DNA Interactions Kinetics of HIV-1 Nucleocapsid Proteins by Single Molecule DNA Stretching

Abstract

The human immunodeficiency virus type 1 (HIV-1) Gag protein is essential for retroviral assembly. During viral maturation, Gag is processed to form matrix (MA), capsid (CA), and nucleocapsid (NC). NC is initially cleaved into NCp15, then NCp9, and finally NCp7. NCp7 functions as a nucleic acid chaperone during retroviral replication, in which it rearranges nucleic acids to facilitate reverse transcription and recombination. The role of Gag cleavage intermediates in facilitating nucleic acid remodeling is not well understood, although it is likely that they also function as chaperones during viral assembly and early reverse transcription steps. We use single molecule stretching to probe the DNA interactions of these three NC protein forms. In the presence of NCp7, the DNA elongates at a lower force almost reversibly, demonstrating that NCp7 facilitates structural transitions in DNA with rapid kinetics. To quantitatively probe the DNA annealing kinetics after the DNA is destabilized by force, we monitor the DNA length at constant force as it relaxes towards equilibrium. The time scale of annealing is found to be tens of seconds, suggesting that, while the kinetics of NCs interactions are faster than many single-stranded DNA binding proteins, the final structural rearrangement is only moderately fast. These results also suggest NCp7 acts as a weak intercalator, allowing the two DNA strands to remain together as the dsDNA structure is destabilized, likely facilitating nucleic acid rearrangements. Somewhat different behavior is observed when DNA is stretched and released in the presence of NCp9 and NCp15, and we will apply this method to quantitatively determine the differences in DNA interaction kinetics for all three HIV-1 NC proteins. This work was funded in part by Federal Funds from NCI, NIH under contract HHSN261200800001E (RJG).