HOST-VIRUS INTERACTION AND GENE EXPRESSION

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The long term goal of the present proposal is to understand the molecular basis of pathogenicity of the respiratory pathogen, human parainfluenza virus 3 (HPIV3), a member of the family paramyxoviridae. The parainfluenza viruses are important respiratory tract pathogens, second to respiratory syncytial virus, as causative agents of common illnesses of children like rhinitis, pharyngitis, and bronchitis. Studies in different parts of the world indicate that these viruses are associated with approximately 40% of severe croup in children. HPIV3 is most pathogenic among the other HPIV types. Effective vaccines to control infection in children have so far been inadequate. Although considerable information regarding the structure and the nucleotide sequence of the genome of HPIV3 is known, our knowledge of the mechanism of gene expression of this important human pathogen is meager. To gain insight into understanding the functions of the viral genes, we recently developed an in vitro transcription system for HPIV3 which efficiently synthesizes virus-specific mRNAs in vitro. More importantly, we demonstrated that cellular actin was needed for effective transcription in vitro to occur, suggesting a possible involvement of cytoskeletal network in the life-cycle of HPIV replication. To delineate the mechanism of gene expression of HPIV3 and study the role of cellular cytoskeletal protein(s) in this process, we propose to study in detail the role of cellular actin and actin-binding proteins in HPIV3 transcription/replication as well as the functional roles of viral RNA polymerase proteins (L and P proteins) in these processes. An in-depth study would be carried out to determine the molecular form of actin required for transcription and whether any other cellular proteins, e.g. actin-binding proteins, are necessary for actin to function. In addition, mode of action of actin in RNA synthesis as well as association of actin with viral nucleocapsid in vitro and in vivo will be studied. The functions of viral RNA polymerase components (L and P proteins) in transcription and replication and their interaction with cellular actin will be studied. Emphasis would be laid on the viral P gene which encodes, in addition to the P protein, C protein and P-D protein, the latter protein arising by a novel editing mechanism. Understanding the functions of these proteins and the unique editing reaction during P gene transcription along with the role of actin in these processes would certainly provide deeper insight into the mechanism of gene expression of HPIV3 and HPIV3-cell protein interactions.