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

Abstract

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 (T _onset1) of ∼55°C and a melting temperature of ∼60°C (which was below the onset temperature of capsid disassembly [T _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 T _onset1 and below T _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.

Keywords: adeno-associated virus; analytical ultracentrifugation; gene therapy; genome release; hydrogen/deuterium exchange mass spectrometry; mass photometry; nano-differential scanning fluorimetry; thermal unfolding.

Graphical abstract

Figure 1

Characterization of rAAV8 particles in low-density and high-density fractions after two cycles of CsCl DG-UC and rAAV8 particles composed of VP3 only (A) CE-SDS electropherograms of VPs incorporated into the capsids for low-density, high-density, and VP3-only particles. (B) VP stoichiometric ratio for low-density, high-density, and VP3-only particles. Each error bar represents the standard deviation (SD) of triplicate measurements. (C) CE-LIF electropherograms of ssDNA encapsidated in low-density, high-density, and VP3-only particles. (D) Orbitrap-based CD-MS distributions for low-density, high-density, and VP3-only particles.

Figure 2

Observation of heat-induced genome release from rAAV8 FP_LD, FP_HD, and FP_VP3only (A) MP histograms of Benzonase-untreated and Benzonase-treated FP_LD and FP_VP3only after incubation on ice (control), and at 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C. The blue and red lines represent areas of EPs and FPs, respectively. (B and C) EP ratio of (B) Benzonase-untreated and (C) Benzonase-treated FP_LD, FP_HD, and FP_VP3only after incubation on ice (control), and at 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C. Each error bar represents the SD of triplicate measurements.

Figure 3

Binding and unbinding properties of rAAV8 FP_LD and FP_VP3only upon heating (A) MP histograms (expanded for the unbinding side in Figure 2A) for FP_LD and (B) FP_VP3only after incubation on ice (control), and at 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C. (C and D) The ratio of unbinding and binding particles to the total particle count for (C) FP_LD and (D) FP_VP3only after incubation on ice (control), and at 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C. Each error bar represents the SD of triplicate measurements.

Figure 4

Thermal stabilities of rAAV8 FP_LD, FP_HD, FP_VP3only, and EP; the binding properties of anti-VP1 antibody to FP_LD, FP_VP3only, and EP; and the thermal-induced structural changes in rAAV8 (A) Nano-DSF thermograms showing the F₃₅₀/F₃₃₀ of FP_LD, FP_HD, and FP_VP3only. (B) The fitting result of the first transition in the FP_LD. (C) Nano-DSF thermogram of EP. (D–F) MP histograms of (D) FP_LD, (E) FP_VP3only, and (F) EP with and without incubation with anti-VP1 (A1) antibody after heating at 60°C. (G–I) The changes in deuterium uptake by the representative peptides of (G) Q101–L131 in the VP1u region, (H) acetylated A205–W229 in the 5-fold axis, and (I) I562–E578. Each error bar represents the SD of triplicate measurements. (J–L) The differences in deuterium uptake between the control and heated rAAV8s were mapped onto (J) the three-dimensional structure of the VP1 N-termini, (K) the shared VP3 region, and (L) capsids. Compared with control rAAV8s, the red regions show a significant increase in deuterium uptake (>3.281 Da) in heated rAAV8s. Regions where peptides were not detected are shown in gray.

Figure 5

Particle distributions in the rAAV8 sample solutions upon heating Sedimentation profiles of (A) Benzonase-untreated and (B) Benzonase-treated rAAV8 solutions after incubation on ice (control) (top), 50°C (middle), and 60°C (bottom). The blue and red lines represent the profiles obtained at an absorbance of 230 and 260 nm, respectively.

Figure 6

Characterization of the commercial rAAV8 sample (A and B) CsCl DG-UC equilibration profiles of the commercial rAAV8 sample after the (A) first cycle and (B) the second cycle. The red and blue lines represent the profiles obtained at an absorbance of 260 and 280 nm, respectively. (C) CE-LIF electropherogram of ssDNA encapsidated in OPs in fractions 41–42 after the first cycle of CsCl DG-UC. (D) MP histograms of fraction 33 after the first cycle (top), fractions 26–27 after the second cycle (upper middle), fractions 28–29 after the second cycle (lower middle), and fractions 41–42 after the first cycle (bottom).

Figure 7

Observation of heat-induced genome release from the fractionated rAAV8 sample (A) MP histograms of Benzonase-treated rAAV8 samples after incubation on ice (control), and at 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C. The blue, red, and green lines represent the areas of EPs, FPs, and OPs, respectively. (B) The ratio of each particle to total particles in the rAAV8 sample with or without Benzonase treatment after incubation on ice (control), and at 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C. Each error bar represents the SD of triplicate measurements. (C) CE-LIF electropherograms of ssDNA encapsidated in FPs and OPs before (top) and after (bottom) incubation at 60°C.