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.
