Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end.

Abstract

The first step in the replication of influenza virion RNAs is the synthesis of full-length transcripts of these RNAs. The synthesis of these transcripts, or template RNAs, requires: (i) unprimed initiation rather than the capped RNAprimed initiation used during viral mRNA synthesis, and (it) antitermination at the polyadenylylation site used during mRNA synthesis. To determine the mechanism of template RNA synthesis, we prepared nuclear extracts from infected cells that were active in the synthesis of both template RNAs and viral mRNAs. By providing the dinucleotide ApG as primer, we circumvented the inefficient unprimed initiation catalyzed by these extracts and, as a consequence, were able to focus on the antitermination step. Antitermination, and hence template RNA synthesis, occurred when ApG but not a capped RNA was used as primer, indicating that the presence of a 5' capped end blocked antitermination at the 3' end of the transcript. Ultracentrifugation of the nuclear extract yielded a pellet fraction that contained viral nucleocapsids active in viral mRNA synthesis but not template RNA synthesis and a supernatant fraction that contained the antitermination factor. When the supernatant, which had essentially no activity by itself, was added to the pellet in the presence of ApG, template RNA synthesis was restored. Depletion experiments in which this supernatant was incubated with protein A-Sepharose containing antibodies to individual viral proteins demonstrated that the viral nucleocapsid protein was required for antitermination. The implications of these results for the control of viral RNA replication are discussed.The synthesis of influenza virus mRNA is primed by capped RNA fragments derived from host-cell RNA polymerase II transcripts (1-3). These primers are generated by a viral cap-dependent endonuclease that cleaves RNA polymerase II transcripts 10-13 nucleotides from their 5' ends preferentially at a purine residue. Viral mRNA chains are elongated until a stretch of uridine residues is reached 17-22 nucleotides before the 5' end of virion RNA (vRNA) is reached, where transcription terminates and poly(A) is added to the mRNAs (4, 5). Viral mRNA synthesis is catalyzed by nucleocapsids from purified virions (2), and some of the roles of the constituent nucleocapsid-associated polymerase (P) proteins (PB1, PB2, and PA) have been delineated by previous experiments (6-8).For replication to occur, an alternative type of transcription is required that results in the production of full-length copies of vRNA. These copies can then serve as the templates for new vRNA. The full-length transcripts, or template RNAs, are initiated without a primer and are not terminated at the poly(A) site used during mRNA synthesis (4, 9). In vivo, the switch from viral mRNA to template RNA synthesis requires the synthesis of one or more virus-specific proteins (9, 10).To identify these proteins and to determine their roles in the switch from viral mRNA to template RNA synthesis, it is necessary to establish an in vit...

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