RESUMO
Viral drug toxicity, resistance, and an increasing immunosuppressed population warrant continued research into new avenues for limiting diseases associated with human cytomegalovirus (HCMV). In this study, a small interfering RNA (siRNA), siX3, was designed to target coding sequences within shared exon 3 of UL123 and UL122 transcripts encoding IE1 and IE2 immediate-early proteins of HCMV. Pretreatment of cells with siX3 reduced the levels of viral protein expression, DNA replication, and progeny virus production compared to control siRNA. Two siRNAs against UL54 and overlapping transcripts (UL55-57) were compared to siX3 in HCMV infection and were also found to be effective at inhibiting HCMV replication. Further investigation into the effects of the siRNAs on viral replication showed that pretreatment with each of the siRNAs resulted in an inhibition in the formation of mature replication compartments. The ability of these siRNAs to prevent or reduce certain cytopathic effects associated with HCMV infection was also examined. Infected cells pretreated with siX3, but not siUL54, retained promyelocytic leukemia (PML) protein in cellular PML bodies, an essential component of this host intrinsic antiviral defense. DNA damage response proteins, which are localized in nuclear viral replication compartments, were reduced in the siX3- and siUL54-treated cells. siX3, but not siUL54, prevented DNA damage response signaling early after infection. Therapeutic efficacy was demonstrated by treating cells with siRNAs after HCMV replication had commenced. Together, these findings suggest that siRNAs targeting exon 3 of the major IE genes or the UL54-57 transcripts be further studied for their potential development into anti-HCMV therapeutics.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas Imediatamente Precoces/genética , Interferência de RNA , Transativadores/genética , Proteínas Virais/genética , Replicação Viral , Linhagem Celular , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Regulação para Baixo , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Virais/metabolismoRESUMO
microRNAs (miRNAs) regulate numerous physiological processes such as cell division and differentiation in many tissue types including stem cells. To probe the role that miRNAs play in regulating processes relevant to embryonic stem cell biology, we used RNA interference to silence DICER and DROSHA, the two main miRNA processing enzymes. Consistent with a role for miRNAs in maintaining normal stem cell division and renewal, we found that perturbation of miRNA pathway function in human embryonic stem cells (hESCs) attenuates cell proliferation. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the CycB/CDK complex and CDKN1A, which encodes p21, a CycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE 1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.
Assuntos
Divisão Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , MicroRNAs/metabolismo , Modelos Biológicos , Sequência de Bases , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , MicroRNAs/genética , Análise em Microsséries , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligonucleotídeos/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , Ribonuclease III/genéticaRESUMO
Stem cells and RNA silencing have emerged as areas of intense interest for both basic and clinical research. Recently these fields have converged with reports implicating small regulatory RNAs in the maintenance and pluripotency of stem cells.
