The translational apparatus in cell-free extracts prepared from L cells infected with reovirus undergoes a time-dependent transition from cap dependence to cap independence. Extracts from uninfected L cells translate capped reovirus mRNA at high efficiency and synthesize the expected three size classes of reovirus polypeptides, and the translation is sensitive to m7G(5')ppp. This same extract translates uncapped mRNA at a much lower efficiency. In contrast, extracts from infected L cells translate uncapped reovirus mRNA at high efficiency and synthesize the correct three size classes of polypeptides, and the translation is not sensitive to inhibition by m7G(5')ppp. Infected cell extracts translate capped mRNA at reduced efficiency (z25%), the translation is not sensitive to inhibition by m7G(5')ppp, and the correct three size classes of viral polypeptides are not synthesized. These observations may explain how reovirus takes over the host translational apparatus.the viral mRNA in this process. The recent development of a method for making efficient cell-free protein-synthesizing systems of extremely low background activity from L cells (14) has enabled us to compare the in vitro translation of exogenously supplied reovirus mRNA in extracts that have been prepared from infected and uninfected L cells. Using extracts from infected cells, we found that the translation of uncapped reovirus mRNA is highly efficient and that the ability of these extracts to translate this form of reovirus mRNA increases with time of infection. Our observations lead us to conclude that the host cell translational machinery becomes modified as a result of viral infection. A model is proposed for the reovirus-induced takeover of the host cell translation machinery, in which uncapped viral mRNAs are preferentially translated at late times during infection.
Reovirus guanylyltransferase, studied as a covalent enzyme-GMP intermediate, was used to guanylate appropriate acceptor molecules in vitro to produce authentic cap structures. Guanylyltransferase activity was associated with A2, the 140-kilodalton product of the L2 gene segment of reovirus serotypes 1 and 3. The proteins of reovirus are arranged as a double capsid structure. The inner capsid, or core, is composed of six viral proteins and contains the 10 double-stranded RNA (dsRNA) genome segments. All of the enzymes required for transcription and capping of viral mRNAs are associated with the viral core particle (1, 2, 7, 9, 11, 20, 22). Assignment of specific enzymatic activities to viral proteins has proven difficult, as solubilization of the core proteins invariably results in the loss of their enzymatic activities (31). Drayna and Fields (6) tentatively assigned the viral transcriptase activity to the core polypeptide A3 by using a genetic approach. In the present report, we used a direct biochemical approach to assign the reovirus guanylyltransferase activity to the core polypeptide X2. Shuman and Hurwitz (24) demonstrated that the guanylyltransferase reaction of vaccinia virus proceeds via a covalent enzyme-guanylate intermediate. A similar reaction mechanism has been demonstrated for the guanylyltransferase enzymes from a variety of organisms (10, 12, 15, 17, 19. 23, 28, 29, 32). Results obtained in our laboratory (35) and results obtained by Shatkin et al. (21) suggested that the * Corresponding author. This work was supported by grant MT-4594 from the Medical Research Council. Ottawa, Ontario. Canada. D.R.C. was a recipient of studentships from Fonds Formation de Chercheurs et Actions Concertees, Quebec. and the McGill McConnell Foundation. H.Z. was a recipient of a studentship from Fonds de la Recherche en Sante du Quebec.
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