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Radioactive pulse chase experiments concerning the mechanism of entry of Semliki Forest viru

University of British Columbia

The mechanism of viral penetration for Semliki Forest Virus into BHK-21 cells was investigated through a series of radioactive pulse-chase experiments. Entry of an enveloped virus such as SF Virus can be visualized to enter host cells both by pinocytosis (viropexis) or by fusion of the viral envelope and plasma membrane. Preliminary experiments were performed to obtain optimum conditions of viral adsorption to host cells. The conditions considered included temperature, time of infection, multiplicity of infection and ionic strength of the inoculum. In subsequent experiments BHK-21 cells were infected one half 35 hour with ³⁵S-Methionine-labeled Semliki Forest Virus. At various time points after removal of unadsorbed ³⁵Met-SF Virus, cells were harvested and fractionated into plasma membrane and endoplasmic reticulum fractions. The fractions were subjected to SDS polyacrylamide gel electrophoresis and analyzed for component proteins of SF Virus. Maximum levels of radioactivity corresponding to the envelope proteins (E₁, E₂ and nucleocapsid protein (NC) were found in the PM fraction at zero minutes of chase. Both E₁, E₂ and NC were found to decline during the chase period (approximately 90% within 60 minutes of chase). On the other hand, high levels of only nucleocapsid protein were observed associated with the endoplasmic reticulum fraction although no general pattern of incorporation was indicated furing the experiment. (There were high levles of NC present in the ER fraction throughout the chase period). The results of these studies are generally inconclusive as they can be rationalized both by the viropexis and fusion mechanisms. The loss of both E₁, E₂ and NC from the PM suggest that viropexis is the mechanism of entry, however, fusion is not eliminated as an alternative since the envelope proteins in the PM may be degraded after internalization of the virus core by host proteases. Although no conclusions have been drawn, this study has demonstrated that radioactive pulse-chase experiments can be performed to augment electron microscopy data concerning the mechanism of viral penetration into animal cells.

Text

2010-02-26

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http://hdl.handle.net/2429/21075

Biochemistry and Molecular Biology

University of British Columbia

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