Jacques Ghysdael

Tyrosine-specific protein kinase activity associated with p105 of avian sarcoma virus PRCII

A protein kinase activity was found to be associated with the transformation-specific polyprotein (p105) of avian sarcoma virus PRCII. The kinase was detected in immune complexes with antisera reactive with the gag sequences of p105. Addition of [γ-32P]ATP to these complexes resulted in phosphorylation of p105 and, with some sera, phosphorylation of immunoglobulin heavy chain. Phosphoamino acid analysis showed that phosphotyrosine was the major product of the in vitro kinase reaction and the major phosphoamino acid of p105 extracted from 32P-labeled cells. The same tryptic peptides of p105 were found to be labeled by the in vitro kinase reaction and by 32P labeling of PRCII-transformed cells. These phosphopeptides were not found in Pr76gag Thus, like Rous sarcoma virus, PRCII has an associated tyrosine-specific protein kinase which may be responsible for its transforming activity.

Esh avian sarcoma virus codes for a gag-linked transformation-specific protein with an associated protein kinase activity

Esh sarcoma virus, initially isolated from a spontaneous tumor of a chicken, transforms fibroblasts in vitro and induces fibrosarcomas in vivo. It is defective for replication, and infectious viral stocks consist of a mixture of a sarcomagenic virus (ESV) and an a avian leukosis virus of subgroup A (EAV) which serves as helper. Cloned stocks of infectious ESV contain two RNA components of Mr, 3 and 1.5 × 106, respectively, as determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The component of Mr 1.5 × 106 appears to be the genome of defective ESV, since it is not detected in preparations of the helper virus EAV. The size of the ESV genome suggests major deletions of replicative genes, and ESV-transformed nonproducer cells fail to express functional translation products of the gag, pol, and env genes. ESV-transformed producer and nonproducer clones also do not express pp60src but contain a gag-related protein of Mr 80,000 (p80). Two-dimensional analyses of the [35S]methionine-labeled tryptic peptides of p80 indicate that this protein contains part of the sequences of gag-p19 covalently linked to additional sequences unrelated to gag, pol, and env gene products. These ESV-specific sequences are also unrelated to pp60src and to gag-linked polyproteins found in cells transformed by defective avian sarcoma viruses PRCII and Fujinami or defective leukemia viruses AEV, MC29, and MH2. P80 is phosphorylated in vivo at two major sites, one involving phosphoserine and the other phosphotyrosine residues. Immunoprecipitates containing ESV-p80 are associated with a protein kinase activity that is specific for tyrosine residues of several acceptor molecules including p80 itself, rabbit immunoglobulin H chain of the immune complex and exogenously added α-casein. p80 is phosphorylated in vitro at the same tyrosine site as in vivo suggesting that the enzyme activity detected in vitro is of physiological significance. The p80-associated protein kinase activity is strictly dependent on the presence of Mg2+ or Mn2+ but was found independent of known effectors of cellular protein kinases Ca2+, cAMP, or cGMP.

Structural similarities of proteins encoded by three classes of avian sarcoma viruses

The structure and location of the phosphorylation sites of a number of avian sarcoma virus polyproteins have been examined by protease cleavage analysis. The PRCIIp and PRCII polyproteins, P170gag-fps and P105gag-fps yield indistinguishable cleavage fragments from an N-terminal region of 65,000 molecular weight, including the gag/non-gagjunction. This provides strong support for the view that PRCII arose directly from PRCIIp by a genomic deletion. For P909agag-yes, P800gag-yes, and P105gag-fps the major tyrosine phosphorylation sites are on C-terminal fragments of 27,000, 26,500, and 36,000 molecular weight, respectively. Further similarities have been shown by partial sequence analysis of the tyrosine phosphorylation sites; the positions of trypsin and staphylococcal V8 protease cleavage sites largely correspond in the src, fps, and yes gene products. The homology between the src and yes products is particularly striking. They yield C-terminal V8-resistant fragments of similar size, containing the major tyrosine phosphorylation sites which are indistinguishable after further cleavage with several proteases. These results suggest structural and functional relatedness between the src, fps, and yes gene products despite the lack of hybridization between their DNA sequences.

The 28 S genomic RNA of avian sarcoma virus PRCII codes for the transformation-specific polyprotein P105☆

We have characterized the genomic RNA of the defective avian sarcoma virus PRCII. Replicating virus which consists of transforming PRCII and a nontransforming but replication-competent helper, PRCII-AV, contains two RNA species. One is identical in size to the 35 S genome of avian leukosis helper viruses. The second component migrates slightly faster than 28 S ribosomal RNA in polyacrylamide gel electrophoresis but co-sediments with this RNA in sucrose gradients. In vitro translation across a gradient of velocity-sedimented poly(A)-containing PRCII virion RNA yielded three major proteins. The virion protein precursors Pr76gag and Pr180gag-pol were translated from 35 S RNA, while the transformation-specific polyprotein P105 was translated from 28 S RNA. P105 may be the only protein coded for by the PRCII genome, although this product would not exhaust the coding capacity of 28 S RNA. Whether translated in vitro or immunoprecipitated from transformed cells, P105 was essentially identical as demonstrated by comparative peptide maps.