RESUMEN
Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.
Asunto(s)
Núcleo Celular/microbiología , Replicación del ADN , ADN Viral/biosíntesis , Virus 40 de los Simios/genética , Animales , Antígenos Transformadores de Poliomavirus , Fraccionamiento Celular , Línea Celular , Cromatina/microbiología , Electroforesis en Gel de Agar , Humanos , Matriz Nuclear/microbiologíaRESUMEN
The replication of influenza virus RNA in vitro has been studied by cell fractionation of MDCK-infected cells and characterization of in vitro synthesized RNA. Analysis of the RNA product polarity by liquid hybridization to excess single-stranded DNA probes shows that only the RNP complexes present in the nuclear matrix fraction are able to synthesize negative-polarity RNA. This RNA product has been characterized as authentic vRNA by size analysis, RNase-protection by unlabelled, positive-polarity riboprobes and T1-fingerprinting. Priming the in vitro reaction with ApG stimulates preferentially the synthesis of positive-polarity RNA, while ApGpU stimulates both positive and negative-polarity RNA synthesis.
Asunto(s)
Virus de la Influenza A/genética , Matriz Nuclear/microbiología , ARN Viral/biosíntesis , Ribonucleoproteínas , Animales , Fraccionamiento Celular , Línea Celular , Clonación Molecular , Sondas de ADN , Electroforesis en Gel de Poliacrilamida , Virus de la Influenza A/enzimología , Hibridación de Ácido Nucleico , Oligorribonucleótidos , ARN Viral/aislamiento & purificación , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
After adenovirus infected HeLa cells were pulse labeled and pulse-chase labeled with 3H-thymidine, the nuclear matrix and DNA remaining tightly bound to the matrix were obtained by sequential cell fractionation. Measuring the radioactivity of labeled DNA indicated that newly synthesized viral DNA specifically attach to the nuclear matrix and the amount of binding DNA is in direct proportion to the viral DNA replication activity: then the DNA gradually detach from the matrix and are involved in viral assembly. Electron microscopic autoradiography of the extracted cells showed the virion and viral DNA associated with the nuclear matrix, and thus further confirmed the anchoring of newly synthesized viral DNA to the nuclear matrix.
Asunto(s)
Adenovirus Humanos/metabolismo , Replicación del ADN , ADN Viral/biosíntesis , Matriz Nuclear/microbiologíaRESUMEN
A previously-described herpes simplex virus type 2 DNA-binding protein with a molecular size of 38 kD has been further characterized. Using purified nucleocapsids, a DNase release assay, and intertypic recombinants, this protein was found to be a component of the nucleocapsid, intimately associated with the nuclear matrix, and encoded between 0.605 and 0.720 on the herpes simplex virus type 2 genome.