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

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.

Detection of Receptor-Induced Glycoprotein Conformational Changes on Enveloped Virions by Using Confocal Micro-Raman Spectroscopy

“…The incorporation of M, F, and G into the virus-like particles was confirmed by electron microscopy (uranyl acetate and phosphotungstic acid staining, as described previously [38]) and by Western blot analysis using specific anti-AU1 and anti-hemagglutinin (HA) tag antibodies at 1:2,000 and 1:4,000 dilutions, respectively, as previously described (28,29).…”

“…PCDNA3.1 plasmids expressing the NiV-M (38), NiV-F, and NiV-G (39) codon-optimized genes were previously constructed and reported to express relatively high levels of their respective proteins (29,34,38,39). The green fluorescent protein (GFP) expression plasmid was also previously reported (13,36).…”

“…NiV VLPs were produced in different cell lines, CHO, 293T, or Vero cells, as previously described (38). Briefly, cells were transfected with equal amounts of codon-optimized NiV-M, -F, and -G expressed in PCDNA3.1 expression plasmids, and viruses were purified from the viral supernatants, as described below.…”

“…We and others previously reported that NiV VLPs can be purified from supernatants of mammalian cells transiently expressing NiV-M, -F, and -G (38,(49)(50)(51). Thus, we produced and purified NiV VLPs by transfecting 293T cells with equal amounts of codon-optimized NiV-M, -F, and -G expression plasmids, just as previously reported (38).…”

A broad-spectrum antiviral targeting entry of enveloped viruses

“…Because the viral envelope appears to remain functionally intact, we investigated if LJ001 inhibits virus-cell fusion. To do so, we developed a NiV matrix-based virus-like particle entry assay in which viruscell fusion is mediated by cognate NIV-F and -G proteins, and entry is monitored only by cytosolic delivery of a reporter protein fused to the NiV matrix protein, circumventing the need for viral transcription or translation (32). Fig.…”