The strategy of replication of influenza virus will be investigated. The mechanism of synthesis of the co-linear and interrupted mRNAs derived from viral genome RNA segments 7 and 8 will be emphasized. Our available evidence indicates that the interrupted mRNAs are produced by a splicing mechanism and we shall attempt to investigate this further using a variety of experimental systems. From these studies significant information should be gained on the biogenesis and control processes involved in the formation of the interrupted mRNAs of influenza virus. In addition, we hope to develop a simple model system for examining mRNA biosynthesis in eukaryotic cells. We will try to learn about the functions of the virus specific polypeptides NS1, NS2, and M2 using ts mutants and cell fractionation techniques and search for a peptide that may be translated from mRNA3 derived from influenza virus RNA segment 7. We will investigate whether protein synthesis is necessary for influenza virus mRNA splicing, determine the site of synthesis of the processed mRNAs and look for the conservation of methyl groups on the precursor mRNAs during the formation of processed mRNAs. We will determine the processing potential of the mRNAs using an SV40/influenza virus recombinant system and attempt to develop an in vitro splicing system. In addition, we will determine the structure and organization of RNA segments 7 and 8 of influenza B virus and their mRNAs and examine the expression of cloned DNA of the neuraminidase to investigate the role of a possible N-terminal membrane attachment region. Important comparative data will also be obtained in the course of these experiments such that we can look for regions of homology of difference between the nucleotide and protein sequences of the influenza A and B viruses.
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