Investigating the genome packaging mechanism of influenza A virus. (360G-Wellcome-086157_Z_08_Z)

Influenza A virus genome consists of eight segments, all of which encode essential gene products; thus, one copy of every segment needs to be incorporated for the virus to be viable (1- 3]. Genome packaging of influenza A virus is thought to rely on interactions between the packaging signals found in the eight segments which have been proposed to be arranged in a 7+1 array within the virion. The ultimate goal of this project is to test the 7+1' array hypothesis, to identify the putative order of segments in the 7+1 conformation and to define the inter-segment interactions important for packaging of the virus genome. Specifically, there are three aims: 1) Each viral segment has packaging signals at the tenmini. Previous work suggests that certain combinations of packaging mutations in different segments affect virus fitness to a much greater degree. Thus, the first aim of the project is to identify which pairwise mutant segment combinations have a greater effect on virus viability in order to determine which interactions are the most important; this will suggest the position of the segments within the 7+1 array. 2) Previous studies have shown that packaging mutants can regain fitness by generating 'pseudorevertants', i.e. viruses which acquire compensatory mutations without reverting the original ones. This project aims to investigate the 'pseudorevertants' from double packaging mutants which would allow identification of important intersegment interactions. 3) Certain packaging double mutants are lethal. It is possible that absence of crucial intersegment interactions would result in disruption of virus budding. This project will examine the hypothesis that certain double packaging mutants have a budding deficiency.