Quaternary structure is critical for protein display on capsid-like particles (CLPs): efficient generation of hepatitis B virus CLPs presenting monomeric but not dimeric and tetrameric fluorescent proteins.

Self-organizing assemblies such as viral capsids may be used as symmetrical molecular platforms for the display of heterologous sequences, with applications ranging from vaccines to structural studies. The 183-amino-acid hepatitis B virus (HBV) core protein assembles spontaneously into icosahedral capsid-like particles (CLPs). The most exposed, and most immunogenic, substructure on the CLPs is a small loop that connects two long antiparallel alpha-helices which act as dimerization interface. Ninety (90) or 120 dimers multimerize into the capsid; the four-helix bundles formed by the dimers protrude as spikes from the surface. We recently demonstrated that the entire enhanced green fluorescent protein (eGFP) can be inserted into this loop, yielding CLPs that natively displayed eGFP on their surface. The central location of the insertion site requires that any insert be fixed to the carrier via both termini, with corresponding restrictions regarding insert size and structure. eGFP obviously satisfied these criteria but, surprisingly, all attempts to produce CLPs with the isostructural red fluorescent proteins DsRed1, DsRed2, and HcRed failed. Suspecting their oligomerization tendency to be responsible, we generated fusions containing instead monomeric yellow, cyan, and red fluorescent proteins (mYFP, mCFP and mRFP1). This strongly increased the yields of YFP and CFP-CLPs, and it allowed for the first time to efficiently generate red fluorescent CLPs. Hence insert quaternary structure is a highly critical factor for CLP assembly. These data have important implications for the rational design of self-assembling fusion proteins.