Electron microscopy of Rous sarcoma virus

Abstract

1. The most appropriate methods were investigated for producing Rous sarcoma virus of suitable quantity and quality for use in the study of the viral RNA by electron microscopy. The roller bottle method of Smith and Bernstein (1973) which was adopted, produced virus yields of up to 5mg per litre of transformed cell culture supernatant after 24 hour incubations, and 0.2mg per litre of culture supernatant after 4 hour incubations. 2. The method of purifying RNA tumour viruses which resulted in the least damage to the virions was found to be isopyncic and velocity sedimentation in Ficoll density gradients containing 5mM tris-HCl and 1mM EDTA pH 8.5. The use of solutions of sucrose or >0.1M salt resulted in both osmotic changes in the virus and viral aggregation. 3. The lipoprotein coat of the Rous sarcoma virus was shown by freeze-fracturing and electron microscopy to have properties similar to those of plasma membranes, except that the number of intramembranous particles was smaller. The hydrated diameter of Rous sarcoma virus was estimated from freeze-fracture replicas of purified virions to be 140nm. 4. Vesicular contaminants, derived from serum, were present in Rous sarcoma virus preparations that had been purified from transformed cell culture supernatants. The isolated contaminants resembled virus when examined by both freeze-fracturing and negative-staining, but were readily distinguished from virus in thin sections. The virus-like serum vesicles were present in sera from several different sources. When treated with detergent and subjected to polyacylamide gel electrophoresis, the vesicles were found to contain polypeptides that possessed similar electrophoretic mobilities to those of Rous sarcoma virus polypeptides. It is probable that extraneous nucleic acid molecules, observed in preparations of Rous sarcoma virus RNA were the result of VLSV contamination of virus suspensions. 5. Contamination of purified virus suspensions by virus-like material derived from serum was reduced by centrifugation of the serum prior to its addition to cell culture medium. Virus suspensions, purified from cell supernatants from which the contaminating vesicles had been removed, were resolved in sharp bands at p = 1.07 g/ml in Ficoll density gradients; in the analytical ultracentrifuge they sedimented as homogenous populations with a sedimentation value of 740s20,w and were observed by electron microscopy to be relatively free of contaminants. 6. The maximum length of molecules from preparations of both 60-70s and 30-40s viral RNA prepared in 80% and 50% formamide respectively was 2.5μm, but both preparations were not homogeneous since they contained other, smaller molecules. 7. A model is proposed in which the difference in physical properties between the native (60-70S) form and the denatured (30-40S) form of the viral RNA is suggested to be the result of two possible conformations of a single RNA molecule. This model is an alternative to the prevailing model in which the RNA tumour virus genome is proposed to contain a number of RNA molecules of equivalent size.