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. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/19824 |
| Date | January 1976 |
| Creators | Richardson, Christopher Donald |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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