Isolation of noninfectious particles containing Rous sarcoma virus RNA from the medium of Rous sarcoma virus-transformed nonproducer cells.

Abstract

Stocks of the Brvan high-titer strain of Rous sarcoma virus (RSV) have been shown to contain avian leukosis viruses as well as RSV.I Chick embryo fibroblasts infected with the leukosis viruses associated with the Bryan stocks, Rous associated virus-I (RAV-1) or Rous assoc iated virus-2 (RAV-2), do not transform but do produce virus, whereas cells infected with RSV transform but do not produce infectious virus.2 When RSV-infected transformed cells are superinfected with RAV-1 or RAV-2, the transformed cells not only produce the superinfecting virus but also produce infectious RSV which has the growth characteristics,3 antigenic specificity,4 and host range of the superinfecting virus.5 The RSV-transformed cells which do not produce virus have been named nonproducer (NP) cells, and the avian leukosis viruses associated with the Bryan stock, helper viruses. Viral coat antigen cannot be detected in NP cells,2 4 and it has been hypothesized that the absence of coat arntigen in RSV-infected cells prevents synthesis of infectious RSV and that the role of the helper virus in the production of infectious RSV is to provide coat antigen.2 The finding that NP cells do not produce infectious virus raised the possibility that the carcinogenic action of RSV nmight be related to the apparent absence of late functions in virus replication in infected cells.2 Recently, Dougherty and DiStefano6 have observed particles with the appearance of avian leukosis viruses in electron micrographs of RSV-transformed NP cells and have suggested that NP cells may be producing non infectious virus. The experiments presented here approach the question of virus production by the NP cell with the aid of radioactive tracers7 and demonstrate that the NP cell is producing noninfectious particles with the physical characteristics of the avian leukosis viruses. The nucleic acid of the particles is characterized and found indistinguisbable in sedimentation velocity and base composition from the RNA of RSV + RAV. Materials and Methods.--Isolation and care of NP cells: Chick embryo cells from 10-day-old embryos of strain 13 or 813 white leghorn chickens which were susceptible to infections by both group 1 and group 2 avian leukosis viruses were used throughout the course of this work. The methods used for culturing chick enmbryo cells9 and assaying for focus-forming units of RSV9 and interfering units of RAVy have been described. Clones of NP cells were isolated according to the method of Trager and Rubin8 from secondary chick embryo fibroblasts infected with the Bryan high-titer straini of RiSV. The isolated clones were grown on X-irradiated chick embryo fibroblast feeder layers (4 X 105 cells/60-mrn plate) in a rich medium: 82 per cent 199A, 10 per cent tryptose phosphate broth, 4 per cent calf serum, 1 per cent chicken serum, 2 per cent 2.8 per cent NaHCO3, 1 per cent beef embryo extract, which had been incubated at 57?C for 45 minutes to inactivate any contaminating leukosis viruses. Culture medium was changed daily and the cultures