Template-competitive inhibitors of HIV-1 reverse transcriptase: design, synthesis and inhibitory activity.

We report the design, synthesis and activity studies on a novel class of template-competitive reverse transcriptase inhibitors (TCRTIs). The TCRTIs are 1,N(6)-etheno analogues of a series of dATP-based template-competitive DNA polymerase inhibitors synthesized in our laboratory (Moore, B. M.; Jalluri, R.; Doughty, M.B. Biochemistry 1996, 35, 11634). Thus, nucleotides 2-(4-azidophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 1, the tetrafluoro analogue 2-(4-azido-2,3,5,6-tetrafluorophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 2 and its analogues were synthesized by alkylation of 2-thio-1,N(6)-etheno-2'-deoxyadenosine 5'-monophosphate with the corresponding chloro- or bromo-alkyl halides and converted to the triphosphate. Kinetically, nucleotides 1 and 2 are both competitive inhibitors of reverse transcriptase versus template/primer with K(i)'s of 8.0 and 7.4 microM, respectively, and non-competitive inhibitors versus TTP with K(i)'s of 15 and 10 microM, respectively. Nucleotide 3, which differs from 1 only in that it lacks the etheno group, non-complementary nucleotide triphosphates, and related monophosphates and nucleosides, are completely inactive as inhibitors of reverse transcriptase at concentrations up to 1 mM. Photoinactivation of RT by 1 was both time- and concentration-dependent, and protected by template/primer but not by dNTPs. The concentration-dependent inactivation data gave a K(D,app) of 17.2 microM and maximum inactivation of 90%, and radiolabeled [beta, gamma-32P]-1 photoincorporated specifically and covalently into the p66 subunit of RT. Thus the photoinactivation data support our main conclusion from the kinetic data that this class of RT inhibitors are non-substrate and template-competitive.

[Molecular mechanisms of the biological effects of weak magnetic fields. V. Inactivation in vitro of recombinant Rous sarcoma virus reverse transcriptase under the combined action of weak direct and low-frequency alternating magnetic fields, adjusted to the cyclotron resonance of polar amino acid ions]. / Molekuliarnye mekhanizmy biologicheskogo deistviia slabykh magnitnykh polei. V. Inaktivatsiia in vitro rekombinantnoi obratnoi transkriptazy virusa sarcoma Rausa pri kombinirovannom deistvii slabykh postoiannogo i nizkochastonogo peremennogo magnitnykh polei, nastroennykh na tsiklotronnyi rezonans ionov poliarnykh aminokislot.

The effect of weak direct and alternative magnetic fields adjusted to cyclotron frequencies of ions of polar amino acids leads to a functional inactivation of the recombinant reverse transcriptase of Rous sarcoma virus, which is coupled with the proteolysis of this enzyme.

Target structures for HIV-1 inactivation by methylene blue and light.

In a photodynamic virus inactivation procedure for human fresh frozen plasma the plasma is exposed to visible light in the presence of 1 microM methylene blue. This procedure is known to inactivate HIV-1 by at least 10(6.32) TCID50/ml within 10 minutes. To elucidate the mechanism of photodynamic inactivation of HIV-1 by methylene blue/light treatment, reverse transcriptase (RT), the HIV-1 associated protein p24, and viral RNA were examined. In the dark, methylene blue up to 10 microM has no inhibitory effect on recombinant RT. In the presence of light, recombinant RT inactivation was dependent on illumination time and the concentration of methylene blue. After photoinactivation of the whole virus by methylene blue/light treatment, RT activity was also almost completely inhibited. Simultaneously, it was found by Western blotting that HIV-1 p24 and gp120 are altered in size, possibly due to protein cross-linking. In addition, it was shown by polymerase chain reaction (PCR) inhibition assay that HIV-1 inactivation leads to destruction of its RNA. In summary, methylene blue/light treatment acts on HIV-1 at different target sites: the envelope and core proteins, and the inner core structures RNA and RT.

Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase.

Packaging of the genomic RNA dimer and replication primer tRNA(Lys,3) into HIV virions are required for the production of infectious virus. The initiation of reverse transcription necessitates the annealing of tRNA(Lys,3) to the primer binding site (PBS) of HIV RNA by nucleocapsid (NC) protein. In this report the interactions of replication primer tRNA(Lys,3) with various forms of reverse transcriptase (RT) and nucleocapsid protein have been analyzed by ultraviolet light (UV) cross-linking and gel retardation assays. We show that of the three forms of RT studied, p66/p51, p66 and p51, only the heterodimer p66/p51 can tightly and stably interact with tRNA(Lys,3). Tight interactions between tRNA(Lys,3) and nucleocapsid protein, either NCp15 or NCp7, were found to take place within the anticodon domain. Interestingly enough, primer tRNA(Lys,3) can interact with RTp66/p51 and NCp15 to form a high molecular weight complex in which RTp66/p51 appears to enhance the binding of NCp15 to tRNA(Lys,3). These findings favor the notion that the RT enzyme and NC protein co-operate to select and package primer tRNA.

Active site labeling of HIV-1 reverse transcriptase.

The human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) heterodimer (M(r) = 66,000 and M(r) = 51,000) has been photoaffinity labeled using 4-thiodeoxyuridine triphosphate (S4-dUTP) as a probe. A nascent polymerization complex was assembled from a single-stranded DNA template, a 12-mer DNA primer, and the necessary dNTPs (one of which was alpha-32P-labeled) to extend the primer to produce the n-1 product. The photoaffinity probe was then uniquely added at the 3'-terminal position of the extended primer bound at the catalytic site and photolyzed. The larger subunit (p66) was exclusively derivatized. The unique radioactive peptide resulting from proteolysis was isolated and identified by amino acid sequencing.

Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1.

We have identified an RNA-dependent DNA polymerase activity in the microsomal fraction of human pluri-potential embryonal carcinoma cells NTera2D1, which are known to express the full length coding strand of the genomic Line-1 (L1) elements. This activity was classified as a reverse transcriptase (RT) based on its utilization of an RT specific synthetic poly(Cm) template in the presence of Mn2+ ions. Treatment of the cell by ultraviolet irradiation (200 erg/mm2) which resulted in a 2- to 3-fold enhancement of the RT activity, was required for the reproducible detection of the activity throughout the entire purification procedure. More than a 100-fold enrichment in RT activity was obtained by centrifugation in a glycerol step gradient and a linear sucrose density gradient followed by Sephacryl S-1000 gel filtration. These experiments demonstrated that the RT activity was associated with a macromolecular complex having the characteristics of a viral-like particle with a major protein component of 37 kd. The presence of L1 mRNA in RT-containing fractions suggests that the activity identified could originate from L1 elements and/or be involved in the mechanism of retroposition.

Hematoporphyrin-sensitized photodynamic inactivation of viral RNA-dependent DNA polymerase.

Visible light (6328A) was shown to inactivate RNA-dependent DNA polymerase activity of Moloney mouse leukemia virus, in the presence of hematoporphyrin. The same treatment on exogenous, template-initiation complex had no effect on enzyme activity. It was suggested that this enzyme may be the target of photodynamic inactivation of this and related viruses.