Sialic acid biophore directed design of enhanced affinity HN protein from Newcastle disease virus.

The hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is a multifunctional protein that mediates virus attachment to host sialic acid-containing receptors, exhibits neuraminidase activity, and promotes membrane fusion. Notably, purified HN protein also targets sialic acids on malaria-parasitised red blood cells (PRBCs), thereby reducing parasite viability, and is thus a potential therapeutic molecule against the malaria parasite. To gain further insights into the structural and functional basis of its sialic acid recognition, we performed comparative analyses of the sialic acid-binding pocket across different viral lectins. It revealed a Type-VI sialic acid-binding module in HN, characterised by fewer interactions with the functional groups of sialic acid compared to Type-I influenza neuraminidases (NA). Introduction of a rationale-based specific mutation (I175Y) in the central binding pocket of HN transformed its sialic acid-binding module from Type-VI to Type-I. It resulted in significantly higher affinity for sialic acid, accompanied by a substantial loss in its neuraminidase activity. This disruption in the delicate functional balance between receptor-binding and enzymatic cleavage highlights a trade-off between optimising one activity at the cost of another. Biologically, the mutant HN exhibited enhanced hemadsorption, or cell-binding activity, and thus showed greater ability to block NDV replication in both cell monolayers and in-ovo systems. The mutant protein also demonstrated an increased capacity to target PRBCs and showed potent anti-plasmodial activity, compared to the wild-type. This enhanced targeting capability highlights the mutant as a promising candidate for developing precision drug delivery systems for malaria.