistance when the catalytic rateis responsible for drug resistance such as in the case of K65Rand dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here,we extend this property to BH3-d4TTP and BH3-3TCTP toward theirclinically relevant mutants Q151M and M184V, respectively. Pre-steady-statekinetics on mutants of the Q151M RT family reveal a 3–5-foldresistance to d4TTP. This resistance is suppressed using BH3-d4TTP.Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184VRT (180-fold) is suppressed using BH3-3TCTP because of a 160-foldacceleration of the catalytic constant kpol. Mechanistic insightsinto the rate enhancement were obtained using various -boranophosphatenucleotides. The presence of the BH3 group renders kpol independentof amino acid substitutions present in RT. Indeed, the 100-folddecrease in polymerase activity caused by the R72A substitutionis restored to wild-type levels using BH3-dTTP. Metal ion titrationstudies show that -boranophosphate nucleoside analogs enhance3–8-fold the binding of Mg2+ ions to the active site ofthe RT·DNA·dNTP complex and alleviate the requirementof critical amino acids involved in phosphodiester bond formation.To our knowledge, this is the first example of rescue of polymeraseactivity by means of a nucleotide analog.* This work was supported in part by the Agence Nationale de Recherchesur le SIDA (AIDS) and Ensemble Contre le SIDA. The costs ofpublication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. Supported by a predoctoral fellowship from the Agence Nationalede Recherche sur le SIDA.¶ Supported by a postdoctoral fellowship from the Ensemble Contrele SIDA.
The Y181C Substitution in 3'-Azido-3'-deoxythymidine-resistant Human Immunodeficiency Virus, Type 1, Reverse Transcriptase Suppresses the ATP-mediated Repair of the 3'-Azido-3'-deoxythymidine 5'-Monophosphate-terminated Primer*
Journal - Journal of Biological Chemistry
Resistance to zidovudine (3'-azido-3'-deoxythymidine, AZT) bythe human immunodeficiency virus, type 1, requires multipleamino acid substitutions such as D67N/K70R/T215F/K219Q in theviral reverse transcriptase (RT). In this background of AZTresistance, additional "suppressive" substitutions such as Y181Crestore sensitivity to AZT. In order to characterize the mechanismof this AZT resistance suppression, the Y181C substitution wasintroduced into both wild-type and AZT-resistant reverse transcriptase.The introduction of the Y181C substitution suppresses the increasedrepair (or unblocking) of the AZTMP-terminated primer providedby the AZT resistance substitutions in RT using either DNA orRNA templates, independently from the RT RNase H activity. Contraryto wild-type RT, the low level of unblocking activity is notdue to inhibition by the next correct nucleotide binding tothe RT/AZTMP-terminated primer complex. When Y181C is addedto the AZT resistance substitutions, ATP binds with less affinityto the AZTMP-terminated primer-RT binary complex. These resultsprovide an insight into one possible molecular mechanism ofre-sensitization of AZT-resistant viruses by suppressive substitutions.* This work was supported in part by the Agence Nationale de Recherchesur le SIDA and Ensemble Contre le SIDA. The costs of publicationof this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact. Supported by a postdoctoral fellowship from the Ensemble Contrele SIDA.¶ Supported by a doctoral fellowship from the Agence Nationalede Recherche sur le SIDA.