Birdwell, Charles Ray (1974) Studies on the infection of animal cells with sindbis virus : adsorption, cell surface modification, and maturation. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-09292005-083645
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The surface replica technique was used to study the distribution of Sindbis virus receptors on chick embryo fibroblasts. Using the position of adsorbed Sindbis virions on the cell surface to indicate the position of Sindbis virus receptors, an even distribution of Sindbis virus receptors over the cell surface was observed if the cells had been previously fixed with glutaraldehyde before adsorption of virus. The number of receptors observed varied from cell to cell, being on the order of [...] per cell. Occasionally areas of the cell surface with different concentrations of virus receptors were seen on the same cell. The virus particle-receptor complexes were found to move laterally within the plasma membrane, and the virus particles appeared clustered on the cell surface, when unfixed cells were adsorbed with Sindbis virus at 4 C.
The insertion of Sindbis virus glycoproteins into the cell surface was studied by an indirect electron microscopic labeling technique, which involved treating infected cells sequentially with rabbit anti-Sindbis IgG followed by hemocyanin-conjugated goat (anti-rabbit IgG) IgG, and examining the cells in the electron microscope by the surface replica technique; the position of hemocyanin on the cell surface was used to indicate the position of viral glycoproteins. Sindbis virus glycoproteins were detected at the surface of chick embryo fibroblasts by 2 hours after infection. When infected cells were prefixed with glutaraldehyde before labeling at 37 C, the distribution of virus glycoproteins in the cell surface was fairly even, although a slight clustering was noticed early in infection. However, when infected cells were labeled at 37 C without prefixation, the hemocyanin was clustered, suggesting that the viral glycoproteins moved laterally within the plasma membrane after treatment with the antibody.
The modification of the cell surface by Sindbis virus was also studied by examining the agglutination of infected cells by two of the plant lectins, concanavalin A and Ricinus communis agglutinin. By 3-3.5 hours after infection, Sindbis virus-infected chick embryo fibroblasts were more agglutinable by these lectins than uninfected cells. Evidence is presented that this increase in agglutination was not due to an increase in the number of lectin binding sites on the cell surface.
Using the surface replica technique, the maturation of Sindbis virus in baby hamster kidney cells and chick embryo fibroblasts was studied. Sindbis virus was found to bud through certain areas of the cell surface in patches, and through virus-specific processes - extending only from the cell periphery; budding virions were also seen on the undersides of infected cells. The virus-specific processes were also seen in cells infected with vesicular stomatitis virus, but budding through processes was not a major mode of virus release in these cells. Electron microscopic examination of thin-sections of Sindbis virus-infected cells revealed that these processes contained viral nucleocapsids in the process of budding. The maturation of Sindbis virus may be somewhat dependent on the structural integrity of cellular microtubules and microfilaments, as both colchicine and cytochalasin B were found to inhibit Sindbis virus release.
A temperature-sensitive mutant of Sindbis virus, ts-103, was found to bud much differently than wild type Sindbis virus. Cells infected with ts-103 contained abnormal virus-specific processes extending from the cell edge; these mutant processes often contained large virus particles containing many viral nucleocapsids.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||24 April 1974|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||29 Sep 2005|
|Last Modified:||26 Dec 2012 03:03|
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