HIV-1 Nucleocapsid Protein Binds Double-Stranded DNA in Multiple Modes to Regulate Compaction and Capsid Uncoating.

Helena Gien,Mark C Williams,Jonathan P Kitzrow,Micah J Mccauley,Robert J Gorelick,Ioulia Rouzina,Karin Musier-Forsyth,Michael Morse

doi:10.3390/v14020235

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. In this study, we use optical tweezers, fluorescence imaging, and atomic force microscopy to observe NC binding a single long DNA substrate in multiple modes. We find 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. When NC is removed from solution, the globule dissipates over time, but specifically-bound NC maintains long-range DNA looping that is less compact but highly stable. Both binding modes are additionally observed using AFM imaging. These results suggest multiple binding modes of NC compact DNA into a conformation compatible with reverse transcription, regulating the genomic pressure on the capsid and preventing premature uncoating.

Highlights

HIV-1 nucleocapsid protein (NC) is essential to viral replication
A previous study utilized thin microchannels (100 by 150 nm) to isolate, straighten, and image single long DNA substrates in the presence of NC protein [23]. Imaging of both the DNA sparsely labeled with an intercalator dye and NC with a covalently-conjugated dye showed the formation of DNA/protein globules along the substrate that were associated with shortening of the DNA’s end-to-end length
We observed that at saturated NC binding, DNA was compacted into a maximum density globule, reaching a minimum volume limited by steric interaction

Summary

Introduction

HIV-1 nucleocapsid protein (NC) is essential to viral replication. The NC domain of Gag and its binding affinity to nucleic acids is essential for the assembly of immature viral particles (Figure 1). As the viral particle matures, Gag is cleaved multiple times, resulting in the release of mature viral proteins including NC (sometimes referred to as NCp7), which binds to and condenses the viral RNA (vRNA) inside the mature capsid. After degradation of the vRNA into 8–17 nucleotide (nt) long oligonucleotides by the RNaseH activity of reverse transcriptase, double-stranded (ds) plus-strand DNA ((+)DNA) is synthesized and the remaining vRNA dissociates from the (−)DNA template, resulting in the full length proviral dsDNA. The mature HIV-1 viral capsid is roughly conical in shape, measuring approximately 100 nm along its long axis and 50 nm in diameter.

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Conclusion