RESUMO
We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.
Assuntos
Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/enzimologia , Adenina/análogos & derivados , Adenina/farmacologia , Sequência de Aminoácidos , Aptâmeros de Nucleotídeos/farmacologia , DNA/biossíntese , DNA/metabolismo , Transcriptase Reversa do HIV/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/enzimologia , Mutação , Nucleotídeos/metabolismo , Organofosfonatos/farmacologia , DNA Polimerase Dirigida por RNA/genética , Inibidores da Transcriptase Reversa/farmacologia , Homologia de Sequência de Aminoácidos , Tenofovir , Zidovudina/farmacologia , beta-Galactosidase/genéticaRESUMO
Transcriptional silencing of tumor suppressor genes by aberrant DNA methylation is a characteristic frequently observed in cancer cells. Therefore, reversing this process is a therapeutic target against cancer. In this study, we established a screening system for silencing inhibitors with cell lines transfected by a retroviral vector containing a luciferase gene. More than 100 nucleosides were tested for antisilencing activity with a selected clone in which the silenced expression of luciferase could be recovered by 5-aza-2'-deoxycytidine. A group of halogenated thymidine analogues was found to reactivate transcription of not only the reporter retrovirus vector but also endogenous glutathione-S-transferase 1 gene, without influence to DNA hypermethylation. Gel mobility shift assay showed that 5-bromo-2'-deoxyuridine (BrdUrd) or 5-iodo-2'-deoxyuridine incorporation did not affect the binding of the methyl-CpG binding protein motif to methylated DNA. Finally, in the retroviral promoter, BrdUrd treatment increased the acetylated histone H3 level and decreased methylation of histone H3 Lys9 in accordance with recovered transcription. This study shows that halogenated thymidines have an antisilencing effect without changing DNA methylation status by interfering with step(s) between DNA methylation and histone acetylation.
Assuntos
Inativação Gênica/efeitos dos fármacos , Timidina/análogos & derivados , Timidina/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Sequência de Bases , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , DNA/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Metilação de DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Histonas/efeitos dos fármacos , Histonas/genética , Humanos , Hidrocarbonetos Halogenados/farmacologia , Células K562 , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Vírus da Leucemia Murina de Moloney/genéticaRESUMO
Basic peptide-mediated protein delivery into living cells is becoming recognized as a potent approach for the understanding of cellular mechanisms and drug delivery. We have prepared the conjugates of the S-peptide (1-15) derived from RNase S with membrane-permeable basic peptides, octaarginine and the human immunodeficient virus (HIV)-1 Rev (34-50). The RNase S complexes, formed among these S-peptide (1-15)-basic peptide conjugates and the S-protein and having a dissociation constant in the range of 10(-5) M, efficiently penetrated into the HeLa cells. These RNase S complexes exerted an anti-HIV replication activity. The time-of-drug-addition assay suggested that the site of action for these complexes would reside in the stages between the viral entry into the cells and reverse transcription. The present study exemplified the applicability of the arginine-rich peptides to the intracellular targeting of non-covalent protein complexes and supramolecular assemblies for the research in chemical and cellular biology.