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.

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Hepatitis B virus core protein self-assembles into icosahedral, highly immunogenic capsid-like particles (CLPs) that can serve as molecular platforms for heterologous proteins. Insertion into the centrally located c/e1 epitope leads to surface display, fusion to the C terminus to internal disposition of the foreign domains. However, symmetry-defined space restrictions on the surface and particularly inside the CLPs limit the size of usable heterologous fusion partners. Further, CLPs carrying differing foreign domains are desirable for applications such as multivalent vaccines, and for structure probing by distance sensitive interactions like fluorescence resonance energy transfer (FRET). Here, we report an in vitro co-assembly system for such mosaic-CLPs allowing successful CLP formation with a per se assembly-deficient fusion protein, and of CLPs from two different fluoroprotein-carrying fusions that exert FRET in an assembly-status dependent way.

Hepatitis B viruses, or hepadnaviruses, are small DNA-containing viruses that replicate through reverse transcription. Their prototype, HBV, causes severe liver disease in humans. The hepadnaviral P protein is an unusual reverse transcriptase (RT) that initiates DNA synthesis by host-factor-dependent protein priming on a specific RNA stem-loop template, epsilon, yielding a short DNA oligonucleotide covalently attached to the RT. This priming reaction can be reconstituted with in vitro-translated duck hepatitis B virus (DHBV) P protein. No direct structural data are available for any P protein. However, P proteins share a number of conserved motifs with other polymerases. Box A contains an invariant bulky residue recently shown to be crucial for dNTP versus NTP discrimination in RTs and some DNA polymerases; its equivalent in DHBV P protein would be phenylalanine 451 (F451). Four mutants, containing glycine (F451G), alanine (F451A), valine (F451V) and aspartate (F451D), were therefore analyzed for their ability to utilize dNTPs and NTPs in in vitro priming. Priming efficiencies with dNTPs decreased with decreasing side chain size but GTP utilization increased; the wild-type enzyme was inactive with GTP. In the context of complete DHBV genomes, all mutant proteins were competent for RNA encapsidation, indicating the absence of global structural alterations. Because the function of the discriminatory residue depends on its specific spatial disposition this strongly suggests a similar architecture for the P protein dNTP-binding pocket as in other RTs.

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