CLONING AND EXPRESSION OF INFLUENZA VIRAL RNA SEGMENTS

流感病毒是正黏液病毒，是人类和动物的主要类群

Influenza viruses orthomyxoviruses, a major group of human and animal pathogens, are a group of segmented enveloped negative strand RNA viruses. These viruses assemble and bud at the level of plasma membrane, specifically on the apical side of the polarized epithelial cells. The long term goal of this project define the processes of viral assembly and budding. The specific objectives in the proposed project are to examine the functions of M1 and NA in the assembly and budding processes. Since influenza virus replication occurs in the cell's nucleus and budding takes place at the plasma membrane, how the viral RNP gets in and out of the nucleus and how dissociation and association of M1 facilitates the nuclear entry and exit of vRNP will be examined. Proposed experiments will determine if M1 becomes dissociated from the viral RNP during uncoating and if the preexisting M1 will interfere with the nuclear transport of the incoming vRNP. In addition, these experiments will determine if vRNP made in the absence of M1 is exported out of the nucleus into the cytoplasm or if M1 is needed for vRNP's exit from the nucleus. Furthermore the domains of M1 involved in interaction with vRNP will be delineated. Since M1 is juxtaposed between the viral envelope and RNP, the protein-protein interactions between M1 and the transmembrane viral proteins like HA, NA or M2 and the domains of each of these proteins involved in the interactions will be defined. Since viral particles bud from the apical domain of plasma membrane in polarized cells, structural features present in these transmembrane proteins, particularly in NA, responsible for targeting the protein to the apical plasma membrane, will be determined. NA is a type II membrane protein. Little is known about the sorting signals for polarized transport of type II protein in general and NA in particular. Chimeric constructions and deletions and mutations will be used to define sorting signals of NA. Experiments proposed here will elucidate the steps involved in uncoating as well as assembly and budding and will be useful in designing the antiviral agents to block these steps in viral replication.