Peptide‐Controlled Access to the Interior Surface of Empty Virus Nanoparticles

Abstract

The structure of Cowpea mosaic virus (CPMV) is known to high resolution, thereby enabling the rational use of the particles in diverse applications, from vaccine design to nanotechnology. A recently devised method for the production of empty virus-like particles (eVLPs) has opened up new possibilities for CPMV capsid-based technologies, such as internal mineralisation of the particle. We have investigated the role of the carboxyl (C) terminus of the small coat (S) protein in controlling access to the interior of CPMV eVLPs by determining the efficiency of internal mineralisation. The presence of the C-terminal 24-amino acid peptide of the S protein was found to inhibit internal mineralisation, an effect that could be eliminated by enzymatic removal of this region. We have also demonstrated the amenability of the C terminus to genetic modification. Substitution with six histidine residues generated stable particles and facilitated external mineralisation by cobalt. These findings demonstrate consistent internal and external mineralisation of CPMV, and will aid the further exploration and development of the use of eVLPs for bionanotechnological and medical applications.

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

“…In freshly prepared samples of eVLPs and virions, which were dialyzed into 50 mM potassium phosphate buffer (pH 7.0), both the slow and fast forms are present (Figure 5A). However, within 2–3 weeks after purification of the eVLPs stored at 4°C, the S subunit contains mostly the fast (cleaved) form (Sainsbury et al, 2011). The extent of proteolysis is also found to be different based on the type of plant host used for the expression of CPMV or eVLPs, such as cowpea ( Vigna unguiculata ) compared with N. benthaminana (Figure 5B).…”

“…More recently, these applications have turned to using eVLPs rather than the infectious virus (Lebedev et al, 2016; Sainsbury et al, 2011, 2014; Wen et al, 2012). Therefore, it is of importance to structurally characterize eVLPs and analyze any significant differences that occur between the wild-type virus and eVLPs of CPMV.…”

“…These can be generated by co-expression of the CP precursor VP60 along with the 24K viral proteinase in Nicotiana benthamiana (Montague et al, 2011; Saunders et al, 2009). CPMV eVLPs generated this way have already proven useful as reagents for bio- and nanotechnology applications (Lebedev et al, 2016; Sainsbury et al, 2011, 2014; Wen et al, 2012). …”

“…The full-length L and S subunits contain 374 and 213 aa, respectively. It has been shown that the intact C terminus of the S subunit is required for the efficient assembly of both CPMV virions and eVLPs (Sainsbury et al, 2011; Taylor et al, 1999). After particle assembly, the surface-exposed C terminus is proteolysed up to residue Leu563 (189) (Taylor et al, 1999).…”

“…In the virion crystal structure (PDB: 1NY7), the last visible C-terminal residue of the S subunit, Leu563 (189) is partially exposed (Lin et al, 1999) (we have chosen to use the continuous numbering system for the S subunit to be consistent with the sequence databases, instead of the old numbering that is shown in parentheses and starts from residue number 1, which was used in the description of the virion structure; Lin et al, 1999). However, the rate of loss of the C-terminal amino acids appears to be slower with eVLPs than with virus (Sainsbury et al, 2011), raising the possibility that some of residues may be visible in the X-ray structure of eVLPs.…”