Shaw (University of Kentucky) EPSCOR 9726966 Uncoating of a Helical Virus: Cotranslational and Coreplicational Disassembly Mechanisms 1. Technical Of all the stages of the infection process by an animal or plant virus, uncoating of the viral genome or disassembly of the virus particle is one of the least understood. This study examines tobacco mosaic virus (TMV), as its positive-sense RNA genome is enclosed (encapsidated) in rod-shaped particles. TMV particles undergo bidirectional disassembly after being introduced into plant protoplasts. The viral RNA is initially uncoated in the 5'-to-3' direction and the process is completed by the removal of coat protein molecules (subunits) in the 3'-to-5' direction. Two hypotheses are set forth to account for the bidirectional disassembly: (1) in vivo uncoating begins by a cotranslational disassembly mechanism in which the removal of subunits in the 5'-to-3' direction is coincident with ribosome translocation during translation of the first ORF of the viral RNA; (2) in vivo uncoating is completed by a coreplicational disassembly mechanism in which the viral polymerase proteins (encoded by the first ORF and/or its readthrough sequence) are involved in the removal of subunits in the 3'-to-5' direction and the synthesis of progeny minus-strand viral RNA molecules. These hypotheses are tested by determining the behavior of various TMV mutants. The possibility that regions in the polymerase proteins that are not involved in replication may be required for 3'-to-5' disassembly will also be explored. The significance of this study lies in its atttempt to understand a stage of life in which the ultimate success or failure of a virus to initiate infection in host cells is determined. 2. Non-technical Particles of tobacco mosaic virus (TMV) disassemble in inoculated plant cells, which allows the genome to be released so that the production of progeny virus particles can proceed, inducing the pathogenic state in the host organism. In this process, about 75% of the subunits is first removed, sequentially, beginning at one end of the viral genome; the remainder is then removed proceeding from the other end. The first step may involve concomitant synthesis of viral proteins, and the other may involve the production of copies of the viral RNA. Experiments are designed to examine the validity of each of these hypothetical mechanisms.
