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A catalytically and genetically optimized β-lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics

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 ful...

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Detalles Bibliográficos
Autores principales: Wolf, Mike C, Wang, Yao, Freiberg, Alexander N, Aguilar, Hector C, Holbrook, Michael R, Lee, Benhur
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727953/
https://www.ncbi.nlm.nih.gov/pubmed/19646266
http://dx.doi.org/10.1186/1743-422X-6-119
Descripción
Sumario: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 β-lactamase-Nipah Matrix (βla-M) fusion protein. We first codon-optimized the bacterial βla and the NiV-M genes to ensure efficient expression in mammalian cells. The βla-M construct was able to bud and form virus-like particles (VLPs) that morphologically resembled paramyxoviruses. βla-M efficiently incorporated both NiV and HeV fusion and attachment glycoproteins. Entry of these VLPs was detected by cytosolic delivery of βla-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 βla increased the sensitivity of our βla-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.