| Autor: |
Holbrook Michael R, Aguilar Hector C, Freiberg Alexander N, Wang Yao, Wolf Mike C, Lee Benhur |
| Jazyk: |
angličtina |
| Rok vydání: |
2009 |
| Předmět: |
|
| Zdroj: |
Virology Journal, Vol 6, Iss 1, p 119 (2009) |
| Druh dokumentu: |
article |
| ISSN: |
1743-422X |
| DOI: |
10.1186/1743-422X-6-119 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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