RESUMEN
Caffeine is an efficient inhibitor of cellular DNA repair, likely through its effects on ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases. Here, we show that caffeine treatment causes a dose-dependent reduction in the total amount of HIV-1 and avian sarcoma virus retroviral vector DNA that is joined to host DNA in the population of infected cells and also in the number of transduced cells. These changes were observed at caffeine concentrations that had little or no effect on overall cell growth, synthesis, and nuclear import of the viral DNA, or the activities of the viral integrase in vitro. Substantial reductions in the amount of host-viral-joined DNA in the infected population, and in the number of transductants, were also observed in the presence of a dominant-negative form of the ATR protein, ATRkd. After infection, a significant fraction of these cells undergoes cell death. In contrast, retroviral transduction is not impeded in ATM-deficient cells, and addition of caffeine leads to the same reduction that was observed in ATM-proficient cells. These results suggest that activity of the ATR kinase, but not the ATM kinase, is required for successful completion of the viral DNA integration process and/or survival of transduced cells. Components of the cellular DNA damage repair response may represent potential targets for antiretroviral drug development.
Asunto(s)
Proteínas de Ciclo Celular/genética , Daño del ADN , Retroviridae/genética , Retroviridae/patogenicidad , Integración Viral/genética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Virus del Sarcoma Aviar/efectos de los fármacos , Virus del Sarcoma Aviar/genética , Virus del Sarcoma Aviar/patogenicidad , Virus del Sarcoma Aviar/fisiología , Secuencia de Bases , Cafeína/farmacología , Proteínas de Ciclo Celular/fisiología , Línea Celular Transformada , Reparación del ADN , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN , Vectores Genéticos , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/patogenicidad , VIH-1/fisiología , Células HeLa , Humanos , Operón Lac , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Retroviridae/fisiología , Transducción Genética , Proteínas Supresoras de Tumor , Integración Viral/efectos de la radiaciónRESUMEN
We have explored a novel strategy for the targeting of retroviral vectors to particular sites or cell types. This strategy involves a method whereby the infectivity of a retroviral vector is neutralized by treatment of viral particles with a photocleavable, biotinylation reagent. These modified viral vectors possess little to no infectivity for target cells. Exposure of these modified viral vectors to long-wavelength UV light induces a reversal of the neutralizing, chemical modification resulting in restoration of infectivity to the viral vector. This infectivity 'trigger' possesses great potential, both as a research tool and as a novel tactic for the targeting of gene-transfer agents, since it would become possible to direct both the time and location of a viral infection in a versatile manner.
