The paramount objective is to generate and purify deletion mutants of poliovirus that have lost part of the 5'-end of the viral chromosome and determine whether the original chromosomal region for the initiation of protein synthesis is conserved. Mutants will be generated by passage of virus at ratios of 300/cell in Human Skin and Muscle Cells. Viral passages will be monitored by measuring infectious yield/cell and densities of progeny virions. Thus far with 40 passages, viral particles less dense than parent virus are produced. Since densities of the deletion mutants (less dense particles) are only 0.01 g/cc less than parent virions further passages will be made in an attempt to isolate deletion mutants at least 0.02 g/cc less than parent virus. Chromosomal locations of deletions will be established. Oligonucleotide mapping with mutants and complete virions will be done. A second series of studies will measure changes in polysome bound protein kinase after infection of HeLa cells and Vero cells with poliovirus. Polivirus blocks cellular protein synthesis within 2 hours after infection and it is possible that protein phosphorylation or dephosphorylation is involved.
NIH ExPorter
NIH ExPorter