Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells

Abstract

Virus-like particles (VLPs) constitute large, polyvalent platforms onto which a wide variety of functional units can be grafted. Their use in biological settings often depends on their specific binding to cells or receptors of interest; this can be compromised by excessive nonspecific association with other cells. We found that lysine residues mediate such nonspecific interactions, presumably by virtue of protonation and interaction with anionic membrane lipid headgroups and/or complementary residues of cell surface proteins and polysaccharides. Chemical acylation of surfaceexposed amines of the Qβ VLP led to a significant reduction in the association of particles with mammalian cells. Single-point mutations of particular lysine residues to either glutamine, glutamic acid, tryptophan, or phenylalanine were mostly well-tolerated and formed intact capsids, but the introduction of double and triple mutants was far less forgiving. Introduction of glutamic acid at position 13 (K13E) led to a dramatic increase in cellular binding, whereas removal of the lysine at position 46 (K46Q) led to an equally striking reduction. Several plasma membrane components were found to specifically interact with the Qβ capsid irrespective of surface charge. These results suggest that specific cellular interactions are engaged or obviated by such mutations and provide us with more "benign" particles to which can be added binding functionality for targeted delivery applications.

Virus-like Particles: Fundamentals and Biomedical Applications

“…For VLP separation, CGE has been manipulated to characterize the process and formulation of the component protein sizes and ratios of VLP-based vaccines derived from Western, Eastern, and Venezuelan equine encephalitis (WEVEE) viruses [ 147 ]. Moreover, microfluidic gel electrophoresis has been used to characterize Q β -VLP mutants (i.e., K16F and K16Y), who manifested varying degrees of self-assembly and interactions with plasma membrane components [ 148 ]. The M w of VLPs is inspected with other analytical techniques such as charge detection mass spectrometry (CDMS).…”

Fitness Landscape-Guided Engineering of Locally Supercharged Virus-like Particles with Enhanced Cell Uptake Properties

“…Thus, the effects of additional positive surface charge on the internalization of MS2 capsids into mammalian cells were both position- and residue-dependent. Previous work on the effects of single mutations on VLP properties found that specific lysine-to-glutamine (K > Q) and lysine-to-glutamate (K > E) point mutants drastically altered Qβ binding to mammalian cells …”

“…Previous work on the effects of single mutations on VLP properties found that specific lysine-to-glutamine (K > Q) and lysine-to-glutamate (K > E) point mutants drastically altered Qβ binding to mammalian cells. 32 To explore the position and residue effects on uptake further, we created MS2 capsid variants representing every single and double mutant combination of arginine and lysine at backbone positions 71 and 73, located at the FG loop. This panel of eight MS2 CP variants was expressed, confirmed to assemble, and labeled with fluorescein for internalization experiments (Figures S3 and S4 and Table S1).…”

Protein Cages: From Fundamentals to Advanced Applications

“…Possible changes might include targeted mutations to provide reactive handles at all symmetry-related sites in the shell or to fine-tune the physicochemical properties of single residues or entire surface patches. 426 Remodeling or replacement of surface loops has also been used to confer novel molecular recognition capabilities to capsids (Figure 15). 427−431 Another common strategy involves appending short peptide tags or entire folded proteins to the N-or C-termini to functionalize capsules with specific binding motifs or catalytic functions (Figure 15).…”

“…Available sequence and structure information typically serves as the basis for identifying potential sites for modification. Possible changes might include targeted mutations to provide reactive handles at all symmetry-related sites in the shell or to fine-tune the physicochemical properties of single residues or entire surface patches . Remodeling or replacement of surface loops has also been used to confer novel molecular recognition capabilities to capsids (Figure ).…”