In vitro replication of poliovirus
Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis of the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.
- Research Organization:
- California Univ., Los Angeles (USA)
- OSTI ID:
- 6478873
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MODELS
DAYS LIVING RADIOISOTOPES
DNA
DNA REPLICATION
ENZYME ACTIVITY
ENZYMES
GENOME MUTATIONS
IN VITRO
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MATHEMATICAL MODELS
MICROORGANISMS
MUTATIONS
NUCLEI
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PARASITES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLIO VIRUS
POLYMERASES
PROTEINS
RADIOISOTOPES
RNA
RNA POLYMERASES
TRACER TECHNIQUES
TRANSFERASES
VIRUSES