APPROACHES TO DETERMINE STOICHIOMETRY OF VIRAL ASSEMBLY COMPONENTS

Due to the rapidity of biological reactions, it is difficult to isolat e intermediates or to determine the stoichiometry of participants in i ntermediate reactions. Instead of determining the absolute amount of e ach component, this study involved the use of relative parameters, suc h as dilution factors, percentages probabilities, and slopes of titrat ion curves, that can be more accurately quantified to determine the st oichiometry of components involved in bacteriophage phi 29 assembly, T his work takes advantage of the sensitive in vitro phage phi 29 assemb ly system, in which 10(8) infectious virions per mi without background can be assembled from eight purified components, It provides a conven ient assay for quantification of the stoichiometry of packaging compon ents, including the viral procapsid, genomic DNA, DNA-packaging pRNA, and other structural proteins and enzymes, The presence of a procapsid binding domain and another essential functional domain within the pRN A makes it an ideal component for constructing lethal mutants for comp etitive procapsid binding. Two methods were used for stoichiometry det ermination, Method 1 was to determine the combination probability of m utant and wild-type pRNAs hound to procapsids. The probability of proc apsids that possess a certain amount of mutant and a certain amount of wild-type pRNA, both with an equal binding affinity, was predicted wi th the binomial equation [GRAPHICS] where Z is the total number of pRN As per procapsid, M is the number of mutant pRNAs bound to one procaps id, and ((Z)(M)) is equal to Z!/M!(Z-M)!. With various ratios of mutan t to wild-type pRNA in in vitro viral assembly, the percent mutant pRN A versus the yield of virions was plotted and compared to a series of predicted curves to find a best fit, It was determined that five or si x copies of pRNA were required for one DNA-packaging event, while only one mutant pRNA per procapsid was sufficient to block packaging, Meth od 2 involved the comparison of slopes of curves of dilution factors v ersus the yield of virions, Components with known stoichiometries serv ed as standard controls, The larger the stoichiometry of the component , the more dramatic the influence of the dilution factor on the reacti on, A slope of 1 indicates that one copy of the component is involved in the assembly of one virion. A slope larger than 1 mould indicate mu ltiple-copy involvement, By this method, the stoichiometry of gp11 in phi 29 particles was determined to be approximately 12, These approach es are useful for the determination of the stoichiometry of functional units involved in viral assembly, be they single molecules or oligome rs, However, these approaches are not suitable for the determination o f exact copy numbers of individual molecules involved if the functiona l unit is composed of multiple subunits prior to assembly.