Transcriptional and Translational Products of Avian Erythroblastosis and Avian Myeloblastosis Viruses

Author

Steven Mathias Anderson

Date of Award

1981

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hanafusa Laboratory

Abstract

The transcription and translation products of two avian acute leukemia viruses, avian erythroblastosisvirus (AEV) and avian myeloblastosisvirus (AMV) , were examined. AEV is defective in virus replication and requires the presence of a helper virus to produce infectious progeny. AEV-transformed nonproducer cells were found to contain no proteins participating in virus replication. Instead, nonproducer cells were found to contain a 75,000 Dalton gag-related polyprotein (p75) which contains the p19 region of the gag gene at its amino terminus. Two-dimensional tryptic peptide mapping indicated that p75 contained novel peptides in addition to those derived from p19. p75 was found to be a phosphoprotein containing a phosphoserine residue. Two-dimensional tryptic phosphopeptide maps of p19 and p75 were identical, indicating that all phosphorylation of AEV p75 occurs in the gag portion of the molecule. Unlike many other transforming proteins, AEV p75 was not found to be a protein kinase using the immune complex protein kinase assay. AEV nonproducer cells were found to contain two viral related RNA species of 28 S and 21 S. Both RNA species contain the AEV unique sequences and the "strong stop" region of the viral genome, as detected by cDNA hybridizatin. In addition to the 28 S RNA, which is the genomic RNA of AEV, the 21 S subgenomic RNA was incorporated into the virus particle. In vitro translation of specific size classes of AEV virion RNA demonstrated that the 28 S RNA encodes p75, and that the 21 S RNA encodes two proteins of 46,000 and 48,000 daltons. These two proteins, p46 and p48, were not immunoprecipitated by antisera directed against the , gag, pol or env gene products. Two-dimensional tryptic peptide mapping indicates these two proteins are closely related to each other. An antiserum specific to the unique sequences in AEV p75 was prepared by immunizing chickens that had regressed from erythroblastic leukemia induced by a temperature-sensitive mutant of AEV, AEVts34. This antiserum did not cross-react with p46/p48 indicating that these proteins are not related to AEVp75. This antibody was used in immunofluorescent staining of AEV-transformed rat cells and staining was obeserved in the cellular membrane, cell ruffles, and in a perinuclear organelle. The possible role of p75 and p46/p48 in oncogenic transformation by AEV is discussed. AMV is a replication-defective acute leukemia virus. Nonproducer clones of AMV-transformed myeloblasts contain pr180^gag-pol, pr76^gag, and the individual gag proteins p19,p27, pI2 and p15. The genome of AMV is 34 S and it produces a subgenomic mRNA of 21 s. This RNA contains the "strong stop" region, the AMV-specific sequences, and the 3' C region. As in the case of AEV, the genomic RNA and the subgenomic RNA of the virus both were incorporated into the virus particle. In vitro translation of AMV virion RNA identified three AMV-specific proteins of 56,000, 48,500, and 47,000 daltons. The 56,000 and the 48,500 dalton proteins were encoded by 20-22 S RNA, which is the same size as the AMV subgenomic RNA. The 47,000 dalton protein was encoded by 22-24 S RNA. ^7mGTP was found to block translation of the 56,000 and 48,500 dalton proteins but not the 47,000 dalton protein, indicating that the 56,000 and 48,500 dalton proteins are translated from capped RNAs but the 47,000 dalton protein is not. These three proteins are not immunoprecipitated by antisera directed against the gag, pol or env gene products. Two-dimensional tryptic peptide mapping had indicated that these proteins gPr92^env, the glycosylated envelope precursor to gp85 and gp37. And antiserum prepared by immunizing rabbits with purified AMV ATPase did not precipitate the 56,000, 48.500 or 47, 000 In vitro translation products of AMV virion RNA. This antiserum did, however, immunoprecipitate AMV ATPase-related polypeptides from AMV-transformed myeloblasts.

Comments

A thesis submitted to the Faculty of the Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Recommended Citation

Anderson, Steven Mathias, "Transcriptional and Translational Products of Avian Erythroblastosis and Avian Myeloblastosis Viruses" (1981). Student Theses and Dissertations. 438.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/438