The Study of the HIV-1 5’-UTR Monomer-Dimer Equilibrium by Native Size Exclusion Chromatography
Briggs, Erran D. II
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In the late stages of its lifecycle, HIV selectively packages two copies of its unspliced RNA genome as a dimer. The mechanism by which this important step in the lifecycle occurs is not fully understood, however, interruption of the dimerization and selective packaging process results in noninfectious virion formation. Dimerization initiates at the 5’-untranslated region (5’-UTR) of the RNA genome. The HIV-1 5’-UTR exists in a concentration-dependent state of equilibrium between the monomer and dimer conformations. In this study, a novel size exclusion chromatography (SEC) methodology was developed to further characterize the monomer-dimer equilibrium by determining the equilibrium constant (Keq). Previous studies of the equilibrium using gel electrophoresis reported differences in dimerization behavior due to inconsistencies in gel and buffer conditions. The SEC method removes these concerns as it allows the equilibrium to be studied under physiological buffer conditions, which are universally defined as 140 mM KCl, 10 mM NaCl, and 1mM MgCl2. Additionally, using SEC allows for the system to be studied without the disruptive effects of applying a current to the sample. With this new method the equilibrium constant of the HIV-1 5’-UTR was determined to be 1.1 ± 0.1. Further development of this methodology will allow for the characterization of other retroviruses with 5’-UTRs, like HIV-2 and SIV.