Mechanism of HIV-1 NC Protein-Induced Condensation of Double Stranded DNA as a Model for DNA Compaction during Reverse Transcription

Abstract

Genomic RNA is packaged into the HIV-1 virion and reverse transcribed within the mature capsid into double stranded proviral DNA (vDNA) in the presence of ∼10 millimolar HIV-1 nucleocapsid protein (NC). NC is a small, highly basic protein known to condense all nucleic acids upon saturated binding. NC-induced condensation of vDNA is critical for maintaining viral capsid integrity during reverse transcription, ensuring its completion. However, how NC condenses double stranded DNA (dsDNA) is unknown. Here, we use optical tweezers to stretch single dsDNA molecules in the presence of wild-type HIV-1 NC and observe NC-induced dsDNA condensation. Above a critical concentration of NC, reducing the extension of the stretched dsDNA molecule at a constant rate reveals stepwise changes in extension along a constant-force plateau. The quantized nature of these steps suggests winding of dsDNA into a compact toroidal globule via stepwise accumulation of dsDNA in loops. We observe changes in the condensing force and kinetics of condensation and decondensation by varying NC concentration and salts, mimicking the process that occurs during capsid uncoating and gradual NC loss. Combining the results of these stretching experiments with fluorescence imaging allows us to visualize the NC-induced formation of DNA toroids in real time. These results help us understand the state of vDNA both within the capsid and after capsid uncoating.