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    Virol J. 2009 Jul 31;6:119.

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

    Wolf MC, Wang Y, Freiberg AN, Aguilar HC, Holbrook MR, Lee B.

    Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA 90095. mikewolf@ucla.edu

    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.

    PMID: 19646266 [PubMed - indexed for MEDLINE]

    PMCID: PMC2727953

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