A catalytically and genetically optimized beta-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 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.
Wolf
Mike C
MC
Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA 90095. mikewolf@ucla.edu
Wang
Yao
Y
Freiberg
Alexander N
AN
Aguilar
Hector C
HC
Holbrook
Michael R
MR
Lee
Benhur
B
D000818:Animals; D003264:Containment of Biohazards / Q000191:economics / Q000379:methods; D005452:Fluoresceins / Q000378:metabolism*; D017930:Genes, Reporter; D045403:Henipavirus / Q000502:physiology*; D007769:Lactams / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D022701:Virosomes / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
C109764:CCF 2; D005452:Fluoresceins; D007769:Lactams; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D022701:Virosomes; D001618:beta-Lactamases