Hybrids between rubella virus and a latent virus of baby hamster kidney cell line BHK21: characterization of rubella virus and type 2 hybrid virus genomes and determination of their physical homology.
The biochemical nature of rubella virus and type 2 hybrid virus, which is a recombinant between rubella virus and a latent retrovirus of BHK21 cells, has been characterized. Type 2 hybrid virus carries DNA polymerase able to copy exogenous DNA. However, disrupted type 2 hybrid virions do not synthesize detectable amounts of DNA using the endogenous viral RNA or synthetic poly(rA)/oligo(dT) primed as a template. Thus, the type 2 hybrid virus DNA polymerase has no detectable reverse transcriptase activity. Rubella virus and type 2 hybrid virus RNA can serve as templates for avian myeloblastosis virus (AMV) reverse transcriptase, although they are inefficient. The addition of oligo(dT) to these viral RNA showed no significant stimulation of their template activity for AMV reverse transcriptase. The oligo(dT)-cellulose affinity column bound neither rubella virus nor type 2 hybrid virus RNA. This suggests that both RNA genomes have a very short poly(A) tail at their 3' end. Thus, complementary DNA (cDNA) synthesis by AMV reverse transcriptase using oligo(dT) primers showed no preferential reverse transcription from the genomic 3' terminus and produced only short cDNA fragments (about 200 nucleotides). We cross-hybridized these short cDNA fragments with their viral RNA, assuming that they are copies of random sites of the genome. These cDNA-RNA hybridization analyses of physical homology between type 2 hybrid virus and rubella virus genomes revealed that about 70% of the type 2 hybrid virus genome is derived from about an 85% portion of the rubella virus genome. These values indicate that the size of the type 2 hybrid virus genome is about 21% larger than that of the rubella virus genome. Co-sedimentation studies of these viral RNA by sucrose density gradient centrifugation confirmed that the molecular weight of type 2 hybrid virus RNA is 20% higher than that of rubella virus RNA. We propose a genomic structure of the type 2 hybrid virus taking into account both physical and biochemical data.
Urade
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M
First Department of Oral and Maxillofacial Surgery, Osaka University Faculty of Dentistry, Japan.
Yamamoto
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N
D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D006224:Cricetinae; D004279:DNA, Viral / Q000096:biosynthesis; D005814:Genes, Viral; D009693:Nucleic Acid Hybridization; D011061:Poly A; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011995:Recombination, Genetic; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D012412:Rubella virus / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic; D014709:Vero Cells
D004279:DNA, Viral; D012367:RNA, Viral; D011061:Poly A; D012194:RNA-Directed DNA Polymerase