ABSTRACT
We describe a new method for the development of a preventive inactivated-HIV vaccine, based on photo-inactivation of HIV reverse transcriptase (RT), which preserves both the conformational and functional integrity of viral surface proteins. The RT of HIV-1 was selectively targeted for inactivation using a photo-labeled compound with specific affinity for HIV-1 RT. The photo-labeled virions were then exposed to UV light causing the photo-labeled compound to form a covalent bond cross-linking the photo-active compound to RT. Replication capacity of the treated virions was significantly reduced when compared to controls suggesting that exposure of treated virions to UV light had caused a stable interaction of RT and the photo-labeling compound.
Subject(s)
AIDS Vaccines/chemistry , HIV Reverse Transcriptase/radiation effects , HIV-1/radiation effects , Reverse Transcriptase Inhibitors/chemistry , Virus Inactivation , Cell Line, Tumor , Cell Survival , Cross-Linking Reagents , HIV Core Protein p24/analysis , HIV Infections , Humans , Inhibitory Concentration 50 , Photoaffinity Labels , Substrate Specificity , Ultraviolet RaysABSTRACT
Frozen solutions of low molecular weight DNA template/primer complexes, in the absence and presence of HIV-1 reverse transcriptase, were irradiated with high-energy electrons. Molecules that survived the radiation exposure were quantified and analyzed using radiation target theory. Transfer of radiation-deposited energy was observed by the damage caused. It was found that damage (as a polynucleotide chain break) was observed in one chain when the radiation interaction occurred in the other chain, suggesting a transfer of energy. In contrast, the target sizes of the DNA template/primers were not altered if bound to HIV-1 reverse transcriptase, signifying that the deposited radiation energy is not transferred between protein and nucleic acid.
Subject(s)
DNA Primers/radiation effects , DNA, Viral/radiation effects , DNA Primers/metabolism , DNA, Viral/metabolism , Dose-Response Relationship, Radiation , Energy Transfer/radiation effects , HIV Reverse Transcriptase/metabolism , HIV Reverse Transcriptase/radiation effects , Templates, GeneticABSTRACT
Analogues of a novel class of template-competitive reverse transcriptase inhibitors (Li, K.; Lin, W.; Chong, K. H.; Moore, B. M.; Doughty, M. B. Bioorg. Med. Chem. 2002, 10, 507) were analyzed as photoprobes of HIV-1 reverse transcriptase (RT) heterodimer. The two photoprobes, 2-(4-azidophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 2 and the tetrafluoro analogue 2-(4-azido-2,3,5,6-tetrafluorophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 3, photodecomposed at 3500 A with half-lives of 4.0 and 2.5 min, respectively. Analysis of the photoproducts of 2m demonstrated that the etheno group is stable but the azido decomposes primarily to the 2-(S-[3H-diazepinon-4-yl]thio)-1,N(6)-etheno-dAMP. Photolysis of both 2 and 3 with RT resulted in a time-dependent loss of activity, with maximum inactivation of 83 and 60%, respectively. Both 2 and 3 showed concentration-dependent photoinactivation of RT in the concentration range from 0 to 100 microM, with EC(50)s of 20 and 25 microM and maximum inactivation of 80 and 60%, respectively. Both the time and concentration dependent photoinactivation were strongly protected by template-primer, but only poorly inhibited by even high concentrations of TTP. Radiolabeled analogues [beta,gamma-(32)P]-2 and [beta,gamma-(32)P]-3 photoincorporated into the p66 subunit, an incorporation also protected by template primer. Identification of the site of incorporation was problematic for both photoprobes, but evidence presented is consistent with labeling sites for the phenacyl side chains of both 2 and 3 in the template grip. Nevertheless, the photoinactivation and incorporation data are consistent with our earlier conclusions from the kinetic data that these inhibitors are specific for the free form of RT in competition with template/primer, and thus represent a novel class of inhibitors.
Subject(s)
HIV Reverse Transcriptase/chemistry , Photoaffinity Labels/chemistry , Reverse Transcriptase Inhibitors/chemistry , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/radiation effects , Binding Sites , Binding, Competitive , HIV Reverse Transcriptase/radiation effects , Humans , Peptide Mapping/methods , Photoaffinity Labels/radiation effects , Photolysis , Reverse Transcriptase Inhibitors/radiation effects , Structure-Activity Relationship , Ultraviolet RaysABSTRACT
In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2',3'-dideoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl) styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([gamma-32P]TDSddUTP) and poly(A).oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3'-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.
Subject(s)
Affinity Labels , Azirines/metabolism , Deoxyuracil Nucleotides/metabolism , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Azirines/radiation effects , Binding Sites , Deoxyuracil Nucleotides/radiation effects , Dideoxynucleotides , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/radiation effects , Recombinant Proteins/metabolism , Recombinant Proteins/radiation effects , Ultraviolet RaysABSTRACT
In order to develop a photoaffinity labeling reagent for HIV-1 reverse transcriptase, we utilized 2',3'-dideoxy-E-5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl] styryl]UTP (TDS-ddUTP) for formation of a photolabile primer. This compound was incorporated into the 3'-terminus of the primer by reverse transcriptase activity, and thus the generated photo-reactive primer was able to bind to the enzyme molecule by photo-irradiation.