title: Increased expression of the N protein of respiratory syncytial virus stimulates minigenome replication but does not alter the balance between the synthesis of mRNA and antigenome.
abstract: A popular model for RNA synthesis by nonsegmented negative-strand RNA viruses is that transcription and RNA replication are executed by the same polymerase complex and that there is a dynamic balance between the two processes that is mediated by the nucleocapsid N protein. According to this model, transcription occurs until sufficient soluble N protein accumulates to initiate encapsidation of the nascent RNA product, which somehow switches the polymerase into a readthrough replicative mode. This model was examined for respiratory syncytial virus (RSV) using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins that are expressed intracellularly from transfected plasmids. Preliminary experiments showed that reconstituted RNA replication was highly productive, such that on average each molecule of plasmid-supplied minigenome that became encapsidated was amplified 10- to 50-fold. N protein was increased on its own or in concert with the phosphoprotein P and in the presence or absence of the M2 ORF1 transcription elongation factor. The maximum level of N and P protein expression achieved from plasmids equalled or exceeded that obtained in RSV-infected cells. Increased levels of N protein stimulated RNA replication. This is consistent with the idea that RNA replication is dependent on the availability of N protein for encapsidation, which is one postulate of the model. The M2 ORF1 protein had no detectable effect on RNA replication under the various conditions of expression of N and P, which confirmed and extended previous results. However, there was no evidence of a significant switch in positive-sense RNA synthesis from transcription (synthesis of mRNAs) to RNA replication (synthesis of antigenome). The synthesis of positive-sense antigenome and mRNA appeared to occur at a fixed ratio, with mRNA being by far the more abundant product.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, 7 Center Drive MSC 0720, Bethesda, Maryland 20892-0720, USA.
authors.forename: R
authors.initials: R
authors.lastname: Fearns
authors.forename: M E
authors.initials: ME
authors.lastname: Peeples
authors.forename: P L
authors.initials: PL
authors.lastname: Collins
mesh_terms: D002213:Capsid / Q000096:biosynthesis; D015500:Chloramphenicol O-Acetyltransferase; D018076:DNA, Complementary; D016679:Genome, Viral; D015707:HN Protein; D006801:Humans; D007700:Kinetics; D019251:Nucleocapsid / Q000096:biosynthesis / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012093:Replicon; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000502:physiology*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000096:biosynthesis / Q000378:metabolism; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D015707:HN Protein; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G; D015500:Chloramphenicol O-Acetyltransferase