COMPOSITION, ASSEMBLY AND REPLICATION OF RNA VIRUSES

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Our laboratory will study the mechanism(s) by which the influenza A virus RNA-dependent RNA polymerase is modified to switch from transcription (synthesis of virus-specific mRNAs which are capped, methylated and polyadenylated) to replication (synthesis of full length (+) and (-) sense RNAs). We will take advantage of a recent major breakthrough which should enable us to reconstitute an in vitro RNA synthesis system containing influenza RNA (full length and truncated T7 transcripts) assembled with NP protein which serves as template for influenza transcriptase subunits purified from either virion RNPs or from Sf9 cells with high levels of expression from baculovirus recombinants. We will study the biochemical functions of the isolated purified polymerase subunits (PB1, PB2, PA), to assign cap endonuclease, casein kinase, ATPase, GTP-binding, initiation and elongation, and promoter binding functions. We will also study the interaction of the holoenzyme with the 3' termini of (+) and (-) sense influenza templates using synthetic oligoribonucleotides and reconstituted RNP complexes, and initiation and elongation by the enzyme. By using chimeric influenza RNA constructs containing the 3' untranslated region of one genome segment and the downstream coding sequence from another we will examine influenza transcriptional and translational controls in vitro. This reconstituted system for influenza RNA synthesis will also enable us to explore the error frequency of the influenza polymerase, and the specificities of influenza A and B which prohibit their reassortment. Using reconstituted RNPs which contain the influenza-specific 3' and 5' termini and an inserted reporter gene (luciferase) as ssRNA (-) sense constructs in RNP complexes we will transfect influenza virus (wt) infected cells to see if this reporter gene-containing construct can be transcribed, replicated and packaged into virions. Nuclear extracts from influenza virus infected-cells, treated with either cycloheximide or alpha-amanatin to inhibit replication or transcription respectively, and anti-sera against polymerase subunits and influenza nonstructural proteins will be utilized in an attempt to determine the nature of the enzyme modification(s) involved in the switch from transcription to replication and the possible involvement of cellular factors in this switch.