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Antisera against the five major polypeptides of the mouse mammary tumor virus (MMTV), gp52, gp36, p28, p14, and p10, were characterized. Anti-gp52, anti-gp36, anti-p28, and anti-p10 reacted exclusively with their respectve antigens. Anti-p14 reacted with p28 and p10 but not with p14. Virions, bald particles, cores, and A particles all reacted with anti-p28 and anti-p10. SDS-PAGE revealed that p28 and p10 were the major polypeptides in virions and bald particles but that p10 was reduced in the cores. No mature MMTV polypeptides were identified in the A particles by SDS-PAGE. A salt-detergent extraction for hydrophobic proteins revealed that p10 was distinctly hydrophobic. These results are consistent with the hypothesis that A particles are precursors to MMTV cores. They also suggest that p28, p14, and p10 come from a common precursor particle but that during maturation p10 is released from the MMTV core to associated with the envelope. A model of MMTV assembly and maturation is offered which integrates these observations.

Polyacrylamide gel electrophoresis of Japanese encephalitis virus (JEV) grown in both LLC-MK2 and chick embryo cell culture revealed three principal polypeptides with molecular weights of 8,700, 13,500, and 53,000 (V-1, V-2, and V-3, respectively). Infected chick cells that were treated with actinomycin D and cycloheximide contained seven polypeptides not present in uninfected cells. In addition to V-2 and V-3, polypeptides with molecular weights of 10,500, 19,000, 45,000, 71,000, and 93,000 (NV-1 through NV-5) were found; V-1 was not regularly detected. A similar pattern of polypeptides was obtained by radioimmune precipitation of soluble antigens from cytoplasmic extracts of infected, actinomycin-D treated, chick cells. When virions were treated with NP-40, a dense, RNA-rich structure was detected which contained V-2. An extracellular, slowly sedimenting, RNA-poor, hemagglutinating particle with a density comparable to the virion was present in virus preparations from cell culture and contained V-1, V-3, and NV-2.

The buoyant density of MTV in several different gradient solutions was determined, using a single pool of MTV-infected milk as the source of virus. Increased ultraviolet absorbance and detectable MTV B-particle antigenicity correlated with the positions of discrete light-scattering bands formed during centrifugation. MTV isodensities were found to be similar to those reported for other RNA tumor viruses. Centrifugation of MTV-positive preparations in preformed gradients containing sucrose or potassium tartrate resulted in the formation of two distinct light-scattering bands. Electron microscopic examination showed that only those bands at the higher buoyant density contained characteristic MTV particles. The bands of lower isodensity consisted of viruslike particles having considerable variability in size and shape, which may represent incomplete MTV particles. The properties of isopycnically centrifuged MTV-free milk samples were clearly distinguishable from those of MTV-positive samples; no discrete light-scattering bands were formed, and no viruslike particles were observed in electron micrographs.

Mouse mammary tumor virus (MMTV) contains an enzyme which catalyzes DNA synthesis in the presence of a preformed initiator without template direction. This type of enzyme activity is characteristic of the class of polymerases called terminal deoxynucleotidyl transferase. The MMTV enzyme is localized in the viral core and its activity depends upon the presence of detergent, reducing agent, preformed initiator, and divalent cation. Optimal conditions for activity include pH 6.85 and the use of a thymidine-containing substrate and initiator. The reaction product is predominantly single stranded. Concentration-dependent inhibition by several salts is observed. Manipulation of the reaction optima and salt concentration allows the functional separation of MMTV terminal transferase activity from RNA-dependent DNA polymerase activity.

A number of different conditions were evaluated to obtain a quantitatively efficient isolation procedure for the production of mouse mammary tumor virus (MMTV) cores. The efficiency of each procedure was determined by the recovery of radiolabeled MMTV-RNA in particles with a density of 1.24 – 1.26 g/cm 3 . Maximum recoveries of 40–60% were obtained with pretreatment of virions with 0.5 – 1% nonionic detergent at 37° for 15 min followed by isopycnic centrifugation. The resulting particles were morphologically identical to the internal core structure of MMTV virions and contained 60–70 S RNA and reverse transcriptase. The isolated cores were analyzed by immunodiffusion and by sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis. The major polypeptides of the cores were p30, p28, and p14. Two polypeptides, p28 and p14, were also major virion polypeptides. The emergence of p30 as a major core polypeptide was considered to be from interconversion and selective retention of other MMTV polypeptides. The envelope glycoproteins could not be identified among the many minor polypeptides.