Exploring the Landscape of the PP7 Virus-like Particle for Peptide Display

Abstract

Self-assembling virus-like particles (VLPs) can tolerate a wide degree of genetic and chemical manipulation to their capsid protein to display a foreign molecule polyvalently. We previously reported the successful incorporation of foreign peptide sequences in the junction loop and onto the C-terminus of PP7 dimer VLPs, as these regions are accessible for surface display on assembled capsids. Here, we report the implementation of a library-based approach to test the assembly tolerance of PP7 dimer capsid proteins to insertions or terminal extensions of randomized 15-mer peptide sequences. By performing two iterative rounds of assembly-based selection, we evaluated the degree of favorability of all 20 amino acids at each of the 15 randomized positions. Deep sequencing analysis revealed a distinct preference for the inclusion of hydrophilic peptides and negatively charged amino acids (Asp and Glu) and the exclusion of positively charged peptides and bulky and hydrophobic amino acid residues (Trp, Phe, Tyr, and Cys). Within the libraries tested here, we identified 4000 to 22,000 unique 15-mer peptide sequences that can successfully be displayed on the surface of the PP7 dimer capsid. Overall, the use of small initial libraries consisting of no more than a few million members yielded a significantly larger number of unique and assembly-competent VLP sequences than have been previously characterized for this class of nucleoprotein particle.

Keywords: molecular library; peptide display; selection; self-assembly; virus-like particles.

Figure 1

Assembly-based selection of PP7 virus-like particles bearing randomized loop insertions or C-terminal peptide extensions.

Figure 2

Representative analyses of particles isolated from loop insert and C-terminal extension PP7 libraries. (a) TEM images: (left, with expanded section) G1 loop NNS library with monomeric and dimeric VLPs labeled in white and yellow arrows, respectively; (middle) G1 C-term NNS extension library; (right) G1 loop VNS library. (b) Mass distributions of particles isolated from the G1 loop NNS library with the mass ranges of monomeric CPs (left) and dimeric CPs (right), shown separately. (c) Microfluidic gel electrophoresis analysis of G1 loop and C-term NNS libraries. (d–f) Mass distributions of particles isolated from G1 C-term NNS and close-up of the 28–32 kDa mass range, G1 loop VNS (e), and G1 C-term VNS (f) libraries. (g) SEC trace of the G1 loop VNS library (in solid lines) compared to wild-type PP7VLP (dotted lines).

Figure 3

Average hydropathy (a) and charge (b) of 15-mer peptides in each VLP library. T1 = generation 1 transformation library (possible sequences expressed in E. coli), C1 = generation 1 cDNA library (particles recovered and sequenced), and T2 and C2 = transformation and cDNA libraries for generation 2, respectively.

Figure 4

Favorability, measured by log₂ of the abundance ratio (cDNA library/transformed library) of each amino acid at each position of the full 15-mer peptides. (a) Generation 1 (G1) loop NNS, (b) G1 C-term NNS, (c) G1 loop VNS, (d) G1 C-term VNS. Amino acids are grouped by their properties indicated by the color of the single-letter code (hydrophobic = black, polar/uncharged = blue, basic and positively charged = green, acidic and negatively charged = red, aromatic = purple, and thiol containing = pink). The data are plotted here on a common heat map scale to enable comparisons between libraries. For representations of the data using individual minimum and maximum values, see Figure S11.

Figure 5

Cryo-EM analysis of the PP7 loop and C-term libraries. (a) PP7 dimer coat proteins with C-term NNS or VNS extension (left), with uninterrupted loop NNS or VNS (middle), or with a stop codon in the loop resulting in two PP7 monomer subunits with C-termini extensions (right). (b) Selected cryo-EM 2D class averages corresponding to T = 4, T = 3, and non-icosahedral cages. (c) Molecular models of T = 4 and T = 3 icosahedral cages built from the EM maps obtained after 3D refinement of selected particles.

Figure 6

Relationship between the assembly state of PP7 dimer variants selected from the G2 libraries and their frequency in the G2 cDNA library (a), hydrophobicity (b), and charge (c). The solid black lines mark the median (yes = isolated in significant quantities upon individual expression; no = little or no particles isolated upon individual expression). (d) Relationship between hydropathy and charge of the added peptide in successful (blue, significant yield) and unsuccessful (orange, insignificant or no yield) particles. The dashed line is an approximate boundary between a successful and unsuccessful combination of properties.