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

Mike C. Wolf, Yao Wang, Alexander Freiberg, Hector C. Aguilar, Michael R. Holbrook, Benhur Lee

Research output: Contribution to journalArticle

22 Citations (Scopus)

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.

Original languageEnglish (US)
Article number119
JournalVirology Journal
Volume6
DOIs
StatePublished - 2009

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Henipavirus
Nipah Virus
Hendra Virus
Virion
Viruses
Virus Attachment
Cell Fusion
GTP-Binding Proteins
Codon
Catalytic Domain
Glycoproteins
Mutation
Antibodies
Infection
Genes
Proteins

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

A catalytically and genetically optimized β-lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics. / Wolf, Mike C.; Wang, Yao; Freiberg, Alexander; Aguilar, Hector C.; Holbrook, Michael R.; Lee, Benhur.

In: Virology Journal, Vol. 6, 119, 2009.

Research output: Contribution to journalArticle

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