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.

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Reovirus progeny subviral particles were isolated from L-cells at late times postinfection. It has been shown (D. Skup and S. Miliward, J. Virol. 34: 490-496, 1980) that these progeny subviral particles have masked capping enzymes, indicating that mRNA synthesized by these particles should be uncapped. When progeny subviral particles were used for mRNA synthesis in vitro, they failed to incorporate the /8-phosphate of [f8-32P]GTP into the 5' terminal. Direct analysis of reovirus mRNA synthesized by progeny subviral particles in the presence of either [a-32P]GTP or [a-32P]CTP indicated that the 5' terminal was uncapped, having the structure pGpC... The implications of this finding to the reovirus replicative cycle are discussed.

BSTRACTMicrococcal nuclease was used to eliminate endogenous protein synthesis in extracts prepared from L cells. The nuclease can be inhibited subsequently with 2'-deoxythymidine-3', 5'-diphosphate. Nuclease-treated extracts primed with exogenous reovirus mRNA, synthesized full length polypeptides with linear kinetics for almost two hours leading to stimulation of the order of 104 times over endogenous background. On the average, between 40 and 50 molecules of polypeptide were synthesized per molecule of mRNA.

We examined the enzyme activities associated with progeny subviral particles isolated from L-cells infected with reovirus at 12 h postinfection. Activities normally present in reovirus cores were also found to be present in the progeny subviral particles, with the exception of the capping enzymes. The methylase and guanyl transferase activities, which constitute the capping system, were present in a masked form that could be activated by chymotrypsin digestion. The appearance of these progeny subviral particles in infected cells coincided with the time when mRNA synthesis was maximal, suggesting that viral mRNA synthesized at later times is uncapped.