Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8

The role of viral protein (VP) 1 and VP2, which comprise the recombinant adeno-associated virus (rAAV) capsid, in heat-induced genome release was investigated using rAAV serotype 8 (rAAV8) samples with a high VP1/VP2 to VP3 ratio, a low VP1/VP2 to VP3 ratio, and VP3 only. The thermal unfolding of the VP1 N-termini was closely monitored by nano-differential scanning fluorimetry with an onset temperature (<i>T</i> _onset1) of ∼55°C and a melting temperature of ∼60°C (which was below the onset temperature of capsid disassembly [<i>T</i> _onset2] >70°C), which is related to genome release upon heating. The folded VP1 N-termini prevented release of the full-length genome at temperatures below 60°C, whereas unfolding of the VP1 N-termini facilitated genome release above 60°C. Above <i>T</i> _onset1 and below <i>T</i> _onset2, most rAAV8 particles remained as monomeric particles in three states: capsids encapsidating their single-stranded DNA (ssDNA), capsids that had fully released their genome, and capsids that had fully ejected the genome while tethering the genome on the capsid surface as evidenced by large frictional ratios in analytical ultracentrifugation. The ratio of VP1 and/or VP2 to total VPs had little effect on the extent of genome release. These findings provide new insights into heat-induced genome release from rAAV at the molecular level.