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Journal article
A catalytically and genetically optimized β-lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics
Virology journal, Vol.6(1), pp.119-119
2009
Handle:
https://hdl.handle.net/2376/102665
PMCID: PMC2727953
PMID: 19646266
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 β la 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.
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Details
- Title
- A catalytically and genetically optimized β-lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics
- Creators
- Mike C Wolf - Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA 90095Yao Wang - Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA 90095Alexander N Freiberg - Department of Pathology, University of Texas, Medical Branch, UTMB, Galveston, TX, USA 77555Hector C Aguilar - Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA 90095Michael R Holbrook - Department of Pathology, University of Texas, Medical Branch, UTMB, Galveston, TX, USA 77555Benhur Lee - Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA 90095
- Publication Details
- Virology journal, Vol.6(1), pp.119-119
- Academic Unit
- Paul G. Allen School for Global Animal Health
- Publisher
- BioMed Central
- Number of pages
- 11
- Grant note
- UCLA Warsaw Fellowship AI069317; AI060694; AI070495; AI059051; AI07323; CA16042; AI28697 / NIH; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA P30CA016042 / NATIONAL CANCER INSTITUTE; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI) U54AI065359 / NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Allergy & Infectious Diseases (NIAID)
- Identifiers
- 99900546794301842
- Language
- English
- Resource Type
- Journal article