Enveloped RNA viruses are the etiological agents for several important human diseases, including acquired immune deficiency syndrome (Human Immunodeficiency Virus), influenza (Influenza Virus), lower respiratory disease in children (Respiratory Syncytial Virus) and dengue fever (Dengue Virus). Although these viruses are distinct with regard to the structure of their RNA genome and employ different strategies in their replication in host cells, they share several activities and properties in common. One of these occurs in the final stages of virus replication when virus nucleocapsids or nucleoproteins interact with virus-specific transmembranal glycoproteins to initiate the assembly of newly replicated viruses. This end-stage of the virus replication cycle is clearly essential for virus growth and spread, yet little is known about the detailed biochemistry of enveloped virus assembly. Much of the experimental work proposed in this grant application focuses on virus assembly and utilizes a model virus system which has proved to be highly amenable to a molecular biology study of virus replication. The virus is Sindbis, an alphavirus whose complete genome of 11,700 nucleotides has been cloned as a cDNA which, upon in vitro transcription, produces a precise copy of the genome RNA that is infectious in tissue culture cells and gives rise to progeny virus. The power of this system lies in the ability to prepare site-directed mutations in those regions of the virus genome which code for proteins that function in virus assembly. Experiments performed thus far with this system have yielded mutants whose phenotypes are indicative of defects in very late stages of virus replication and affect the budding process. I propose to continue and extend these studies in order to obtain a molecular description of virus nucleocapsid interactions with glycoproteins. A new collaborative project will be initiated. Its objective is to obtain a detailed atomic structure of intact Sindbis virus derived from analyses of diffraction patterns from virus crystals. Results from these studies are expected to reveal new information about enveloped virus structure and assembly and should offer new insights into the design of effective antiviral agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019494-20
Application #
3128844
Study Section
Experimental Virology Study Section (EVR)
Project Start
1982-04-01
Project End
1994-12-31
Budget Start
1992-01-01
Budget End
1992-12-31
Support Year
20
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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Harrison, S C; Strong, R K; Schlesinger, S et al. (1992) Crystallization of Sindbis virus and its nucleocapsid. J Mol Biol 226:277-80
Collier, N C; Knox, K; Schlesinger, M J (1991) Inhibition of influenza virus formation by a peptide that corresponds to sequences in the cytoplasmic domain of the hemagglutinin. Virology 183:769-72
Gaedigk-Nitschko, K; Schlesinger, M J (1991) Site-directed mutations in Sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding. Virology 183:206-14
Gaedigk-Nitschko, K; Ding, M X; Levy, M A et al. (1990) Site-directed mutations in the Sindbis virus 6K protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure. Virology 175:282-91
Gaedigk-Nitschko, K; Schlesinger, M J (1990) The Sindbis virus 6K protein can be detected in virions and is acylated with fatty acids. Virology 175:274-81
Ding, M X; Schlesinger, M J (1989) Evidence that Sindbis virus NSP2 is an autoprotease which processes the virus nonstructural polyprotein. Virology 171:280-4
Wen, D; Ding, M X; Schlesinger, M J (1986) Expression of genes encoding vesicular stomatitis and Sindbis virus glycoproteins in yeast leads to formation of disulfide-linked oligomers. Virology 153:150-4
Wen, D Z; Schlesinger, M J (1986) Regulated expression of Sindbis and vesicular stomatitis virus glycoproteins in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 83:3639-43