Subgenomic mRNA in OK10 Defective Leukemia Virus-Transformed Cells

Abstract

OK10, a defective leukemia virus, is produced as a defective particle by so-called nonproducer transformed quail fibroblasts. OK10 defective viral particles contain an 8-kilobases (kb)-long genomic RNA, lack any detectable reverse transcriptase activity, and are not infectious. We studied the genetic content of OK10 RNA extracted from both virions and infected cells. As shown by RNA-cDNA hybridizations in stringent conditions, about 77% (6.4 kb) of the OK10 8.0kb RNA was related to avian leukosis viruses in the three structural genes gag, pol , and env , as well as in the c region. The remainder of the OK10 genome-encoding capacity (≤1.6 kb) was homologous to the MC29-specific transforming sequence myc (m) and therefore has been named myc (o). Eco RI restriction analysis of the OK10 integrated proviral DNA with different probes indicated the presence of only one provirus in the OK10 QB5 clone, which agreed with the gene order: 5′- gag -Δ pol - myc (o)-Δ env - c - 3′. Heteroduplex molecules formed between the viral OK10 8.0-kb RNA and the 6.8-kb Sac I DNA fragment of the Prague A strain of Rous sarcoma virus confirmed that structure and indicated that the myc (o) sequence formed a continuous RNA stretch of 1.4 to 1.6 kb long between Δ pol and Δ env . We also examined the myc (o)-containing mRNA's transcribed in OK10-transformed cells. OK10-transformed quail fibroblasts (OK10 QB5) transcribed two mRNA species of 8.0 and 3.6 kb containing the myc (o) sequence. The genetic content of the 3.6-kb species made it a possible maturation product of the genome size 8-kb species by splicing out the gag and pol sequences. In OK10-transformed bone marrow cells (OK10 BM), a stable bone marrow-derived cell line producing OK10, the myc (o) sequence was found in four RNA species of 11.0, 8.0, 7.0, and 3.6 kb. Again, the genetic content of these mRNA's indicated that (i) the 3.6-kb species could be spliced out of the 8.0-kb-genome size mRNA and (ii) the 11.0-kb-long mRNA could represent a read-through of the OK10 provirus, the corresponding maturation product being, then, a 7.0-kb mRNA. The 7.0- and 3.6- kb mRNA's both contained the myc (o) sequence, but no sequences related to the gag or pol gene. In conclusion, whereas the myc sequences have been generally thought to be expressed through a gag-onc fusion protein, as for MC29 and CMII viruses, our experiments indicate that they could also be expressed as a non- gag -related product made from a subgenomic mRNA in the OK10-transformed cells.