Methodology

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

Mike C Wolf¹ , Yao Wang¹ , Alexander N Freiberg⁴ , Hector C Aguilar¹ , Michael R Holbrook⁴ and Benhur Lee^1,2,3

¹  Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA 90095

²  Department of Pathology and Laboratory Medicine, UCLA, Los Angeles, CA, USA 90095

³  UCLA AIDS Institute, UCLA, Los Angeles, CA, USA 90095

⁴  Department of Pathology, University of Texas, Medical Branch, UTMB, Galveston, TX, USA 77555

author email corresponding author email

Virology Journal 2009, 6:119doi:10.1186/1743-422X-6-119

Published:	31 July 2009

Abstract

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.