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Characterization of viral polyproteins in cells transformed and producing Moloney murine sarcoma virus-124
Abstract:
The viral proteins of Moloney murine sarcoma virus-transformed mouse cells (G8-124), that overproduce the sarcoma virus relative to helper leukemia virus, were examined by immunoprecipitation, sizing by gel electrophoresis and peptide mapping. Using antisera to the viral core proteins, a p10-deficient core polyprotein of 63 000 daltons (P63gag) was detected which was found to be a product of the MuSV-124 genome. Helper virus proteins Pr67gag, gPr85gag, Pr200gag-pol, and gPr83env were also detected and characterized. An unusual helper virus precursor polyprotein of 93 000 daltons was also detected and characterized. It was labeled with [3H]fucose, suggesting that it was an intermediate precursor containing gp70 on which further modification of the core oligosaccharide had occurred relative to gPr83env. The usual viral proteins were also found in sarcoma virus particles and they were undistinguishable in size from those of Moloney murine leukemia virus (cl-1 strain). These experiments together with pulse-labeling experiments performed in the presence of the arginine analog canavanine, which prevents proteolytic cleavage, suggest that the product derived from the Mo-MuSV-specific sequences must be expressed as a separate gene product since we were unable to detect in transformed cells a polyprotein containing both viral (e.g., 'gag', 'pol' or 'env') and non-viral components. Cell-free protein synthesis studies performed with Mo-MuSV-124 genomic RNA have confirmed this interpretation and have identified three size classes of polypeptides as translation products of the sarcoma virus genome. They are P63gag, P42-P38 and P23. Intracellular p63gag and cell-free synthesized P63gag, appear to have identical sizes, antigenic determinants and tryptic peptides. The P42-P38 proteins contain core protein determinants. P23 is not related to the products of the replication genes of MuLV. Thus, P23 is a candidate for the 'src' gene product of Mo-MuSV-124.
Insights
Researchers identified Moloney murine sarcoma virus (Mo-MuSV) gene products in transformed mouse cells. They found P63gag, P42-P38, and P23 as translation products, with P23 being a candidate for the src gene product.
Area of Science:
- Molecular Virology
- Oncogenic Viruses
- Gene Expression
Background:
- Moloney murine sarcoma virus (Mo-MuSV) transforms mouse cells and overproduces sarcoma virus relative to helper leukemia virus.
- Understanding the viral protein products of Mo-MuSV is crucial for elucidating its oncogenic mechanisms.
Purpose of the Study:
- To characterize the viral proteins produced by Mo-MuSV-transformed mouse cells (G8-124).
- To identify the specific gene products encoded by the Mo-MuSV genome, particularly the sarcoma virus-specific sequences.
Main Methods:
- Immunoprecipitation and gel electrophoresis to size viral proteins.
- Peptide mapping to analyze protein structure.
- Pulse-labeling experiments with canavanine to study protein precursors.
- Cell-free protein synthesis using Mo-MuSV-124 genomic RNA.
Main Results:
- A p10-deficient core polyprotein (P63gag) was identified as a product of the MuSV-124 genome.
- Helper virus proteins (Pr67gag, gPr85gag, Pr200gag-pol, gPr83env) and an unusual precursor polyprotein (93,000 daltons) were characterized.
- Cell-free synthesis identified three polypeptide classes: P63gag, P42-P38, and P23.
- P63gag showed identical characteristics to intracellular P63gag.
- P42-P38 proteins contain core protein determinants, while P23 is unrelated to MuLV replication genes, making it a candidate for the src gene product.
Conclusions:
- Mo-MuSV-specific sequences are expressed as a separate gene product.
- P23 is identified as a strong candidate for the src gene product of Mo-MuSV-124.
- The study provides insights into the translation and processing of viral proteins in Mo-MuSV-transformed cells.