Inhibition of henipavirus infection by Nipah virus attachment glycoprotein occurs without cell-surface downregulation of ephrin-B2 or ephrin-B3

Abstract

Nipah virus (NiV) and Hendra virus (HeV) are newly identified members of the family Paramyxoviridae and have been classified in the new genus Henipavirus based on unique genetic characteristics distinct from other paramyxoviruses. Transgenic cell lines were generated that expressed either the attachment protein (G) or the fusion protein (F) of NiV. Functional expression of NiV F and G was verified by complementation with the corresponding glycoprotein, which resulted in the development of syncytia. When exposed to NiV and HeV, expression of NiV G in Crandall feline kidney cells resulted in a qualitative inhibition of both cytopathic effect (CPE) and cell death by both viruses. RT-PCR analysis of surviving exposed cells showed a complete absence of viral positive-sense mRNA and genomic negative-sense viral RNA. Cells expressing NiV G were also unable to fuse with cells co-expressing NiV F and G in a fluorescent fusion inhibition assay. Cell-surface staining for the cellular receptors for NiV and HeV (ephrin-B2 and ephrin-B3) indicated that they were located on the surface of cells, regardless of NiV G expression or infection by NiV. These results indicated that viral interference can be established for henipaviruses and requires only the expression of the attachment protein, G. Furthermore, it was found that this interference probably occurs at the level of virus entry, as fusion was not observed in cells expressing NiV G. Finally, expression of NiV G by either transient transfection or NiV infection did not alter the cell-surface levels of the two known viral receptors. INTRODUCTIONNipah virus (NiV) and Hendra virus (HeV) are newly emergent members of the family Paramyxoviridae and have been classified in the newly created genus Henipavirus (Murray et al., 1995;Chua et al., 2000;Mayo, 2002). HeV was first described in 1995 in connection with an outbreak of severe pneumonia among stabled horses and also caused the deaths of two horse handlers in northern Australia (Rogers et al., 1996). NiV was subsequently identified in 1999 as the causative agent of an outbreak of severe encephalitis in peninsular Malaysia and Singapore (Philbey et al., 1998;Chua et al., 1999;Paton et al., 1999). A total of 265 human cases was documented, of which 105 patients succumbed to the disease. Recently, NiV has been identified as the causative agent of several smaller outbreaks of severe encephalitis in Bangladesh (Anonymous, 2003;Butler, 2004;Hsu et al., 2004) and India (Chadha et al., 2006). Under experimental conditions, NiV is able to infect cats, pigs, guinea pigs and hamsters (Middleton et al., 2002), whilst HeV infects horses, cats and bats (Williamson et al., 1998(Williamson et al., , 2000(Williamson et al., , 2001. In a natural setting, fruit bats from the genus Pteropus appear to be the reservoir species for NiV and HeV (Halpin et al., 2000). In the Malaysian outbreak, NiV was transmitted to pigs via an uncharacterized route, resulting in a high risk of NiV exposure for both pig farmers and abattoir workers from infected...

Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly

“…CDV H genes or gene fragments were amplified from the expression plasmid pCG-H5804PZeo (von Messling et al, 2005), while NiV G genes or gene fragments were amplified from the plasmid pczCFG5-NiV G (Sawatsky et al, 2007) and cloned into the Bam HI and Sph I sites of the pCG1-IRESzeomut expression plasmid (von Messling et al, 2003). FLAG epitope tags (DYKDDDDK) were introduced into the 39 primer immediately upstream of the stop codon of each gene to facilitate detection (Plemper et al, 2001).…”

“…The 39 primers for the NiV M gene contained the coding sequence for the c-myc epitope tag (EQKLISEEDL) immediately upstream of the stop codon (Salditt et al, 2010), while the corresponding primer for CDV M contained no tag. The CDV F gene was amplified from the expression plasmid pCG-F5804PZeo (von Messling et al, 2005), while the NiV F genes or gene fragments were amplified from the plasmid pczCFG5-NiV F (Sawatsky et al, 2007). The 39 primers for the CDV and NiV F expression plasmids both contained the coding sequence for the haemagglutinin epitope tag (HA, YPYDVPDYA).…”

Canine Distemper Virus Epithelial Cell Infection Is Required for Clinical Disease but Not for Immunosuppression

“…For the biochemical analysis of recombinant proteins, 12-well plates of VerodogSLAMtag cells were transfected at ap-proximately 90% confluence as described previously (26,27). Briefly, each well was transfected with 2 g each of the H expression plasmid pCG-H5804Pzeo (34) or the respective mutant H plasmid and a plasmid expressing either the MeV Edmonston (3) or CDV 5804P F protein by use of Turbofect (Fermentas).…”

Canine Distemper Viruses Expressing a Hemagglutinin without N-Glycans Lose Virulence but Retain Immunosuppression

“…For the biochemical analysis of recombinant proteins, 12-well plates of Vero dogSLAMtag cells were transfected at approximately 90% confluence as described previously (37). Briefly, each well was transfected with 2 g of the H expression plasmid pCG-H5804Pzeo or the respective mutant H plasmids using Lipofectamine 2000 (Invitrogen).…”