Strategies for Packaging Macromolecules onto or into Virus-Like Particles

There is an unmet need to develop vehicles for delivering protein cargo and ribonucleoprotein complexes to cells. Virus capsids, or virus-like particles, have numerous advantages as self-assembling protein platforms, but there remains a need to develop robust methods for the specific internal packaging of cargo. In this work, we use the Hepatitis B Virus (HBV) capsid as our delivery vehicle. To specifically associate protein cargo with the capsid, we covalently attached a derivative of an HBV sulfamoylbenzamide (SBA) antiviral to green fluorescent protein (GFP). We show that SBA-tagged GFP can associate with HBV capsids, but efficient internal packaging requires engineering the assembly of capsids around a cargo. Under standard co-assembly conditions, SBA-tagged GFP predominantly displayed on the capsid exterior, reaching ∼40 GFP cargo proteins per capsid. We tested two strategies to drive encapsidation of protein cargo. First, remodeling the capsid interior was nominally effective. Conversely, His-tag/Ni2+-mediated clustering converts the cargo into a multivalent capsid-binding scaffold that templates assembly, enabling internal packaging of up to ∼30 cargo proteins per capsid. Based on capsid geometry, we suggest the practical limit is approximately 30 cargo proteins per T = 4 HBV capsid, indicating that this strategy approaches maximal loading. Together, these results provide a highly effective method for packaging proteins inside VLPs.