Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis

Abstract

A structure-function analysis of the icosahedral RNA bacteriophage fr coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis. Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid. This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles indistinguishable morphologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their assembly properties investigated. The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of the fr CP. A third type of assembled structure was formed by a variant with a single amino acid substitution I104T. The alpha A-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E. coli. The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed.

The True Story and Advantages of RNA Phage Capsids as Nanotools

“…Recombinant fr CPs containing 2- to 12-aa-long additions to their N- or C-termini or insertions at position 50 in the RNA-binding region were capable of self-assembly, but this was not the case at positions 97-111 in the αA-helix [140]. The majority of other fr CP mutants demonstrated reduced self-assembly capabilities and formed either CP dimers (aa exchanges at residues 2, 10, 63, or 129) or both dimer and capsid structures (residue 2 or 69) [141]. The FG loop of the fr CP was also recognized as a potential target for insertions/replacements and was initially modified by a 4-aa-long deletion [142].…”

Evaluation of HBs, HBc, and frCP Virus-Like Particles for Expression of Human Papillomavirus 16 E7 Oncoprotein Epitopes

“…The recombinant HBs, HBc, and frCP proteins assemble into VLPs [28][29][30][31][32][33][34], and potentially can be configured as chimeric VLPs for expression of HPV16 E7 epitopes. To insert the E7 epitopes, we chose the regions within the HBs, HBc, and frCP proteins, which were predicted to be compatible with efficient self-assembly of the chimeric protein subunits and to allow surface localisation of the inserted heterologous epitopes ( fig.…”

“…It is known that inserting heterologous epitopes at the C-terminus of the HBc molecule (for a review, see Pumpens et al [41]) or the N-terminus of the frCP [34] is compatible with the self-assembly of chimeric VLPs. In the HBc carrier, the E7(35-98) fragment was introduced downstream from aa residue 144 in place of the Arg-rich C-terminus of the HBc molecule.…”

“…2E). It is likely that self-assembly of the frCP-E7(35-98) was blocked at the dimerization stage, as some of the N-terminal mutants of the frCP exist in dimeric form [34].…”

“…Our objective was to compare the expression of the HPV16 E7 protein region , which contains murine and human B cell [12,18], Th cell [12,[18][19][20], and CTL [21][22][23][24][25][26][27] epitopes, on three different VLP carriers: HBs [28,29], HBc [30][31][32], and frCP [33,34] for developing vaccine candidates.…”