Studies concerning the mechanism for the membrane assembly of Semliki Forest virus
Creators: name: Richardson, Christopher Donald
Publisher: name: University of British Columbia
Year: 2010

description: The data from chemical studies and electron microscopy-suggest that Semliki Forest virus obtains its envelope by budding into the medium from the plasma membrane of the host cell. Biochemical evidence for this phenomenon, however, has not been published. Therefore, we undertook a series of pulse-chase studies so that we might quantitatively evaluate the importance of the budding mechanism in the morphogenesis of Semliki Forest virus. Baby hamster kidney cells (clone 13) were grown in culture and infected with Semliki Forest virus. The cells were exposed to [4.5-³H] leucine for 20 min and the subsequent incorporation of the label into virus proteins associated with cytoplasmic membrane and extracellular virus was determined. Initial experiments had been conducted previously with microsomes and a precursor-product relationship demonstrated between viral proteins in the microsomes and extracellular virus (CD. Richardson and D.E. Vance, J. Biol. Chem. 251, 5544-5550). Further studies were performed with endoplasmic reticulum and plasma membrane preparations. Maximum incorporation of [³H]leucine was observed in the viral proteins located in the endoplasmic reticulum at the end of a 20-min pulse period; greater than 50% of this radioactivity had disappeared within 2 hr. The plasma membrane fraction contained no radioactivity at the end of the pulse period; subsequently, maximal labeling of the viral proteins in the plasma membrane occurred 3 hr into the chase period, and these labeled proteins disappeared from this membrane 8.5 hr after the pulse. At 8.5 hr chase, maximum incorporation of the labeled proteins into extracellular virus was observed. These data are consistent with a precursor-product relationship between viral proteins in the endoplasmic reticulum, plasma membrane, and extracellular media. Viral proteins migrate to the plasma membrane and are subsequently incorporated into extracellular virus. All the radioactivity in the extracellular virus appears to have been derived from viral proteins associated with the plasma membrane of the cell. Therefore, mechanisms for the morphogenesis of Semliki Forest virus (in baby hamster kidney cells), other than budding from the plasma membrane, are unlikely to be of quantitative importance. The possibility that an intact cytoskeletal system might be required for the assembly of Semliki Forest virus was investigated. The microtubules and microfilaments of baby hamster kidney cells (BHK-21) were disassembled with specific drugs and the effect on production of extracellular virus was determined. Colchicine, Nocodazole, dibucaine, and cytochalasin B reduced the production of extracellular virus by 75-90%. Lumicolchicine had no effect on virus growth. Other control experiments showed no effect by these drugs on the incorporation of [³H]leucine of [³⁵S] methionine. At various times after addition of one of these drugs, the incorporation of the labeled precursors into viral proteins associated with endoplasmic reticulum or plasma membrane of the cell was evaluated. The results clearly show that the envelope and nucleocapsid proteins of the virus move to the plasma membrane of the cell where they accumulated. These studies strongly suggested that the cytoskeletal system was involved in the final stages of membrane assembly of Semliki Forest virus at the plasma membrane. Studies were also performed with the cross-linking agents -dimethylsuberimidate (DMS), dithiobis(succinimidyl propionate) (DSP), and dimethylthiobi's(propionimidate) (DTBP) . The proteins of purified virus and infected cells reacted with these agents and the cross-linked proteins were evaluated by one- and two-dimensional SDS electrophoresis. Nucleocapsid protein cross-linked to form up to pentameric complexes, and envelope proteins reacted to yield dimeric species. Nucleocapsid protein did not crosslink with envelope proteins. Cross-linking agents were also utilized to determine the effects of colchicine and dibucaine on the proximity of viral proteins to each other in the plasma membrane of the host cell. Colchicine (which disrupts microtubules) appeared to have no effect on the degree to which [³⁵S]-labeled virus proteins reacted with the agents, while dibucaine (which supposedly disrupts both microtubules and microfilaments) abolished envelope protein dimers dramatically. This result was interpreted to mean that microtubules may not be required for the formation of patches of virus proteins in the plasma membrane prior to budding, while microfilaments may play a more dominant role in this process.