MACROMOLECULAR SYNTHESIS IN VIRUS-INFECTED CELLS

将试图回答几个有关的性质的问题

Attempts will be made to answer several questions concerning the nature of the functions encoded in the reovirus genome and concerning reovirus multiplication. They are: a. Reovirus morphogenesis will be estudied in vivo by analyzing the nature of the RNA and protein components of progressively more mature immature reovirus particles, starting with the first complexes into which the ten individual species of ssRNA molecules are assembled. It will also be studied in vitro by reacting various combinations of native reovirus proteins with ssRNA molecules and isolating and characterizing the complexes that result. b. Native reovirus proteins will be examined for possession of the five enzyme activities that are exhibited by reovirus cores. Such proteins will be isolated from bacteria transformed by expression vectors into which reovirus genes have been cloned. c. Attempts will be made to elucidate the functions of reovirus proteins by examining in detail the multiplication cycles of ts mutants at nonpermissive temperatures. Mutants with lesions in each of the ten genes are available. Multiplication cycles will be analyzed in terms of the kinetics of synthesis of ss and dsRNA species and reovirus proteins, as well as of the various particles on the pathway of reovirus morphogenesis. d. The various reovirus messenger RNA species are not translated with equal efficiency. The importance of regions upstream from and around initiation codons as elements that control efficiency of translation will e investigated by introducing mutations, deletions and insertions into them. be. The evolutionary and functional relationships of the three reovirus serotypes will be studied by sequencing cognate sets of their genes and examining cognate sets of their proteins for similarity of tertiary structure and for the nature of their antigenic and functional domains. f. The mode of the anti-reovirus activity of ribavirin, and various pure interferon species will be examined by determining which reactions of the reovirus multiplication cycle they inhibit. Hopefully, these results can be extended and applied to infections by rotaviruses and orbiviruses of medical importance.