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
- Resource Relation:
- Other Information: Thesis (Ph. D)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
POLIO VIRUS
DNA REPLICATION
RNA POLYMERASES
ENZYME ACTIVITY
BIOLOGICAL MODELS
DNA
GENOME MUTATIONS
IN VITRO
MATHEMATICAL MODELS
PHOSPHORUS 32
PROTEINS
RNA
TRACER TECHNIQUES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
MUTATIONS
NUCLEI
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PARASITES
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
TRANSFERASES
VIRUSES
550201* - Biochemistry- Tracer Techniques