RESUMO
Pull-down assay and co-immunoprecipitation of cell extracts in which the integrase or reverse transcriptase of Moloney murine leukemia virus was transiently expressed showed that both enzymes interacted with PML proteins. In infected cells, interaction between the integrase and PML was also observed. Transient expression of PIASy and SUMO proteins facilitated SUMOylation of the integrase but had no apparent effects on the interaction with PML. A FLAG-tagged integrase co-localized with PML protein possibly in the PML body. Knockdown of PML by small interfering RNA resulted in reduced viral cDNA levels and integration efficiency. This suggested that PML proteins activated reverse transcription.
Assuntos
Integrases/metabolismo , Vírus da Leucemia Murina de Moloney/enzimologia , Proteínas Nucleares/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Estruturas do Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunoprecipitação , Integrases/genética , Camundongos , Células NIH 3T3/virologia , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , DNA Polimerase Dirigida por RNA/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to -3861 bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning -3861 to -1569 bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately -1800 bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (-2112 to -1569 bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene.
Assuntos
Galinhas/genética , Elementos Facilitadores Genéticos , Ovalbumina/genética , Oviductos/metabolismo , Transgenes , Animais , Animais Geneticamente Modificados , Embrião de Galinha , Galinhas/metabolismo , Feminino , Hormônio do Crescimento Humano/genética , Humanos , Ovalbumina/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Retroviral integrases associate during the early viral life cycle with preintegration complexes that catalyze the integration of reverse-transcribed viral cDNA into the host chromosomes. Several cellular and viral proteins have been reported to be incorporated in the preintegration complex. This study demonstrates that transcription factor Yin Yang 1 binds to Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. The results of coimmunoprecipitation and in vitro pulldown assays revealed that Yin Yang 1 interacted with the catalytic core and C-terminal domains of Moloney murine leukemia virus and human immunodeficiency virus type 1 integrases, while the transcriptional repression and DNA-binding domains of the Yin Yang 1 molecule interacted with Moloney murine leukemia virus integrase. Immunoprecipitation of the cytoplasmic fraction of virus-infected cells followed by Southern blotting and chromatin immunoprecipitation demonstrated that Yin Yang 1 associated with Moloney murine leukemia virus cDNA in virus-infected cells. Yin Yang 1 enhanced the in vitro integrase activity of Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. Furthermore, knockdown of Yin Yang 1 in host cells by small interfering RNA reduced Moloney murine leukemia virus cDNA integration in vivo, although viral cDNA synthesis was increased, suggesting that Yin Yang 1 facilitates integration events in vivo. Taking these results together, Yin Yang 1 appears to be involved in integration events during the early viral life cycle, possibly as an enhancer of integration.