UBC Theses and Dissertations
Studies on the entry of alphaviruses into BHK-21 cells Talbot, Pierre
The nature of the infectious mechanism of entry of alphaviruses into cultured BHK-21 cells was studied. The importance of cellular lysosomes in a productive Sindbis virus infection was determined. Chloroquine (100 μM) and NH₄Cl (10 mM), known inhibitors of lysosomal function, decreased the production of infectious Sindbis virions in BHK-21 cells by 10- and 12-fold respectively, when present during the 1 h infection period of a plaque assay. There were no apparent toxic effects on cells exposed to these chemicals, with the exception of a reversible 2-fold inhibition of cellular protein synthesis by chloroquine. In order to determine if the decrease in the virus titer was correlated with a similar decrease in the number of virus particles, the reduction in the formation of Sindbis virions was monitored by incorporation of Ḻ-[³⁵S]methionine and shown to be 2-fold at 8 and 11 h post-infection. Shedding of envelope glycoproteins into the culture medium was indirectly demonstrated by the fact that much more of these proteins were released that can be accounted for by progeny virions. Finally, the importance of a lysosomal pathway of infection was studied in more details. Cells were infected with Sindbis virus labelled with Ḻ-[³⁵S]methionine and the fate of radiolabeled viral proteins into subcellular fractions followed up to 120 min post-infection. Viruses were transiently associated with a cell fraction enriched in lysosomes. Capsid proteins were preferentially released into the cytoplasm after 60 min post-infection and viral proteins started to be significantly degraded between the first and second hour after infection. Chloroquine produced an initial accumulation of viruses in the lysosomes at 20 min post-infection and these trapped particles appeared to be degraded thereafter. Unexpectedly, NH₄Cl blocked an early infection step between binding of the virus on the cell surface and penetration into lysosomes, possibly receptor clustering into coated pits or receptor recycling. Results were identical with virus preparations which contained either 147 or 36 particles per infective unit. In conclusion, the results of these studies strongly suggest the involvement of lysosomes and thus a receptor-mediated endocytic pathway (viropexis) as the main infectious mechanism for entry of Sindbis virus into BHK-21 cells. The mechanism of entry of radiolabeled Semliki Forest virus was also studied. After a 15 min pulse, whole virus particles migrated within 60 min from a fraction of the cells enriched in plasma membranes into an intracellular fraction (endoplasmic reticulum). This result is consistent with an endocytic mechanism of entry.
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