HIV-1 nucleocapsid protein binds double-stranded DNA in multiple modes to regulate compaction and capsid uncoating.

Abstract

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral DNA genome is stably contained within a narrow capsid with diameter similar to the persistence length of double-stranded (ds) DNA, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates the DNA substrate in a non-specific binding mode that triggers uniform DNA self-attraction, condensing the DNA into a tight globule at a constant force up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to observe the activity of basic residue mutations of NC. All basic residue mutations lead to a defective interaction with the DNA substrate. Notably, the constant force “plateau” observed with wild-type NC is missing or largely diminished. However, the NC mutants that are still able to compact dsDNA, at least to some extent, create a DNA condensate that persists even at high forces in our stretching experiments. We investigate the nature of this high kinetic barrier for DNA decompaction, which may come from either direct NC-induced DNA crosslinking or, alternatively, from the steric entanglement of DNA in its compacted state. In either case, the slow kinetics of DNA decompaction from its NC-condensed state is likely responsible for the few hours delay between the completion of HIV-1 DNA synthesis inside the mature capsid, and subsequent capsid breaking, i.e. uncoating.