Vesicular Stomatitis Virus N Protein-specific Single-domain Antibody Fragments Inhibit Replication

Hanke L, et al., 18(6):1027-1037, EMBO Rep, 2017

Vesicular stomatitis virus (VSV) single-stranded RNA genome is tightly encapsidated by nucleoprotein N to form a nucleocapsid (N-RNA). N-RNA serves as the template for RNA synthesis. In the absence of nucleoprotein N, full-length RNAs will not be produced and therefore nucleoprotein N is considered as a target of intervention of the virus life cycle. Described herein is the use of cytosolically expressed variable region of the heavy chain of camelid heavy-chain-only antibodies (VHHs) to target the viral nucleoprotein N. Four of the VHHs identified are specific for VSV. Affinity and kinetic parameters were determined by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 instrument equipped with Streptavidin Biosensor probes was used to perform all BLI experiments. Biosensor tips were loaded with biotinylated WT and escape mutant versions of N-RNA variants. Association and dissociation of VHHs were studied over a series of concentrations. Experimental data obtained were fitted using the 2:1 heterogeneous ligand binding global fit model. kon, koff, and KD values were determined. The binding sites on nucleoprotein N for some of the identified VHHs were characterized and the information was used to present a molecular explanation for the inhibitory effects of the N-specific VHHs. Overall, these findings reveal new features on the N protein surface that may have the potential to be used as an antiviral intervention options.

Read More