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Molecular biology ofhuman pathogenicviruses 1993 год

Molecular biology of human pathogenic viruses
статья из журнала

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Аннотация: Negative stranded RNA virus genomes require not only an RNA dependent RNA polymerase for infectivity but also the RNA genome itself must be encapsidated with the viral nucleocapsid protein in order to be a functional template for the polymerase.These considerations have severely hindered the application of reverse genetic methods to the analysis of cis-acting signals in regulation of gene expression of these viruses.We have carried out transcription of a complete negative stranded genomic RNA from a cDNA clone of a VSV DI RNA directly in cells simultaneously expressing the five VSV proteins from separately transfected cDNA clones.By this means, t h e negative stranded transcript was encapsidated with N protein to form a functional template and was replicated by the VSV polymerase.Furthermore, the replicated RNAs were assembled and budded to yield infectious DI virions.No helper VSV was required.Replication occured a t high levels and could be assayed by direct biochemical means.An exact 3' terminus of the initial transcript which was generated by autolytic cleavage using a ribozyme from hepatitis delta virus was critical for replication.In contrast, up t o four additional, heterologous nucleotides could be present at the 5' end of t h e initial transcript without compromising replication.Importantly, however, these extra nucleotides were not present at the 5' ends of the RNA products replicated from the initial transcript, indicating repair by the RNA polymerase either by termination of synthesis of the initial positive strand RNA prior to these nucleotides or by initiation of synthesis of the new negative strand internally, directly at the first VSV specific nucleotide.The sequences at the 3' and 5' termini essential for encapsidation and replication have been assayed by mutagenesis of the terminal 50 nucleotides of the DI cDNA clone with subsequent assay of the effects of these changes on efficiency of encapsidation and replication.The results of these experiments will be discussed.In addition, the minimum requirements for assembly and budding of infectious RNA-containing particles were analyzed by deletion of increasing regions of the genomic RNA.The implications of these results for virus assembly will be discussed.
Год издания: 1993
Издательство: Wiley
Источник: Journal of Cellular Biochemistry
Ключевые слова: Viral gastroenteritis research and epidemiology, Animal Virus Infections Studies, Virus-based gene therapy research
Другие ссылки: Journal of Cellular Biochemistry (HTML)
Europe PMC (PubMed Central) (PDF)
Europe PMC (PubMed Central) (HTML)
PubMed Central (HTML)