A catalytically and genetically optimized beta-lactamase-matrix based assay for sensitive, specific, and higher...

Nipah virus (NiV) and Hendra virus (HeV) are the only paramyxoviruses requiring Biosafety Level 4 (BSL-4) containment. Thus, study of henipavirus entry at less than BSL-4 conditions necessitates the use of cell-cell fusion or pseudotyped reporter virus assays. Yet, these surrogate assays may not fully emulate the biological properties unique to the virus being studied. Thus, we developed a henipaviral entry assay based on a beta-lactamase-Nipah Matrix (betala-M) fusion protein. We first codon-optimized the bacterial betala and the NiV-M genes to ensure efficient expression in mammalian cells. The betala-M construct was able to bud and form virus-like particles (VLPs) that morphologically resembled paramyxoviruses. betala-M efficiently incorporated both NiV and HeV fusion and attachment glycoproteins. Entry of these VLPs was detected by cytosolic delivery of betala-M, resulting in enzymatic and fluorescent conversion of the pre-loaded CCF2-AM substrate. Soluble henipavirus receptors (ephrinB2) or antibodies against the F and/or G proteins blocked VLP entry. Additionally, a Y105W mutation engineered into the catalytic site of betala increased the sensitivity of our betala-M based infection assays by 2-fold. In toto, these methods will provide a more biologically relevant assay for studying henipavirus entry at less than BSL-4 conditions.