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Simian sarcoma virus-encoded gag-related protein: in vitro cleavage by Friend leukemia virus-associated proteolytic
Abstract:
The simian sarcoma virus (SSV) encodes a gag-related 65,000-Da protein (SSV p65) which is not processed in SSV nonproducer cells (SSV-NP cells) (H.-J. Thiel, T. J. Matthews, E. M. Broughton, K. J. Weinhold, D. P. Bolognesi, T. Graf, and H. Beug (1981a), Virology 114, 124-131). In order to cleave SSV p65, retroviral particles containing this antigen were incubated with extracts from the heterologous helper virus Friend leukemia virus (FLV). Superinfection of SSV-NP cells by FLV has been previously shown to result in processing of SSV p65 in vivo (H.-J. Thiel, F. Weiland, R. Hafenrichter, T. J. Matthews, and K. J. Weinhold (1982), Virology 123, 229-234). In vitro cleavage was most efficient in the presence of a nonionic detergent (greater than 0.1% Nonidet-P40) and a reducing agent (greater than 5 mM dithiothreitol) at a pH of 7.0. The products, termed SSV p55 (p15, p12, p30), SSV p30, SSV p25 (p15, p12), and SSV p10, were characterized by (1) molecular weight, (2) kinetics experiments, (3) incorporation of different radiolabeled amino acids, and (4) comparison with SSAV structural proteins. Kinetics experiments with two amino acids ([3H]leucine, [35S]cysteine) revealed that initial processing of SSV p65 produced SSV p55 and SSV p10, with subsequent processing of SSV p55 occurring thereafter. In contrast to the Moloney system, the major intermediate p40 (p30, p10) could not be clearly demonstrated. A direct comparison of SSAV p10 and the cleavage product SSV p10 by SDS-PAGE suggests that SSAV pr65gag and SSV p65 differ slightly by molecular weight.
Insights
Simian sarcoma virus (SSV) p65 protein processing was achieved in vitro using Friend leukemia virus (FLV) extracts. This study characterizes the cleavage products and conditions, advancing retroviral protein research.
Area of Science:
- Retroviral research
- Molecular biology
- Virology
Background:
- Simian sarcoma virus (SSV) encodes a gag-related 65,000-Da protein (SSV p65) that requires processing.
- SSV p65 is not processed in SSV nonproducer cells (SSV-NP cells).
- In vivo processing of SSV p65 occurs upon superinfection of SSV-NP cells by Friend leukemia virus (FLV).
Purpose of the Study:
- To investigate the in vitro cleavage of SSV p65 using FLV extracts.
- To characterize the resulting cleavage products and optimal conditions for processing.
- To compare SSV p65 processing with other retroviral systems.
Main Methods:
- Incubation of retroviral particles containing SSV p65 with FLV extracts.
- Optimization of cleavage conditions including detergent, reducing agent, and pH.
- Characterization of cleavage products by molecular weight, kinetics, radiolabeling, and comparison with SSAV proteins.
Main Results:
- In vitro cleavage of SSV p65 was efficient under specific conditions (Nonidet-P40, dithiothreitol, pH 7.0).
- Identified cleavage products include SSV p55, SSV p30, SSV p25, and SSV p10.
- Kinetics revealed initial processing to SSV p55 and SSV p10, followed by SSV p55 processing.
- A minor difference in molecular weight was observed between SSAV pr65gag and SSV p65.
Conclusions:
- Friend leukemia virus (FLV) extracts can mediate the in vitro cleavage of simian sarcoma virus (SSV) p65.
- The study defines optimal conditions and characterizes the products of SSV p65 processing.
- Findings contribute to understanding retroviral gag protein maturation and structural differences.