Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation

Abstract

For a virus-like particle (VLP) to serve as a delivery platform, the VLP must be able to release its cargo in response to a trigger. Here, we use a chemical biology approach to destabilize a self-assembling capsid for a subsequent triggered disassembly. We redesigned the dimeric hepatitis B virus (HBV) capsid protein (Cp) with two differentially addressable cysteines, C150 for reversibly crosslinking the capsid and C124 to react with a destabilizing moiety. The resulting construct, Cp150-V124C, assembles into icosahedral, 120-dimer VLPs that spontaneously crosslink via the C-terminal C150, leaving C124 buried at a dimer–dimer interface. The VLP is driven into a metastable state when C124 is reacted with the bulky fluorophore, maleimidyl BoDIPY-FL. The resulting VLP is stable until exposed to modest, physiologically relevant concentrations of reducing agent. We observe dissociation with FRET relaxation of polarization, size exclusion chromatography, and resistive-pulse sensing. Dissociation is slow, minutes to hours, with a characteristic lag phase. Mathematical modeling based on the presence of a nucleation step predicts disassembly dynamics that are consistent with experimental observations. VLPs transfected into hepatoma cells show similar dissociation behavior. These results suggest a generalizable strategy for designing a VLP that can release its contents in an environmentally responsive reaction.

Chemically Tagging Cargo for Specific Packaging inside and on the Surface of Virus-like Particles

“…In Cp150, all native cysteines are mutated to alanine (C48A, C61A, and C107A) and a C-terminal cysteine is appended; C150 can form disulfide cross-links to neighboring subunits. 24 Assembly of Cp150 is similar to that of Cp149, though yielding a higher fraction of T = 3 capsids (∼30%). 25 With HAP-GFP PEG4 and HAP-GFP PEG2 , the average binding maxima were 23 and 21, respectively, assuming 120-dimer capsids (by focusing on the 8.5 to 9.5 mL elution, in particular, the peak around 8.68 mL, we minimize contamination by T = 3 capsids).…”

“…To prevent capsid subunit rearrangement, we used Cp150 cross-linked capsids for subsequent experiments with preassembled capsids (Figures , S3). In Cp150, all native cysteines are mutated to alanine (C48A, C61A, and C107A) and a C-terminal cysteine is appended; C150 can form disulfide cross-links to neighboring subunits . Assembly of Cp150 is similar to that of Cp149, though yielding a higher fraction of T = 3 capsids (∼30%) .…”

“…32 A strategy has been developed that enables dissociation of an otherwise stable capsid to be triggered by intracellular redox potential. 24 Cp has two well-defined sites for epitope display: the exposed loops of the four-helix bundle at the intradimer interface and appended to the C-terminus. 33 A "split-core" strategy has been developed to enable the addition of very large inserts into the four-helix bundle.…”

Energetics and Kinetic Assembly Pathways of Hepatitis B Virus Capsids in the Presence of Antivirals

“…The ability to chemically trigger the dissociation of virus-like particles derived from HBV Core proteins has already been demonstrated. 45 Therefore, the assembly of highly stable cargo capsids used to deliver therapeutic molecules could be regulated by these assembly modulators.…”

“…Since the HBV Core protein can self-assemble in vitro , it constitutes a good cargo protein engineering system for the transport and delivery of large and small molecules. The ability to chemically trigger the dissociation of virus-like particles derived from HBV Core proteins has already been demonstrated . Therefore, the assembly of highly stable cargo capsids used to deliver therapeutic molecules could be regulated by these assembly modulators.…”