Crystal Structure of Měnglà Virus Nucleoprotein Bound by a Poorly Cross-Reactive Anti-Marburg Virus Nanobody Highlights a Single Amino Acid Affinity Switch, a Feature Also Evident in Dehong Virus

Although Marburg and Měnglà viruses occupy different geographies and genera of the family <i>Filoviridae</i>, genetic and host similarities suggest spillover potential for the latter. While Marburg virus causes transmissible and often fatal hemorrhagic fever in humans, the pathogenicity of Měnglà virus is unknown. Understanding antibody cross-reactivity between the two viruses appears prudent in preparation for detecting the new virus and facilitating component-based studies of replication. Previously, while nanobodies to the monomeric nucleoprotein C-terminal domain (NPCTD) of Marburg virus recognized Měnglà virus NPCTD, cross-reactivity was too weak to quantify monovalent equilibrium concentrations. Here, using oligomeric NP in a nanobody-driven sandwich assay, the cross-reactivity deficit was essentially negated, suggesting we would be able to detect both viruses equally. Curious as to why monovalent reactivity was so disparate, we crystallized the Měnglà virus NPCTD-nanobody complex for X-ray crystal structure determination. Comparative analysis of the antibody-antigen interfaces revealed bonded and nonbonded opportunities at one location in the Marburg complex that were absent in the Měnglà complex. Mutagenesis of the NPCTDs, to make Marburg more Měnglà-like (H690N) and Měnglà more Marburg-like (N692H), resulted in dramatic ablation and restoration of nanobody binding, respectively, via changes in off-rates. Similar trends were observed for the more recently discovered Dehong virus, and dimeric enzymatic and fluorescent reporter fusions improved NP recognition potency within blotting and cell probing assays. Understanding the structural basis for cross-reactivity helps predict the likelihood of detecting viral variants based upon genomic sequence information and can inform the design of antibodies with broader recognition potential.