Construction and characterization of poliovirus subgenomic replicons

Journal Article · Sun May 01 04:00:00 EDT 1988 · Journal of Virology; (USA)

OSTI ID:6528920

Kaplan, G; Racaniello, V R ^[1]

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1. Columbia Univ. College of Physicians Surgeons, New York, NY (USA)

Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3,064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of ({sup 35}S-labeled) viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2A{sup pro}, 2C, and 3D{sup pol} and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs.

• OSTI ID: 6528920
• Journal Information: Journal of Virology; (USA), Journal Name: Journal of Virology; (USA) Vol. 62:5; ISSN JOVIA; ISSN 0022-538X
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DNA
DNA BASE TRANSITIONS
DNA HYBRIDIZATION
DRUGS
ENZYMES
EVEN-ODD NUCLEI
GENES
HELA CELLS
HYBRIDIZATION
IMMUNOASSAY
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
METHIONINE
MICROORGANISMS
MOLECULAR BIOLOGY
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
PHOSPHORUS-GROUP TRANSFERASES
POLIO VIRUS
POLYMERASES
RADIOISOTOPES
RECOMBINANT DNA
REPLICONS
RNA POLYMERASES
SULFUR 35
SULFUR ISOTOPES
TRANSCRIPTION
TRANSFERASES
VIRUSES