Efficient transcription, not translation, is dependent on adenovirus tripartite leader sequences at late times of infection

Abstract

To determine whether the tripartite leader is required for efficient translation in adenovirus-infected cells at late times of infection, we constructed recombinant adenoviruses containing the influenza virus nucleocapsid protein (NP) gene expressed under the control of the adenovirus major late promoter (MLP). We chose the NP gene because previous results showed that the influenza virus NP mRNA was an extremely effective initiator of translation in cells which were superinfected with influenza virus at late times of adenovirus infection (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986). The NP gene in the adenovirus recombinants was inserted downstream of an MLP that replaced part of the early (E1A) region. The resulting NP mRNAs either lacked any tripartite leader sequences or contained at their 5' ends various portions of the tripartite leader: 33, 172, or all 200 nucleotides of the leader. The relative amounts of the NP protein synthesized by the recombinants were directly proportional to the amounts of the NP mRNA made, indicating that the presence of 5' tripartite leader sequences did not enhance the translation of NP mRNA. In addition, the sizes of the polysomes containing NP mRNA were not increased by the presence of tripartite leader sequences, indicating that the initiation of translation was not enhanced by these sequences. On the other hand, the presence of tripartite leader sequences immediately downstream of the MLP did enhance the transcription of the inserted NP gene, as shown by Northern (RNA) analysis of in vivo NP mRNA levels and by in vitro runoff assays with isolated nuclei. Our results indicate that more than 33 nucleotides of the first leader segment of the tripartite leader are required for optimal transcription from the MLP.