X-ray crystallography and cryo-electron microscopy are to be used for structural studies of enveloped viruses and their self-assembled cores. The work will concentrate on alpha- (Sindbis, Semliki Forest, Ross River, Aura), rubella and flavi- (hepatitis C and yellow fever) viruses. Rubella virus is a major problem to the human fetus in pregnant mothers. Hepatitis C virus infections are a worldwide problem, affecting as much as 20% of the population in some countries. Alphaviruses can sometimes cause fatal diseases such as encephalitis fever and arthritis. Currently, little structural information is available about enveloped viruses. Structural studies are likely to provide understanding of the mechanisms by which these positive-stranded RNA viruses disassemble on cell entry and assemble prior to cell exit. Many laboratories have attempted to crystallize alphaviruses, but only very poorly diffracting crystals have been produced. We suspect that the probable cause for the crystalline disorder may be the presence of heterogeneous carbohydrate moieties. Hence, site-directed mutations are being made (in collaboration with Richard Kuhn) to eliminate or reduce the number of potential glycosylation sites on the surface glycoproteins of Sindbis virus. The most difficult and time-consuming part of the work will be in sample preparation involving site-specific mutagenesis to deglycosylate the surface glycoproteins to obtain homogeneous samples of in vitro assembled cores. Although the electron microscopic, image reconstruction and crystallographic studies will take time and effort, the technologies are well established. We have attained good expression of Sindbis virus, Ross River virus, rubella virus and hepatitis C virus capsid protein in E. coli, although crystallization of the capsid protein has so far been achieved for only Sindbis virus. Mutational and structural studies of these proteins will be pursued similar to those previously used in the analysis of Sindbis virus. Self-assembled core-like particles using various nucleic acids, including genomic RNA and a DNA 48-mer, have been obtained for all the above mentioned capsid proteins. Cryo-electron microscopy studies and crystallization trials are underway. Reproducible small crystals (less than or equal to 0.05 mm diameter) of the in vitro assembled Sindibis cores can be produced routinely.
Showing the most recent 10 out of 52 publications
