RESUMO
Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.
Assuntos
Adenovírus Humanos/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Regulação para Baixo/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Infecções por Adenovirus Humanos/tratamento farmacológico , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/enzimologia , Adenovírus Humanos/genética , DNA Polimerase Dirigida por DNA/biossíntese , Humanos , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Terapêutica com RNAi , TransfecçãoRESUMO
Human adenoviruses (HAdV) are used as a model system to investigate tumorigenic processes in mammalian cells where the viral oncoproteins E1A and E1B-55K are absolutely required for oncogenic transformation, because they simultaneously accelerate cell cycle progression and inhibit tumor suppressor proteins such as p53, although the underlying mechanism is still not understood in detail. In our present study, we provide evidence that E1B-55K binding to the PML-NB component Sp100A apparently has an essential role in regulating adenovirus-mediated transformation processes. Specifically, when this E1B-55K/Sp100A complex recruits p53, Sp100A-induced activation of p53 transcriptional activity is effectively abolished. Hence, Sp100A exhibits tumor-suppressive activity, not only by stabilizing p53 transactivation but also by depressing E1A/E1B-55K-mediated transformation. E1B-55K counteracts this suppressive activity, inducing Sp100A SUMOylation and sequestering the modified cellular factor into the insoluble matrix of the nucleus or into cytoplasmic inclusions. These observations provide novel insights into how E1B-55K modulates cellular determinants to maintain growth-promoting activity during oncogenic processes and lytic infection.
Assuntos
Proteínas E1B de Adenovirus/fisiologia , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Transformação Celular Viral/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas E1B de Adenovirus/genética , Transformação Celular Viral/genética , Humanos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.