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1 National Microbiology Laboratory, Canadian Science Centre for Human and Animal Health, 1015 Arlington Street, Winnipeg, MB R3E 3R2, Canada
2 Department of Medical Microbiology, University of Manitoba, 730 William Avenue, Winnipeg, MB R3E 0W3, Canada
3 National Centre for Foreign Animal Disease, Canadian Science Centre for Human and Animal Health, 1015 Arlington Street, Winnipeg, MB R3E 3R2, Canada
Correspondence
Markus Czub
m.czub{at}ucalgary.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; 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, 2000, 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 pigs (Chew et al., 2000; Parashar et al., 2000). In the more recent outbreaks in Bangladesh, NiV infection appears to have been acquired from sources other than pigs, involving fruit bats or another unidentified intermediary, and person-to-person transmission was also a prominent feature of this outbreak (Hsu et al., 2004), in contrast to the Malaysian outbreak in 1998–1999 (Mounts et al., 2001).
NiV and HeV have two major surface glycoproteins, a typical feature of members of the family Paramyxoviridae. The attachment glycoprotein (G) is responsible for attachment to the host-cell receptors, two of which have been identified as ephrin-B2 and ephrin-B3 (Bonaparte et al., 2005; Negrete et al., 2005, 2006). The fusion glycoprotein (F) mediates fusion between the viral and host-cell membranes. In other related paramyxoviruses, close interaction between these two glycoproteins generally is required for fusion to occur (Sakai & Shibuta, 1989; Ebata et al., 1991; Morrison et al., 1991; Wild et al., 1991; Horvath et al., 1992; Hu et al., 1992; Tanabayashi et al., 1992; Cattaneo & Rose, 1993; Bousse et al., 1994; Heminway et al., 1994; Nishio et al., 1994; Bagai & Lamb, 1995; Bar-Lev Stern et al., 1995), although this requirement is not absolute for some species (Kahn et al., 1999; Mizuguchi et al., 1999; Seth & Shaila, 2001). NiV and HeV glycoproteins presumably share a certain number of functional domains with other paramyxovirus glycoproteins, and previous studies have demonstrated that heterologous NiV and HeV glycoproteins are able to complement each other functionally in the induction of membrane fusion and that neither glycoprotein on its own is fusogenic (Bossart et al., 2001, 2002; Tamin et al., 2002). However, when any of the NiV or HeV glycoproteins are co-expressed with other complementary paramyxovirus glycoproteins, such as those of Measles virus (MeV), no fusion results (Bossart et al., 2002). The lack of fusogenic activity for any of the henipavirus glycoproteins on their own, coupled with the lack of compatibility with other paramyxovirus glycoproteins, indicates that there are specific domains within the NiV and HeV glycoproteins that interact and allow the glycoproteins to carry out fusion between the host cell and viral membranes.
Expression of glycoproteins from some retroviruses such as Avian leukosis virus or Murine leukemia virus has been shown to render otherwise susceptible cells refractive to infection (Czub et al., 1995; Hunt et al., 1999; Ponferrada et al., 2003). This phenomenon is known as viral receptor interference. Due to interactions between the viral attachment glycoprotein and the endogenous host-cell receptor, functional receptor molecules appear to be neither on the cell surface nor available for virus attachment (Delwart & Panganiban, 1989; Welstead et al., 2004). Receptor interference has been characterized for gammaherpesviruses (Geraghty et al., 2000) and a number of retroviruses (Czub et al., 1995; Potash & Volsky, 1998; Lyu et al., 1999; Ponferrada et al., 2003), as well as for several other virus families (Karpf et al., 1997; Marschall et al., 1997) but among paramyxoviruses has only been investigated in some detail for Human parainfluenza virus 3 (Horga et al., 2000), Mumps virus (Hishiyama et al., 1996) and MeV (Schneider-Schaulies et al., 1995a, b; Welstead et al., 2004). Recently, it has also been demonstrated that a soluble HeV G molecule consisting of the ectodomain of the protein is able to bind to the cell surface and competitively inhibit NiV and HeV infection (Bossart et al., 2005). As viral interference may be a useful mechanism to combat viral infections, possibly including those with NiV and HeV, we generated cell lines expressing the viral proteins G and F. After exposure to henipaviruses, transgenic cells expressing the NiV G attachment glycoprotein were resistant to infection with NiV and HeV. Our results demonstrated that a viral interference system can be established for these members of the family Paramyxoviridae and that expression of the attachment glycoprotein did not result in cell-surface downregulation of the viral receptors.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Generation of NiV F and G expression plasmids.
The NiV F and G genes (GenBank accession no. AF212302
[GenBank]
) were amplified by PCR from viral cDNA, cloned into pBK-CMV (Stratagene) using the NheI and HindIII (New England Biolabs) sites downstream of the multiple cloning site and sequenced. The retroviral vectors pczCFG5 IEGZ and pHITBE were kindly provided by D. Lindemann (Würzburg, Germany) (Lindemann et al., 2001). pczCFG5 IEGZ and pHITBE both have multiple cloning sites immediately upstream of an internal ribosome entry site (IRES) derived from either Encephalomyocarditis virus for pczCFG5 IEGZ or immunoglobulin heavy chain-binding protein (BiP) for pHITBE. Green fluorescent protein (GFP) genes are located immediately downstream of the IRES sites, so cells expressing the NiV glycoproteins from these vectors also express GFP. For ligation into pczCFG5 IEGZ, both NiV genes were excised from pBK-CMV by digestion with NheI and HindIII, followed by Klenow (Gibco) fill-in of the overhanging ends. pczCFG5 IEGZ was digested with SwaI and ligated to the filled-in NiV genes. For ligation into pHITBE, both NiV genes in pBK-CMV were digested with NheI, followed by Klenow fill-in and digestion with HindIII. pHITBE was digested with EcoRI, followed by Klenow fill-in and digestion with HindIII and ligation to the filled-in NiV genes. pczCFG5 IEGZ was digested with SwaI (New England Biolabs) and the filled-in NiV genes were ligated into pczCFG5 IEGZ. Non-fluorescent versions of pHITBE-NiV F and pHITBE-NiV G were generated by excising the GFP gene. Briefly, pHITBE-NiV F and pHITBE-NiV G were digested with AgeI and NotI, which flank the GFP gene. After gel extraction of the plasmids, the resulting overhanging ends were filled in with Klenow fragment and religated to give the plasmids pHIT
GFP-NiV F and pHITGFP-NiV G.
Transfection.
293T cells at 80–90 % confluence were transfected in six-well dishes using serum-free OptiMEM (Gibco) and Lipofectamine 2000 (Invitrogen). Briefly, plasmid DNA (4 µg) and Lipofectamine 2000 were mixed separately in OptiMEM and incubated at room temperature for 5 min. Following incubation, an equal volume of OptiMEM : Lipofectamine 2000 was added to the OptiMEM : DNA mixture. The transfection mixtures were mixed gently and then incubated at room temperature for 15 min to allow DNA : Lipofectamine 2000 complexes to form. Following incubation, an equal volume of OptiMEM was added and mixed, and 1 ml was pipetted onto monolayers of 293T cells. Transfections were incubated overnight at 37 °C and 5 % CO2.
Production of retroviral particles and generation of transgenic cell lines.
Retroviral particles for transductions were produced by adaptation of a previously described method (Soneoka et al., 1995). The pczCFG5 IEGZ vector contains a replication-deficient retroviral genome with a 
packaging sequence from Moloney murine leukemia virus. Plasmids (4 µg each of pczCFG5 IEGZ, pczCFG5-NiV F or pczCFG5-NiV G) were co-transfected with separate plasmids encoding vesicular stomatitis virus glycoprotein (VSV G) and murine retroviral gag–pol open reading frames as described above. After transfection, the newly transcribed plasmid genomes are packaged by the gag and pol protein products and viral particles are pseudotyped with VSV G. VSV G mediates binding and entry via a phospholipid receptor that is present on many mammalian cells. Retroviral particles were either used in transductions immediately or were harvested and stored at –70 °C until further use. Target CRFK cell monolayers were transduced with undiluted retroviral particles as described above. Monolayers were transduced twice at 16–24 h intervals with retroviral particles to ensure high levels of integrated provirus. The pczCFG5 IEGZ vector also contains a GFP–Zeocin resistance fusion gene. Transgenic cells were selected for this drug marker by treatment with 400 µg Zeocin (Invitrogen) ml–1 for 2 weeks. Transgene expression was assessed by FACS analysis for GFP expression in transgenic cells.
RT-PCR of NiV- and HeV-exposed transgenic cells.
Wild-type (wt) CRFK, CRFK-pcz, CRFK-NiV F and CRFK-NiV G cells were seeded into two 24-well dishes. Cells were exposed to HeV and NiV as described above at dilutions ranging from 5x104 to 5 TCID50 per well. At 5 days post-exposure, surviving cells were trypsinized and RNA was extracted using an RNeasy mini kit (Qiagen). Extracted total cellular RNA was first subjected to first-stand cDNA synthesis using a SensiScript reverse transcriptase kit (Qiagen) and a reverse transcriptase primer. The resulting cDNA was amplified using a Master Mix PCR kit (Qiagen) and primers that were designed to target HeV and NiV positive-sense mRNA from the N, M and G genes and negative-sense genomic viral RNA (vRNA) at the N/P, M/F and F/G gene junctions. RNA extracts from all CRFK cells and CRFK-derived cells were verified with an internal mRNA control using primers for feline glyceraldehyde 3-phosphate dehydrogenase mRNA (fGAPDH; GenBank accession no. AB038241
[GenBank]
): fGAPDH1 fwd (5'-TTCCACGGCACAGTCAAGGCTGAGA-3') and fGAPDH1 rev (5'-GGTGCAGGAGGCATTGCTGACAATC-3'). Amplification of mRNA in all samples gave the expected 294 bp RT-PCR product.
Fluorescent fusion inhibition assay.
293T cells were seeded into six-well dishes. When the cells were approximately 80–90 % confluent, they were transfected with 4 µg each of pczCFG5 IEGZ, pczCFG5-NiV F, pczCFG5-NiV G or pHITGFP-NiV F+pHITGFP-NiV G. After 8 h, the cells were trypsinized and the pczCFG5 IEGZ-, pczCFG5-NiV F- and pczCFG5-NiV G-transfected cells were mixed separately in a 1 : 1 ratio with pHITGFP-NiV F+pHITGFP-NiV G-transfected cells in a fresh 12-well dish. The cells were incubated overnight, fixed with 3.7 % PBS-buffered formaldehyde and examined for green fluorescent syncytia the next day.
Production of polyclonal NiV antisera.
Two female guinea pigs (Hartley, 500 g; Charles River Laboratories) were inoculated intraperitoneally with 105 p.f.u. live NiV per guinea pig. The guinea pigs were boosted intraperitoneally with a further 105 p.f.u. per guinea pig at 14 days post-inoculation and were terminally bled at 28 days post-inoculation. Guinea pigs were anaesthetized prior to inoculation, bleeding or exsanguination by intramuscular administration of xylozine (5 mg kg–1) and ketamine (40 mg kg–1). The generation of swine antisera against NiV G has been described elsewhere (Weingartl et al., 2005, 2006). All animal work was performed under BSL4 conditions and according to Canadian Council of Animal Care guidelines.
Western blots.
For Western blots, cells were lysed in 1x SDS gel loading buffer [50 mM Tris/HCl (pH 7.5), 1 % SDS, 8.75 % glycerol and 0.125 % bromophenol blue] with 4 % 2-mercaptoethanol. Lysates were boiled for 5 min before being run on 10 % resolving SDS-polyacrylamide gels. Protein gels were transferred to PVDF membranes (Amersham) using a Mini-PROTEAN 3 Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) overnight at 30 V in transfer buffer [25 mM Tris/HCl (pH 8.3), 192 mM glycine and 20 % methanol]. Membranes were blocked for at least 1 h at room temperature in blocking buffer (5 % skimmed milk, 0.1 % Tween 20 in PBS). Membranes were washed three times for 5 min in PBS/0.1 % Tween 20 (PBS-T) and then probed with a guinea pig anti-NiV immune serum as the primary antibody; primary antibodies were diluted 1 : 800–1 : 1000 in blocking buffer containing 1 % normal rabbit serum (Sigma). Primary antibody incubation was performed at room temperature for 1–2 h on a rocker. Membranes were washed three times for 10 min each in PBS-T. A rabbit anti-guinea pig or goat anti-swine horseradish peroxidase-conjugated antibody (Sigma) was used as the secondary antibody at a dilution of 1 : 10 000 in blocking buffer. Secondary antibody incubation was performed at room temperature for 1 h, followed by washing with PBS-T three times for 10 min each. Blots were developed using the ECL Plus kit (Amersham) and exposed to Hyperfilm (Amersham) to visualize the bands.
Cell-surface staining using fluorescence activated cell sorting (FACS).
In order to assess the effect of NiV glycoprotein expression and NiV infection on cell-surface ephrin-B2 and ephrin-B3, cell-surface staining was performed and stained cells were analysed by FACS on a FACSCalibur flow cytometer (BD). For infected cells, a 75 cm2 flask of 293T cells was infected with approximately 105 TCID50 ml–1 of NiV and incubated overnight at 37 °C. Approximately 18–20 h after infection, cells were removed from the flask with Versene (Invitrogen), pelleted by centrifugation at 500 g for 10 min, resuspended in 5 ml Mg2+/Ca2+-free PBS (Gibco) and fixed in an equal volume of PBS-buffered 4 % paraformaldehyde (PFA) overnight at 4 °C. The following day, infected cells were pelleted by centrifugation (10 min, 500 g), resuspended in 5 ml Mg2+/Ca2+-free PBS, diluted with an equal volume of PBS-buffered 4 % PFA and removed from BSL4 containment. Rabbit polyclonal antibodies against human ephrin-B2 and human ephrin-B3 were purchased from Genex Biosciences, diluted in Mg2+/Ca2+-free PBS to a concentration of 1 mg ml–1 and then used as described below. Polyclonal goat anti-rabbit R-phycoerythrin conjugate was purchased from Jackson ImmunoResearch. Mock and transfected cells were removed from 75 cm2 flasks using 10 ml Versene per flask and resuspended into a single-cell suspension by pipetting. The resuspended cells were fixed with an equal volume of PBS-buffered 4 % PFA overnight at 4 °C. The cells were centrifuged the following day for 5 min at 500 g, the supernatant was discarded and the cells were resuspended in 5 ml fresh Mg2+/Ca2+-free PBS. For FACS staining, 2.5x105 cells per tube were blocked with 10 µl human gammaglobulin for 10 min at room temperature. Primary antibody diluted in Mg2+/Ca2+-free PBS (100 µl per tube) was added to cells, mixed and incubated at 4 °C for 30 min. Cells were washed twice with Mg2+/Ca2+-free PBS (1 ml per tube) with centrifugation for 5 min at 500 g between each wash. Secondary antibody diluted in Mg2+/Ca2+-free PBS was added to cell pellets, mixed and incubated for 30 min at 4 °C, followed by two washes with Mg2+/Ca2+-free PBS (1 ml per tube). Final cell pellets were resuspended in 500 µl Mg2+/Ca2+-free PBS and fixed overnight at 4 °C with an equal volume of PBS-buffered 4 % PFA. Primary antibodies were used at a dilution of 1 : 100, whilst secondary antibody dilutions were generally used at dilutions of 1 : 200 to 1 : 400.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); the 
19 kDa cleavage fragment F2 was not visible on the blot. NiV G was detected as one band of 75–80 kDa (Fig. 1). We then attempted to assess the fusogenic behaviour of the glycoproteins in cell culture. When NiV F or G was transfected alone into 293T cells, there was no formation of syncytia (Fig. 2). When NiV F and G were co-transfected, extensive fusion resulted and large syncytia developed (Fig. 2). However, when NiV F and G were transfected singly into separate cell populations followed by mixing of the cell populations, no syncytia were detected (Fig. 2). These results confirmed the observations by other groups that NiV F and G are both required for fusion to occur (Bossart et al., 2002; Tamin et al., 2002). The lack of fusion in the mixed populations of singly transfected NiV F and NiV G cells indicated that there is a requirement for a specific interaction between NiV F and G in the same cell for fusion to occur.
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, left and right panels). These data indicated that we obtained a high level of transgene expression in the transgenic cells. In our experience, the read-through rate of the IRES between the first and second genes was >90 % and was a very reliable indicator of upstream gene expression. In order to verify functional expression of the glycoproteins, CRFK-NiV F or CRFK-NiV G cells were transduced with the complementary retroviral particle (CRFK-NiV F+NiV G particles or CRFK-NiV G+NiV F particles). Following incubation for 48 h, cells were examined for fusion. Syncytia developed containing approximately 15–20 nuclei per syncytium (Fig. 3a, bottom left and right). No syncytia developed in any of the singly transduced transgenic cells (Fig. 3a, top left and right). These data demonstrated the functional expression of NiV F and G in transgenic cells. The GFP levels in the CRFK-NiV F and CRFK-NiV G transgenic cells were verified by FACS analysis to assess transgene expression.
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At all doses of NiV and HeV, all control cells (wt CRFK, CRFK-pcz and CRFK-NiV F not shown) died by 5 days post-exposure. Transgenic CRFK-NiV G cells survived NiV exposure at all doses over the course of the experiment (Fig. 4), as did CRFK-NiV G cells exposed to HeV (Fig. 4). These data indicated that there was specific protection conferred by expression of the NiV G attachment glycoprotein. In contrast, the lack of protection in cells expressing NiV F also demonstrated that this is a phenomenon specific to NiV G expression and was not due to non-specific interference from NiV genes or gene products. The lack of protection in vector control cells (CRFK-pcz) also demonstrated the specificity of NiV G-conferred protection by excluding any components of the pczCFG5 IEGZ-based vector backbone. We also checked transgenic cells exposed to NiV and HeV for viral mRNA and genomic vRNA. We were not able to detect vRNA in CRFK-NiV G cells exposed to 5 TCID50 of virus, indicating that nucleic acid from NiV and HeV could not be detected in cells that had survived 5 days of exposure to NiV or HeV (data not shown). In contrast, CRFK-NiV F and wt CRFK cells exposed to HeV and NiV were positive for all HeV and NiV mRNA and vRNA (data not shown). Mock-infected cells did not have positive PCR results with any primer set. Total cellular RNA was tested for each sample using fGAPDH primers. The inability to detect viral nucleic acid in NiV- and HeV-exposed CRFK-NiV G cells seemed to indicate that virus was unable to enter these cells or was able to enter but was unable to replicate.
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GFP-NiV F and pHITGFP-NiV G (Fig. 5a, right). As controls, cells singly transfected with pczCFG5 IEGZ or pczCFG5-NiV F were also mixed in the same manner (Fig. 5a, left and centre). Approximately 16 h after mixing, pczCFG5-NiV F-transfected cells had fused with cells expressing NiV F and G and formed green fluorescent syncytia (Fig. 5b, centre), indicating that NiV F expression alone was not able to inhibit fusion induced by NiV F and NiV G co-expression. However, pczCFG5-NiV G-transfected cells did not fuse with cells expressing NiV F and G, and therefore did not form green fluorescent syncytia; they remained as single green cells (Fig. 5b, right). This indicated that expression of NiV G was sufficient to inhibit fusion induced by NiV F and G co-expression. The lack of fusion in NiV G-expressing cells suggested that NiV G had downregulated the expression of the cellular receptor on the surface of the cell, thereby rendering these cells resistant to fusion and infection. If NiV G interfered with replication steps downstream of virus attachment or entry, it would be expected that these NiV G-expressing cells would fuse in this assay; this did not occur.
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; Negrete et al., 2005) and ephrin-B3 (Negrete et al., 2006) as receptors for NiV and HeV, we therefore endeavoured to determine whether this phenomenon was occurring in 293T cells expressing NiV G. Untransfected control cells stained positive for cell-surface expression of ephrin-B2 and ephrin-B3 (Fig. 6, green line). Cells transfected with pczCFG5 IEGZ (empty vector) or pczCFG5-NiV F showed no differences in the cell-surface levels of ephrin-B2 or ephrin-B3 (not shown), indicating that expression of NiV F had no effect on cell-surface levels of ephrin-B2 or ephrin-B3 ligand. Interestingly, when 293T cells were transfected with pczCFG5-NiV G there was no difference in the cell-surface level of ephrin-B2 or ephrin-B3 (Fig. 6, blue line). There was also no cell-surface downregulation of ephrin-B2 or ephrin-B3 in cells infected with NiV (Fig. 6, brown line). Expression of NiV G, whether by a recombinant plasmid-based system or by NiV infection, had no effect of the cell-surface levels of these two known NiV receptors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In contrast, cell–cell fusion has been observed in similar experiments in which cells separately expressing the F and HN glycoproteins of Human parainfluenza virus 2 were mixed together (Hu et al., 1992). Cellular expression of the NiV G glycoprotein led to a high degree of resistance towards infection with either NiV or HeV. Several lines of evidence indicated that protection was conferred specifically by expression of NiV G. First, the vector control cells (CRFK-pcz) were not resistant to NiV infection and showed the same degree of CPE as wt CRFK cells. This indicated that components of the pczCFG5 IEGZ vector backbone (GFP–Zeocin resistance fusion protein) were not playing a role in resistance to NiV. Secondly, CRFK-NiV F cells died following exposure to virus, indicating that NiV F had no protective effect. The lack of protection seen in CRFK-NiV F cells also demonstrated that resistance was not due to antisense RNA interference from cellular NiV F positive-sense gene transcripts with the incoming negative-sense viral genome. If antisense inhibition were the primary mechanism, then it would be expected that both CRFK-NiV F and CRFK-NiV G cells should show approximately the same level of resistance to NiV, which was clearly not the case. Thirdly, viral nucleic acid (mRNA and vRNA) was not detected in CRFK-NiV G cells that had been exposed to 5 TCID50 HeV and NiV per well. Lastly, expression of NiV G rendered cells resistant to NiV F- and G-mediated fusion. This indicated that the blockage occurred at the level of virus binding and/or entry. If NiV G had inhibited downstream steps in virus replication, we would have expected to see fusion occurring with NiV G-expressing cells, a phenomenon that clearly was not observed.
All of the above lines of evidence point towards authentic receptor interference as the predominant mechanism of resistance to NiV and HeV infection in CRFK-NiV G cells, where newly synthesized NiV G in the transgenic cells interacts with the cognate cellular receptor. As the functional cellular receptor is no longer available to interact with incoming viral glycoprotein, these cells are refractive to infection with NiV. Many viral proteins are known to interact with and downregulate (Marschall et al., 1997; Breiner et al., 2001) or perhaps even induce degradation of their cellular receptors (Horga et al., 2000), leading to the phenomenon of receptor interference. Expression of NiV G specifically inhibited the ability of cells to fuse with other fusogenic cells that expressed both NiV F and G, which indicated a block at the level of interaction with the cellular receptor. This was supported by the lack of any viral nucleic acid in CRFK-NiV G cells that had been exposed to low doses of NiV and HeV, and the accompanying lack of CPE in these cells. We have also observed that the haemagglutinin protein of Canine distemper virus does not protect cells from NiV infection, further confirming the specificity of NiV G-mediated protection (data not shown).
In prior studies, it has been suggested that NiV and HeV may share a cellular receptor (Bossart et al., 2002), which was recently confirmed by the identification of ephrin-B2 as a receptor for both NiV and HeV (Bonaparte et al., 2005; Negrete et al., 2005). Ephrin-B3 has also been identified as a receptor for NiV (Negrete et al., 2006), and it seems reasonable to expect that it can also act as a receptor for HeV. The inhibition of NiV and HeV infection by NiV G expression is therefore likely to occur via the same mechanism, namely interaction with either ephrin-B2 or ephrin-B3, or both. Previous studies have also indirectly indicated a common mechanism of inhibition by binding of a soluble HeV G to cells, which resulted in competitive inhibition of NiV and HeV binding and infection (Bossart et al., 2005). One intriguing aspect of our study is the result that the two known NiV receptors, ephrin-B2 and ephrin-B3, are clearly present on the surface of cells expressing NiV G. We had expected that ephrin-B2 and ephrin-B3 would exhibit decreased cell-surface expression in NiV G-expressing cells; this was clearly not the case. This was also observed in cells infected with NiV, where there was no change in the cell-surface levels of ephrin-B2 and ephrin-B3. Based on our data, we conclude that expression of NiV G and infection by NiV have no effect on the cell-surface levels of ephrin-B2 and ephrin-B3.
In our studies, the result of NiV G expression was resistance to NiV- and HeV-induced CPE. We hypothesize that this resistance is due to viral receptor interference, although neither one of the known viral receptors, ephrin-B2 and ephrin-B3, is downregulated from the cell surface. Further work will focus on identification of the receptor-binding domain(s) within the attachment protein and regions responsible for interaction with the fusion protein, which are probably encoded by separate regions of the protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Bagai, S. & Lamb, R. A. (1995). Quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus. J Virol 69, 6712–6719.[Abstract]
Bar-Lev Stern, L., Greenberg, M., Gershoni, J. M. & Rozenblatt, S. (1995). The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis. J Virol 69, 1661–1668.[Abstract]
Bonaparte, M. I., Dimitrov, A. S., Bossart, K. N., Crameri, G., Mungall, B. A., Bishop, K. A., Choudhry, V., Dimitrov, D. S., Wang, L.-F. & other authors (2005). Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc Natl Acad Sci U S A 102, 10652–10657.
Bossart, K. N., Wang, L.-F., Eaton, B. T. & Broder, C. C. (2001). Functional expression and membrane fusion tropism of the envelope glycoproteins of Hendra virus. Virology 290, 121–135.[CrossRef][Medline]
Bossart, K. N., Wang, L.-F., Flora, M. N., Chua, K. B., Lam, S. K., Eaton, B. T. & Broder, C. C. (2002). Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins. J Virol 76, 11186–11198.
Bossart, K. N., Crameri, G., Dimitrov, A. S., Mungall, B. A., Feng, Y. R., Patch, J. R., Choudhary, A., Wang, L. F., Eaton, B. T. & Broder, C. C. (2005). Receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble G glycoprotein of Hendra virus. J Virol 79, 6690–6702.
Bousse, T., Takimoto, T., Gorman, W. L., Takahashi, T. & Portner, A. (1994). Regions on the hemagglutinin-neuraminidase proteins of human parainfluenza virus type-1 and Sendai virus important for membrane fusion. Virology 204, 506–514.[CrossRef][Medline]
Breiner, K. M., Urban, S., Glass, B. & Schaller, H. (2001). Envelope protein-mediated down-regulation of hepatitis B virus receptor in infected hepatocytes. J Virol 75, 143–150.
Butler, D. (2004). Fatal fruit bat virus sparks epidemics in southern Asia (news). Nature 429, 7.[Medline]
Cattaneo, R. & Rose, J. K. (1993). Cell fusion by the envelope glycoproteins of persistent measles viruses which caused lethal human brain disease. J Virol 67, 1493–1502.
Chadha, M. S., Comer, J. A., Lowe, L., Rota, P. A., Rollin, P. E., Bellini, W. J., Ksiazek, T. G. & Mishra, A. C. (2006). Nipah virus-associated encephalitis outbreak, Siliguri, India. Emerg Infect Dis 12, 235–240.[Medline]
Chew, M. H. L., Arguin, P. M., Shay, D. K., Goh, K.-T., Rollin, P. E., Shieh, W.-J., Zaki, S. R., Rota, P. A., Ling, A.-E. & other authors (2000). Risk factors for Nipah virus infection among abattoir workers in Singapore. J Infect Dis 181, 1760–1763.[CrossRef][Medline]
Chua, K. B., Goh, K. J., Wong, K. T., Kamarulzaman, A., Tan, P. S., Ksiazek, T. G., Zaki, S. R., Paul, G., Lam, S. K. & Tan, C. T. (1999). Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 354, 1257–1259.[CrossRef][Medline]
Chua, K. B., Bellini, W. J., Rota, P. A., Harcourt, B. H., Tamin, A., Lam, S. K., Ksiazek, T. G., Rollin, P. E., Zaki, S. R. & other authors (2000). Nipah virus: a recently emergent deadly paramyxovirus. Science 288, 1432–1435.
Czub, M., McAtee, F. J., Czub, S., Lynch, W. P. & Portis, J. L. (1995). Prevention of retrovirus-induced neurological disease by infection with a nonneuropathogenic retrovirus. Virology 206, 372–380.[CrossRef][Medline]
Delwart, E. L. & Panganiban, A. T. (1989). Role of reticuloendotheliosis virus envelope glycoprotein in superinfection interference. J Virol 63, 273–280.
Ebata, S. N., Côté, M.-J., Kang, C. Y. & Dimock, K. (1991). The fusion and hemagglutinin-neuraminidase glycoproteins of human parainfluenza virus 3 are both required for fusion. Virology 183, 437–441.[CrossRef][Medline]
Geraghty, R. J., Jogger, C. R. & Spear, P. G. (2000). Cellular expression of alphaherpesvirus gD interferes with entry of homologous and heterologous alphaherpesviruses by blocking access to a shared gD receptor. Virology 268, 147–158.[CrossRef][Medline]
Halpin, K., Young, P. L., Field, H. E. & Mackenzie, J. S. (2000). Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol 81, 1927–1932.
Heminway, B. R., Yu, Y. & Galinski, M. S. (1994). Paramyxovirus mediated cell fusion requires co-expression of both the fusion and hemagglutinin-neuraminidase glycoproteins. Virus Res 31, 1–16.[CrossRef][Medline]
Hishiyama, M., Tanabayashi, K., Takeuchi, K. & Yamada, A. (1996). Establishment of cell lines stably expressing mumps virus glycoproteins. Jpn J Med Sci Biol 49, 29–38.[Medline]
Horga, M.-A., Gusella, G. L., Greengard, O., Poltoratskaia, N., Porotto, M. & Moscona, A. (2000). Mechanism of interference mediated by human parainfluenza virus type 3 infection. J Virol 74, 11792–11799.
Horvath, C. M., Paterson, R. G., Shaughnessy, M. A., Wood, R. & Lamb, R. A. (1992). Biological activities of paramyxovirus fusion proteins: factors influencing formation of syncytia. J Virol 66, 4564–4569.
Hsu, V. P., Hossain, M. J., Parashar, U. D., Ali, M. M., Ksiazek, T. G., Kuzmin, I., Niezgoda, M., Rupprecht, C., Bresee, J. & Breiman, R. F. (2004). Nipah virus encephalitis reemergence, Bangladesh. Emerg Infect Dis 10, 2082–2087.[Medline]
Hu, X. L., Ray, R. & Compans, R. W. (1992). Functional interactions between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses. J Virol 66, 1528–1534.
Hunt, H. D., Lee, L. F., Foster, D., Silva, R. F. & Fadly, A. M. (1999). A genetically engineered cell line resistant to subgroup J avian leukosis virus infection (C/J). Virology 264, 205–210.[CrossRef][Medline]
Kahn, J. S., Schnell, M. J., Buonocore, L. & Rose, J. K. (1999). Recombinant vesicular stomatitis virus expressing respiratory syncytial virus (RSV) glycoproteins: RSV fusion protein can mediate infection and cell fusion. Virology 254, 81–91.[CrossRef][Medline]
Karpf, A. R., Lenches, E., Strauss, E. G., Strauss, J. H. & Brown, D. T. (1997). Superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with Sindbis virus. J Virol 71, 7119–7123.[Abstract]
Lindemann, D., Pietschmann, T., Picard-Maureau, M., Berg, A., Heinkelein, M., Thurow, J., Knaus, P., Zentgraf, H. & Rethwilm, A. (2001). A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity. J Virol 75, 5762–5771.
Lyu, M. S., Nihrane, A. & Kozak, C. A. (1999). Receptor-mediated interference mechanism responsible for resistance to polytropic leukemia viruses in Mus castaneus. J Virol 73, 3733–3736.
Marschall, M., Meier-Ewert, H., Herrler, G., Zimmer, G. & Maassab, H. F. (1997). The cell receptor level is reduced during persistent infection with influenza C virus. Arch Virol 142, 1155–1164.[CrossRef][Medline]
Mayo, M. A. (2002). A summary of taxonomic changes recently approved by ICTV. Arch Virol 147, 1655–1656.[CrossRef][Medline]
Middleton, D. J., Westbury, H. A., Morrissy, C. J., van der Heide, B. M., Russell, G. M., Braun, M. A. & Hyatt, A. D. (2002). Experimental Nipah virus infection in pigs and cats. J Comp Pathol 126, 124–136.[CrossRef][Medline]
Mizuguchi, H., Nakanishi, T., Kondoh, M., Nakagawa, T., Nakanishi, M., Matsuyama, T., Tsutsumi, Y., Nakagawa, S. & Mayumi, T. (1999). Fusion of Sendai virus with liposome depends on only F protein, but not HN protein. Virus Res 59, 191–201.[CrossRef][Medline]
Morrison, T., McQuain, C. & McGinnes, L. (1991). Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion. J Virol 65, 813–822.
Mounts, A. W., Kaur, H., Parashar, U. D., Ksiazek, T. G., Cannon, D., Arokiasamy, J. T., Anderson, L. J., Lye, M. S. & Nipah Virus Nosocomial Study Group. (2001). A cohort study of health care workers to assess nosocomial transmissibility of Nipah virus, Malaysia, 1999. J Infect Dis 183, 810–813.[CrossRef][Medline]
Murray, K., Selleck, P., Hooper, P., Hyatt, A., Gould, A., Gleeson, L., Westbury, H., Hiley, L., Selvey, L. & other authors (1995). A morbillivirus that caused fatal disease in horses and humans. Science 268, 94–97.
Negrete, O. A., Levroney, E. L., Aguilar, H. C., Bertolotti-Ciarlet, A., Nazarian, R., Tajyar, S. & Lee, B. (2005). EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus. Nature 436, 401–405.[Medline]
Negrete, O. A., Wolf, M. C., Aguilar, H. C., Enterlein, S., Wang, W., Mühlberger, E., Su, S. V., Bertolotti-Ciarlet, A., Flick, R. & Lee, B. (2006). Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus. PLoS Pathog 2, e7.[CrossRef][Medline]
Nishio, M., Tsurudome, M., Komada, H., Kawano, M., Tabata, N., Matsumura, H., Ikemura, N., Watanabe, N. & Ito, Y. (1994). Fusion properties of cells constitutively expressing human parainfluenza virus type 4A haemagglutinin-neuraminidase and fusion glycoproteins. J Gen Virol 75, 3517–3523.
Parashar, U. D., Sunn, L. M., Ong, F., Mounts, A. W., Arif, M. T., Ksiazek, T. G., Kamaluddin, M. A., Mustafa, A. N., Kaur, H. & other authors (2000). Case-control study of risk factors for human infection with a new zoonotic paramyxovirus, Nipah virus, during a 1998–1999 outbreak of severe encephalitis in Malaysia. J Infect Dis 181, 1755–1759.[CrossRef][Medline]
Paton, N. I., Leo, Y. S., Zaki, S. R., Auchus, A. P., Lee, K. E., Ling, A. E., Chew, S. K., Ang, B., Rollin, P. E. & other authors (1999). Outbreak of Nipah-virus infection among abattoir workers in Singapore. Lancet 354, 1253–1256.[CrossRef][Medline]
Philbey, A. W., Kirkland, P. D., Ross, A. D., Davis, R. J., Gleeson, A. B., Love, R. J., Daniels, P. W., Gould, A. R. & Hyatt, A. D. (1998). An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats. Emerg Infect Dis 4, 269–271.[Medline]
Ponferrada, V. G., Mauck, B. S. & Wooley, D. P. (2003). The envelope glycoprotein of human endogenous retrovirus HERV-W induces cellular resistance to spleen necrosis virus. Arch Virol 148, 659–675.[CrossRef][Medline]
Potash, M. J. & Volsky, D. J. (1998). Viral interference in HIV-1 infected cells. Rev Med Virol 8, 203–211.[CrossRef][Medline]
Rogers, R. J., Douglas, I. C., Baldock, F. C., Glanville, R. J., Seppanen, K. T., Gleeson, L. J., Selleck, P. N. & Dunn, K. J. (1996). Investigation of a second focus of equine morbillivirus infection in coastal Queensland. Aust Vet J 74, 243–244.[Medline]
Sakai, Y. & Shibuta, H. (1989). Syncytium formation by recombinant vaccinia viruses carrying bovine parainfluenza 3 virus envelope protein genes. J Virol 63, 3661–3668.
Schneider-Schaulies, J., Dunster, L. M., Kobune, F., Rima, B. & ter Meulen, V. (1995a). Differential downregulation of CD46 by measles virus strains. J Virol 69, 7257–7259.[Abstract]
Schneider-Schaulies, J., Schnorr, J.-J., Brinckmann, U., Dunster, L. M., Baczko, K., Liebert, U. G., Schneider-Schaulies, S. & ter Meulen, V. (1995b). Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains. Proc Natl Acad Sci U S A 92, 3943–3947.
Seth, S. & Shaila, M. S. (2001). The fusion protein of Peste des petits ruminants virus mediates biological fusion in the absence of hemagglutinin-neuraminidase protein. Virology 289, 86–94.[CrossRef][Medline]
Soneoka, Y., Cannon, P. M., Ramsdale, E. E., Griffiths, J. C., Romano, G., Kingsman, S. M. & Kingsman, A. J. (1995). A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res 23, 628–633.
Tamin, A., Harcourt, B. H., Ksiazek, T. G., Rollin, P. E., Bellini, W. J. & Rota, P. A. (2002). Functional properties of the fusion and attachment glycoproteins of Nipah virus. Virology 296, 190–200.[CrossRef][Medline]
Tanabayashi, K., Takeuchi, K., Okazaki, K., Hishiyama, M. & Yamada, A. (1992). Expression of mumps virus glycoproteins in mammalian cells from cloned cDNAs: both F and HN proteins are required for cell fusion. Virology 187, 801–804.[CrossRef][Medline]
Weingartl, H., Czub, S., Copps, J., Berhane, Y., Middleton, D., Marszal, P., Gren, J., Smith, G., Ganske, S. & other authors (2005). Invasion of the central nervous system in a porcine host by Nipah virus. J Virol 79, 7528–7534.
Weingartl, H. M., Berhane, Y., Caswell, J. L., Loosmore, S., Audonnet, J.-C., Roth, J. A. & Czub, M. (2006). Recombinant Nipah virus vaccines protect pigs against challenge. J Virol 80, 7929–7938.
Welstead, G. G., Hsu, E. C., Iorio, C., Bolotin, S. & Richardson, C. D. (2004). Mechanism of CD150 (SLAM) down regulation from the host cell surface by measles virus hemagglutinin protein. J Virol 78, 9666–9674.
Wild, T. F., Malvoisin, E. & Buckland, R. (1991). Measles virus: both the haemagglutinin and fusion glycoproteins are required for fusion. J Gen Virol 72, 439–442.
Williamson, M. M., Hooper, P. T., Selleck, P. W., Gleeson, L. J., Daniels, P. W., Westbury, H. A. & Murray, P. K. (1998). Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats. Aust Vet J 76, 813–818.[Medline]
Williamson, M. M., Hooper, P. T., Selleck, P. W., Westbury, H. A. & Slocombe, R. F. (2000). Experimental hendra virus infection in pregnant guinea-pigs and fruit bats (Pteropus poliocephalus). J Comp Pathol 122, 201–207.[CrossRef][Medline]
Williamson, M. M., Hooper, P. T., Selleck, P. W., Westbury, H. A. & Slocombe, R. F. S. (2001). A guinea-pig model of Hendra virus encephalitis. J Comp Pathol 124, 273–279.[CrossRef][Medline]
Received 28 July 2006;
accepted 8 October 2006.
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