Synthesis of the Templates for Influenza Virion RNA Replication in vitro

To elucidate the mechanism(s) of influenza viral RNA replication, we have developed an in vitro system in which the templates for viral RNA replication as well as the viral messenger RNAs (mRNAs) are synthesized. Because the synthesis of both the viral mRNAs and the template RNAs occurs in the nucle...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1984-08, Vol.81 (15), p.4682-4686
Main Authors: Beaton, Ann R., Krug, Robert M.
Format: Article
Language:English
Subjects:
Animals
Cell extracts
Cell nucleus
Cell Nucleus - metabolism
Cells, Cultured
Cricetinae
Gels
Hybridity
Infections
Messenger RNA
Nucleocapsid
Orthomyxoviridae - genetics
Protein synthesis
RNA
RNA, Messenger - genetics
RNA, Viral - genetics
Templates, Genetic
Transcription, Genetic
Virions
Virus Replication
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Summary:To elucidate the mechanism(s) of influenza viral RNA replication, we have developed an in vitro system in which the templates for viral RNA replication as well as the viral messenger RNAs (mRNAs) are synthesized. Because the synthesis of both the viral mRNAs and the template RNAs occurs in the nucleus of infected cells, we determined whether infected cell nuclei are active in the synthesis of these two types of transcripts in vitro. Nuclei isolated as early as 1-2 hr after infection catalyze the in vitro synthesis of both the viral mRNAs and template RNAs. The time course of appearance of these activities indicates that they most likely represent the transcriptional complexes functioning in vitro. Template RNA synthesis catalyzed by the nuclei in vitro is independent of concomitant protein synthesis; rather, it utilizes preformed proteins present in the nuclear preparations. This protein pool can be depleted by treating the infected cells with a protein synthesis inhibitor prior to the isolation of the nuclei, thereby rendering the nuclei inactive in template RNA synthesis in vitro. This activity can be restored by the addition of infected cell cytoplasmic extracts or of the high-speed supernatant fraction from these extracts. These results indicate that the cytoplasmic fraction from infected cells enables the viral transcription complex to continue transcription past the site at which termination occurs during viral mRNA synthesis and also suggest that this fraction enables the transcription complex to initiate transcription without the capped primer used in viral mRNA synthesis.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.81.15.4682