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Radioactive pulse chase studies concerning the synthesis of viral proteins : and the membrane assembly of Semliki forest virus Richardson, Christopher Donald

Abstract

The mechanism of membrane assembly for Semliki Forest Virus, a Group A Togavirus, was investigated through a series of radioactive pulse chase experiments. Initially a time course for the appearance of virus specified proteins in the microsomal fraction of infected BHK (baby hamster kidney) cells and mature virions was performed. Infected cells were harvested and fractionated at 0, 1, 2, 4, 6, 8 and 11 hours post-infection. Plaque assays were performed on the virus released into the growth medium at these times. It was found that virus production was maximal between 4 and 6 hours. Nucleocapsid was clearly evident at 6, 8 and 11 hours when the microsomal proteins were separated by SDS polyacrylarnide gel electrophoresis. In the next set of experiments infected cells were pulsed 3 for 20 minutes at 5 hours post-infection with H-Leu. Microsomes were prepared from the cells at 0 min., 20 min., 40 min. and 60 min. after removal of H-Leu and subjected to SDS polyacrylarnide gel electrophoresis. Virus was also isolated from the cell medium by sucrose gradient centrifugat-ion. Nucleocapsid protein radioactivity was at levels much greater than the combined peaks of radioactivity due to the membrane proteins E₁ and E₂. Little if any radioactive virus was released into the media during this time of chase. A similar experiment to the one just outlined was performed except that the radioactive chase was extended over the range of 0 hrs., 0.75 hrs., 1.50 hrs., 2.25 hrs. and 3.00 hrs. Levels of ³H- labelled nucleocapsid were again initially higher' than those of the combined E₁ and E₂ radioactive peak. The radioactivity of E₁ E₂ plateaus between 0.75 hrs. to 3.00 hrs. while that in the nucleocapsid continued to increase. This data appears to support the contention that nucleocapsid is synthesized prior to the viral membrane proteins. In hope of chasing the ³H-Leu label into and then out of the microsomes, infected BHK cells were pulsed at 3 hours and chased for 0, 1, 2, 3, 4, 5, and 6 hours after removal of labelled medium. Levels of ³H-Leu increased in both the nucleocapsid and E₁ E₂ protein bands of the SDS acrylamide gels until about 2 hours and then declined over the following time range. Loss of ³H-Leu in the microsomes appeared to correlate with the increase of label incorporated into the virus. Finally, after devising a method for separating plasma membrane (PM) ghosts and endoplasmic reticulum (ER) fragments, another pulse chase experiment was performed. Infected BHK cells were again radioactively pulsed at 3 hours infection and the level chased for 0, 1, 2, 4, 6, 8 and 11 hours following removal of the ³H-Leu. At the various time points labelled cells were harvested and fractionated into PM and ER. The samples of ER and PM were applied to SDS acrylamide gels and the radioactivity incorporated into the virus protein band was quantitated. Virus released into the medium was purified by sucrose gradient sedimentation, assayed for ³H-Leu, and also fractionated by SDS electrophoresis. Label was initially very high in the ER in the form of precursor proteins (NVPI65, NVP97, PE₂), envelope proteins (E₁, E₂), and nucleocapsid protein. This radioactivity was chased from the ER to the PM and then into mature virus. These results appear to indicate that Semliki Forest Virus nucleocapsid does indeed "bud" from the host cell membrane, thus obtaining its envelope.

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