Genetic structure, transforming sequence, and gene product of avian sarcoma virus UR1

Abstract

We analyzed the genetic structure and gene products of the newly isolated avian sarcoma virus UR1, which recently has been shown to be replication defective and to contain no sequences homologous to the src gene of Rous sarcoma virus. The sizes of the genomic RNAs of UR1 and its associated helper virus, UR1AV, were determined to be 29S and 35S (5.9 and 8.5 kilobases), respectively, by gel electrophoresis and sucrose gradient sedimentation. RNase T1 oligonucleotide mapping of purified viral RNAs indicated that UR1 RNA contains eight unique oligonucleotides in the middle of the genome and shares four 5'-terminal and three 3'-terminal oligonucleotides with UR1AV RNA. The unique sequences of UR1 and Fujinami sarcoma virus were found to be closely related to each other by molecular hybridization of UR1 RNA with DNA complementary to the unique sequence of Fujinami sarcoma virus RNA, but minor differences were found by oligonucleotides fingerprinting. In the regions flanking the unique sequences, UR1 and Fujinami sarcoma viral RNAs contain distinct oligonucleotides, which are shared with oligonucleotides of the respective helper viral RNAs. Cell transformed with UR1 produce a single 29S RNA species which contains a UR1 unique sequence; this species is most likely the mRNA coding for the transforming protein. In UR1-transformed cells, a phosphoprotein fo 150,000 daltons (p150) was detected by immunoprecipitation with antiserum against gag proteins. p150 was associated with a protein kinase activity that was capable of phosphorylating p150 itself, immunoglobulin G of antiserum, and a soluble substrate, alpha-casein. This enzyme transferred phosphate exclusively to tyrosine residues of substrates in vitro, but p 150 labeled in vivo with 32P contained both phosphoserine and phosphotyrosine. The in vitro kinase reaction was not affected by the presence of cyclic AMP or cyclic GMP and strongly preferred Mn2+ over Mg2+. Thus, the properties of UR1 protein are almost identical to those of Fujinami sarcoma virus protein.

Cellular Sequences Related to Three New onc Genes of Avian Sarcoma Virus ( fps, yes , and ros ) and Their Expression in Normal and Transformed Cells

“…This is consistent with our previous results obtained from fingerprinting analyses of viral RNAs (55). It is of interest to note that all of the gene products of these four onc sequences have been reported to be associated with protein kinase activity specific for phosphorylation of tyrosine residues on substrate proteins (13,14,30,38,41,54). In addition we showed that none of the cDNAs specific to these four onc sequences hybridized with RNA of Abelson murine leukemia virus, whose genome has also been reported to contain a gene for tyrosinespecific protein kinase (57).…”

“…Viral RNAs were extracted from virions and hybridized to cDNA probes under conditions of moderate stringency. The data summarized in Table 1 include data on the cross-homology of fps of FSV to unique sequences of PRCII, UR1, and two strains of feline sarcoma viruses which have been reported elsewhere (44,54). The absence of homology of yes to RSV, FSV, or avian acute leukemia viruses confirms the observations described by Yoshida et al (59).…”

“…RNA-cDNA hybridization experiments have shown that among these viruses at least two nonhomologous onc genes are present. One is fps, which is shared with FSV, PRCII, and UR1 (44,54), and the other is yes of Y73 (59). By comparison of tryptic peptides of transforming proteins, Esh sarcoma virus was shown to be related to Y73 in the unique sequences (19).…”

“…These viruses appear to be different from RSV but similar to one another in several characteristics. First, they are all defective in virus replication, because their viral genomes contain unique sequences unrelated to avian C-type retroviruses, in place of sequences required for virus replication (19,23,31,37,54,55,60). Second, their genomes encode unique proteins ranging from 68,000 to 140,000 daltons which are precipitable by antisera against viral structural proteins coded by the gag gene of avian retroviruses.…”

“…These ASV proteins are read-through products of gag and unique sequences in their genomes. Third, their unique sequences are nonhomologous to src; however, all of these polyproteins in immunoprecipitated forms are associated with an enzymatic activity of protein kinase specific for tyrosine residues similar to p60src of RSV (13,14,30,38,41,54). RNA-cDNA hybridization experiments have shown that among these viruses at least two nonhomologous onc genes are present.…”