Cloning of the rat endogenous helper leukemia virus DNA sequence and expression of the helper activity encoded by the cloned DNA sequence in normal rat kidney cells by microinjection.
By the use of recombinant DNA technology and microinjection in cultured cells, the molecular genetic elements involved in the evolution of a retrovirus with the multipotential to infect, transform and replicate in host cell, have been critically examined in this investigation. Recently we have identified and purified the integrated and proviral DNA sequences specific for two rat endogenous helper leukemia viruses, WR- RaLV , originated from a chemically induced wild rat fibrosarcoma, and RHHV , isolated from a chemically induced rat hepatoma, HTC-H1 (1). By using a multidisciplinary approach combining restriction endonuclease analysis, reverse phase V-column chromatography, agarose gel electrophoresis, Southern blot transfer and filter nucleic acid hybridization, we were able to demonstrate that the rat helper leukemia viral DNA sequence was approximately 8.4-8.8 kb. The 8.8 kb RHHV DNA was molecularly cloned via the EK-1 certified vector pBR 322 plasmid into E. coli RRI cells. A successful recombinant clone, 8/32, that carried one entire RHHV 8.8 kb DNA sequence was mapped by restriction endonuclease analyses. Restricted DNA fragments of various sizes throughout the complete RHHV genome were isolated and purified for intranuclear microinjection into normal rat kidney cells. Release of type C infectious helper virus in these microinjected cells was investigated by superinfection on K-NRK, Kirsten sarcoma transformed non-producer cells. Recombination of the helper viral DNA sequence, en toto or of subgenomic sizes, carried in microinjected cells, with the sarcomagenic DNA sequence, carried in K-NRK cells, was also studied by genome-rescue and cell-transformation experiments. Our observations led to the conclusion that all critical genetic elements including the 5' LTR helper DNA sequence, gag, pol, and env genes, encoded for the biological activity of the type C helper virus resided within the 6.0 kb proximal to the 5' terminus of the endogenous rat type C helper virus DNA. They proved vitally essential for the recombination with the Src sequence during the evolution of an infectious, transforming and replication-competent retrovirus.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics*; D007668:Kidney; D008114:Liver Neoplasms, Experimental / Q000382:microbiology*; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D051381:Rats; D012190:Retroviridae / Q000235:genetics* / Q000302:isolation & purification
D004279:DNA, Viral; D004247:DNA; D004262:DNA Restriction Enzymes