Ephrin-B2 and ephrin-B3 as functional henipavirus receptors

Members of the ephrin cell-surface protein family interact with the Eph receptors, the largest family of receptor tyrosine kinases, mediating bi-directional signaling during tumorogenesis and various developmental events. Surprisingly, ephrin-B2 and -B3 were recently identified as entry receptors for henipaviruses, emerging zoonotic paramyxoviruses responsible for repeated outbreaks in humans and animals in Australia, Southeast Asia, India and Bangladesh. Nipah virus (NiV) and Hendra virus (HeV) are the only two identified members in the henipavirus genus. While the initial human infection cases came from contact with infected pigs (NiV) or horses (HeV), in the more recent outbreaks of NiV both food-borne and human-to-human transmission were reported. These characteristics, together with high mortality and morbidity rates and lack of effective anti-viral therapies, make the henipaviruses a potential biological-agent threat. Viral entry is an important target for the development of anti-viral drugs. The entry of henipavirus is initiated by the attachment of the viral G envelope glycoprotein to the host cell receptors ephrin-B2 and/or -B3, followed by activation of the F fusion protein, which triggers fusion between the viral envelop and the host membrane. We review recent progress in the study of henipavirus entry, particularly the identification of ephrins as their entry receptors, and the structural characterization of the ephrin/Henipa-G interactions.     

Single amino acid changes in the Nipah and Hendra virus attachment glycoproteins distinguish ephrinB2 from ephrinB3 usage

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are lethal emerging paramyxoviruses. EphrinB2 and ephrinB3 have been identified as receptors for henipavirus entry. NiV and HeV share similar cellular tropisms and likely use an identical receptor set, although a quantitative comparison of receptor usage by NiV and HeV has not been reported. Here we show that (i) soluble NiV attachment protein G (sNiV-G) bound to cell surface-expressed ephrinB3 with a 30-fold higher affinity than that of sHeV-G, (ii) NiV envelope pseudotyped reporter virus (NiVpp) entered ephrinB3-expressing cells much more efficiently than did HeV pseudotyped particles (HeVpp), and (iii) NiVpp but not HeVpp entry was inhibited efficiently by soluble ephrinB3. These data underscore the finding that NiV uses ephrinB3 more efficiently than does HeV. Henipavirus G chimeric protein analysis implicated residue 507 in the G ectodomain in efficient ephrinB3 usage. Curiously, alternative versions of published HeV-G sequences show variations at residue 507 that can clearly affect ephrinB3 but not ephrinB2 usage. We further defined surrounding mutations (W504A and E505A) that diminished ephrinB3-dependent binding and viral entry without compromising ephrinB2 receptor usage and another mutation (E533Q) that abrogated both ephrinB2 and -B3 usage. Our results suggest that ephrinB2 and -B3 binding determinants on henipavirus G are distinct and dissociable. Global expression analysis showed that ephrinB3, but not ephrinB2, is expressed in the brain stem. Thus, ephrinB3-mediated viral entry and pathology may underlie the severe brain stem neuronal dysfunction seen in fatal Nipah viral encephalitis. Characterizing the determinants of ephrinB2 versus -B3 usage will further our understanding of henipavirus pathogenesis.     

Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection.     

Modes of paramyxovirus fusion: a Henipavirus perspective

Henipavirus is a new genus of Paramyxoviridae that uses protein-based receptors (ephrinB2 and ephrinB3) for virus entry. Paramyxovirus entry requires the coordinated action of the fusion (F) and attachment viral envelope glycoproteins. Receptor binding to the attachment protein triggers F to undergo a conformational cascade that results in membrane fusion. The accumulation of structural and functional studies on many paramyxoviral fusion and attachment proteins, including the recent elucidation of structures of Nipah virus (NiV) and Hendra virus (HeV) G glycoproteins bound and unbound to cognate ephrinB receptors, indicate that henipavirus entry and fusion could differ mechanistically from paramyxoviruses that use glycan-based receptors.     

Nipah virus infects specific subsets of porcine peripheral blood mononuclear cells

Nipah virus (NiV), a zoonotic paramyxovirus, is highly contagious in swine, and can cause fatal infections in humans following transmission from the swine host. The main viral targets in both species are the respiratory and central nervous systems, with viremia implicated as a mode of dissemination of NiV throughout the host. The presented work focused on the role of peripheral blood mononuclear cells (PBMC) in the viremic spread of the virus in the swine host. B lymphocytes, CD4-CD8-, as well as CD4+CD8- T lymphocytes were not permissive to NiV, and expansion of the CD4+CD8- cells early post infection was consistent with functional humoral response to NiV infection observed in swine. In contrast, significant drop in the CD4+CD8- T cell frequency was observed in piglets which succumbed to the experimental infection, supporting the hypothesis that antibody development is the critical component of the protective immune response. Productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells by recovery of infectious virus in the cell supernatants. Virus replication was supported by detection of the structural N and the non-structural C proteins or by detection of genomic RNA increase in the infected cells. Infection of T cells carrying CD6 marker, a strong ligand for the activated leukocyte cell adhesion molecule ALCAM (CD166) highly expressed on the microvascular endothelial cell of the blood-air and the blood-brain barrier may explain NiV preferential tropism for small blood vessels of the lung and brain.     

Infection of hepatitis B virus in extrahepatic endothelial tissues mediated by endothelial progenitor cells

Background

Nipah virus (NiV) is an emerging paramyxovirus distinguished by its ability to cause fatal disease in both animal and human hosts. Together with Hendra virus (HeV), they comprise the genusHenipavirus in theParamyxoviridae family. NiV and HeV are also restricted to Biosafety Level-4 containment and this has hampered progress towards examining details of their replication and morphogenesis. Here, we have established recombinant expression systems to study NiV particle assembly and budding through the formation of virus-like particles (VLPs).

Results

When expressed by recombinant Modified Vaccinia virus Ankara (rMVA) or plasmid transfection, individual NiV matrix (M), fusion (F) and attachment (G) proteins were all released into culture supernatants in a membrane-associated state as determined by sucrose density gradient flotation and immunoprecipitation. However, co-expression of F and G along with M revealed a shift in their distribution across the gradient, indicating association with M in VLPs. Protein release was also altered depending on the context of viral proteins being expressed, with F, G and nucleocapsid (N) protein reducing M release, and N release dependent on the co-expression of M. Immunoelectron microscopy and density analysis revealed VLPs that were similar to authentic virus. Differences in the budding dynamics of NiV proteins were also noted between rMVA and plasmid based strategies, suggesting that over-expression by poxvirus may not be appropriate for studying the details of recombinant virus particle assembly and release.

Conclusion

Taken together, the results indicate that NiV M, F, and G each possess some ability to bud from expressing cells, and that co-expression of these viral proteins results in a more organized budding process with M playing a central role. These findings will aid our understanding of paramyxovirus particle assembly in general and could help facilitate the development of a novel vaccine approach for henipaviruses.     

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.     

Clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection

Nipah virus (NiV) and Hendra virus (HeV) are emerging zoonotic viruses and the causative agents of severe respiratory disease and encephalitis in humans. Little is known about the mechanisms that govern the development of respiratory and neurological disease. Using a hamster model of lethal NiV and HeV infection, we describe the role of the route and dose of infection on the clinical outcome and determine virus tropism and host responses following infection. Infection of hamster with a high dose of NiV or HeV resulted in acute respiratory distress. NiV initially replicated in the upper respiratory tract epithelium, whereas HeV initiated infection primarily in the interstitium. In contrast, infection with a low dose of NiV or HeV resulted in the development of neurological signs and more systemic spread of the virus through involvement of the endothelium. The development of neurological signs coincided with disruption of the blood-brain barrier (BBB) and expression of tumor necrosis alpha (TNF-α) and interleukin 1 β (IL-1β). In addition, interferon-inducible protein 10 (IP-10) was identified as playing an important role in NiV and HeV pathogenesis. These studies reveal novel information on the development and progression of NiV and HeV clinical disease, provide a mechanism for the differences in transmission observed between NiV and HeV outbreaks, and identify specific cytokines and chemokines that serve as important targets for treatment.     

Cedar Virus: A Novel Henipavirus Isolated from Australian Bats

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.     

Regulation of adeno-associated virus DNA replication by the cellular TAF-I/set complex

The Rep proteins of the adeno-associated virus (AAV) are required for viral replication in the presence of adenovirus helper functions and as yet poorly characterized cellular factors. In an attempt to identify such factors, we purified Flag-Rep68-interacting proteins from human cell lysates. Several polypeptides were identified by mass spectrometry, among which was ANP32B, a member of the acidic nuclear protein 32 family which takes part in the formation of the template-activating factor I/Set oncoprotein (TAF-I/Set) complex. The N terminus of Rep was found to specifically bind the acidic domain of ANP32B; through this interaction, Rep was also able to recruit other members of the TAF-I/Set complex, including the ANP32A protein and the histone chaperone TAF-I/Set. Further experiments revealed that silencing of ANP32A and ANP32B inhibited AAV replication, while overexpression of all of the components of the TAF-I/Set complex increased de novo AAV DNA synthesis in permissive cells. Besides being the first indication that the TAF-I/Set complex participates in wild-type AAV replication, these findings have important implications for the generation of recombinant AAV vectors since overexpression of the TAF-I/Set components was found to markedly increase viral vector production.     

Antibodies to henipavirus or henipa-like viruses in domestic pigs in Ghana, West Africa

Henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), have Pteropid bats as their known natural reservoirs. Antibodies against henipaviruses have been found in Eidolon helvum, an old world fruit bat species, and henipavirus-like nucleic acid has been detected in faecal samples from E. helvum in Ghana. The initial outbreak of NiV in Malaysia led to over 265 human encephalitis cases, including 105 deaths, with infected pigs acting as amplifier hosts for NiV during the outbreak. We detected non-neutralizing antibodies against viruses of the genus Henipavirus in approximately 5% of pig sera (N = 97) tested in Ghana, but not in a small sample of other domestic species sampled under a E. helvum roost. Although we did not detect neutralizing antibody, our results suggest prior exposure of the Ghana pig population to henipavirus(es). Because a wide diversity of henipavirus-like nucleic acid sequences have been found in Ghanaian E. helvum, we hypothesise that these pigs might have been infected by henipavirus(es) sufficiently divergent enough from HeVor NiV to produce cross-reactive, but not cross-neutralizing antibodies to HeV or NiV.     

Receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble G glycoprotein of Hendra virus

Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae, which are distinguished by their ability to cause fatal disease in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) glycoproteins. Previously, we reported on HeV- and NiV-mediated fusion activities and detailed their host-cell tropism characteristics. These studies also suggested that a common cell surface receptor, which could be destroyed by protease, was utilized by both viruses. To further characterize the G glycoprotein and its unknown receptor, soluble forms of HeV G (sG) were constructed by replacing its cytoplasmic tail and transmembrane domains with an immunoglobulin kappa leader sequence coupled to either an S-peptide tag (sG(S-tag)) or myc-epitope tag (sG(myc-tag)) to facilitate purification and detection. Expression of sG was verified in cell lysates and culture supernatants by specific affinity precipitation. Analysis of sG by size exclusion chromatography and sucrose gradient centrifugation demonstrated tetrameric, dimeric, and monomeric species, with the majority of the sG released as a disulfide-linked dimer. Immunofluorescence staining revealed that sG specifically bound to HeV and NiV infection-permissive cells but not to a nonpermissive HeLa cell line clone, suggesting that it binds to virus receptor on host cells. Preincubation of host cells with sG resulted in dose-dependent inhibition of both HeV and NiV cell fusion as well as infection by live virus. Taken together, these data indicate that sG retains important native structural features, and we further demonstrate that administration of sG to rabbits can elicit a potent cross-reactive neutralizing antibody response against infectious HeV and NiV. This HeV sG glycoprotein will be exceedingly useful for structural studies, receptor identification strategies, and vaccine development goals for these important emerging viral agents.     

Molecular determinants of antiviral potency of paramyxovirus entry inhibitors

Hendra virus (HeV) and Nipah virus (NiV) constitute the Henipavirus genus of paramyxoviruses, both fatal in humans and with the potential for subversion as agents of bioterrorism. Binding of the HeV/NiV attachment protein (G) to its receptor triggers a series of conformational changes in the fusion protein (F), ultimately leading to formation of a postfusion six-helix bundle (6HB) structure and fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions, the first (the N-terminal heptad repeat [HRN]) adjacent to the fusion peptide and the second (the C-terminal heptad repeat [HRC]) immediately preceding the transmembrane domain. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing activated F molecules from forming the 6HB structure that is required for fusion. We previously reported that a human parainfluenza virus 3 (HPIV3) F peptide effectively inhibits infection mediated by the HeV glycoproteins in pseudotyped-HeV entry assays more effectively than the comparable HeV-derived peptide, and we now show that this peptide inhibits live-HeV and -NiV infection. HPIV3 F peptides were also effective in inhibiting HeV pseudotype virus entry in a new assay that mimics multicycle replication. This anti-HeV/NiV efficacy can be correlated with the greater potential of the HPIV3 C peptide to interact with the HeV F N peptide coiled-coil trimer, as evaluated by thermal unfolding experiments. Furthermore, replacement of a buried glutamic acid (glutamic acid 459) in the C peptide with valine enhances antiviral potency and stabilizes the 6HB conformation. Our results strongly suggest that conserved interhelical packing interactions in the F protein fusion core are important determinants of C peptide inhibitory activity and offer a strategy for the development of more-potent analogs of F peptide inhibitors.     

The Distribution of Henipaviruses in Southeast Asia and Australasia: Is Wallace’s Line a Barrier to Nipah Virus?

Nipah virus (NiV) (Genus Henipavirus) is a recently emerged zoonotic virus that causes severe disease in humans and has been found in bats of the genus Pteropus. Whilst NiV has not been detected in Australia, evidence for NiV-infection has been found in pteropid bats in some of Australia’s closest neighbours. The aim of this study was to determine the occurrence of henipaviruses in fruit bat (Family Pteropodidae) populations to the north of Australia. In particular we tested the hypothesis that Nipah virus is restricted to west of Wallace’s Line. Fruit bats from Australia, Papua New Guinea, East Timor and Indonesia were tested for the presence of antibodies to Hendra virus (HeV) and Nipah virus, and tested for the presence of HeV, NiV or henipavirus RNA by PCR. Evidence was found for the presence of Nipah virus in both Pteropus vampyrus and Rousettus amplexicaudatus populations from East Timor. Serology and PCR also suggested the presence of a henipavirus that was neither HeV nor NiV in Pteropus alecto and Acerodon celebensis. The results demonstrate the presence of NiV in the fruit bat populations on the eastern side of Wallace’s Line and within 500 km of Australia. They indicate the presence of non-NiV, non-HeV henipaviruses in fruit bat populations of Sulawesi and Sumba and possibly in Papua New Guinea. It appears that NiV is present where P. vampyrus occurs, such as in the fruit bat populations of Timor, but where this bat species is absent other henipaviruses may be present, as on Sulawesi and Sumba. Evidence was obtained for the presence henipaviruses in the non-Pteropid species R. amplexicaudatus and in A. celebensis. The findings of this work fill some gaps in knowledge in geographical and species distribution of henipaviruses in Australasia which will contribute to planning of risk management and surveillance activities.     

The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.     

Production of the virus‐like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus‐like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti‐myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high‐performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038–1045, 2016     

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.     

Nipah Virus Entry and Egress from Polarized Epithelial Cells

Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both primary and systemic infection phases. NiV entry and spread from polarized epithelial cells therefore determine virus entry and dissemination within a new host and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were detected only after infection with high virus doses, at time points when the integrity of the cell monolayer was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein, M. Studies with stably M-expressing and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal.     

Simulating Henipavirus Multicycle Replication in a Screening Assay Leads to Identification of a Promising Candidate for Therapy

Nipah (NiV) and Hendra (HeV) viruses are emerging zoonotic paramyxoviruses that cause encephalitis in humans, with fatality rates of up to 75%. We designed a new high-throughput screening (HTS) assay for inhibitors of infection based on envelope glycoprotein pseudotypes. The assay simulates multicycle replication and thus identifies inhibitors that target several stages of the viral life cycle, but it still can be carried out under biosafety level 2 (BSL-2) conditions. These features permit a screen for antivirals for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. The screening of a small compound library identified several effective molecules, including the well-known compound chloroquine, as highly active inhibitors of pseudotyped virus infection. Chloroquine inhibited infection with live HeV and NiV at a concentration of 1 μM in vitro (50% inhibitory concentration, 2 μM), which is less than the plasma concentrations present in humans receiving chloroquine treatment for malaria. The mechanism for chloroquine''s antiviral action likely is the inhibition of cathepsin L, a cellular enzyme that is essential for the processing of the viral fusion glycoprotein and the maturation of newly budding virions. Without this processing step, virions are not infectious. The identification of a compound that inhibits a known cellular target that is important for viral maturation but that had not previously been shown to have antiviral activity for henipaviruses highlights the validity of this new screening assay. Given the established safety profile and broad experience with chloroquine in humans, the results described here provide an option for treating individuals infected by these deadly viruses.Nipah (NiV) and Hendra (HeV) viruses are newly emerging zoonoses that cause encephalitis in humans, with fatality rates of up to 75% (3, 7, 8, 12, 13, 30). NiV has caused at least nine significant outbreaks in Bangladesh and India since its emergence in Malaysia in 1998 (3, 7, 8, 12, 13, 30). The virus emerged from the fruit bat (flying fox) mammalian reservoir, via the pig, into the human population. However, direct transmission from bats to humans can bypass the pig host, and person-to-person transmission also has now become a primary mode of NiV spread (2, 5). HeV, via the same bat host, has caused disease in horses, with transmission to horse-handlers and veterinarians, and since 1995 has caused sporadic illness in Australia (12). Both viruses, in addition to acute disease, may cause asymptomatic infection in up to 60% of exposed people and may lead to late-onset disease or the relapse of encephalitis years after initial infection (25), as well as persistent or delayed neurological sequelae (11). The vast geographic range of the fruit bat mammalian reservoir raises the possibility of a wide spread of these human diseases, which presently have no clinical treatment or vaccine.The first step in infection with HeV or NiV is binding to the target cells, via the interaction of the viral envelope protein (G) with specific receptor molecules on the cell surface. The receptor for HeV is Ephrin B2 (EFNB2) and for NiV is either EFNB2 or EFNB3 (11). The fusion of the viral envelope with the plasma membrane of the cell is then mediated by the viral fusion protein (F). The F protein is synthesized as a precursor protein (F0) that is proteolytically processed posttranslationally to form a trimer of disulfide-linked heterodimers (F1 + F2). This cleavage event places the fusion peptide at the F1 terminus in the mature F protein and is essential for membrane fusion activity. During viral entry, the fusion peptides, which are buried in the F trimer, must be exposed transiently so that they can insert into the target cell membrane. The conformational change that leads to the exposure of the fusion peptides requires an activation step (22), which is initiated by the interaction of G with its receptor. Only virions bearing the mature, cleaved F can undergo activation and thus are infectious (4, 14, 15).We introduce here a biosafety level 2 (BSL-2)-amenable high-throughput screening (HTS) assay (9) for inhibitors that target several stages of the henipavirus viral cycle, based on envelope glycoprotein pseudotypes. The cell-based assay allows for the simultaneous evaluation of antiviral activity and the cytotoxicity of compounds. We have validated the method with several different classes of henipavirus entry inhibitors as well as protease inhibitors. For this assay, HeV envelope glycoproteins were pseudotyped onto a recombinant vesicular stomatitis virus (VSV) that expresses red fluorescent protein (RFP) but lacks its attachment protein, G (19, 20). The resulting pseudotyped virus bears the HeV binding and fusion proteins. The infection of target cells by pseudotyped virus in the absence and presence of compounds is quantified by assessing the production of red fluorescence. This pseudotyped viral entry assay, unlike previous ones (31), simulates multicycle replication because the monolayer cells, which express viral glycoproteins, will generate more pseudotyped particles when infected. Compounds found to be active in this assay may be those that either block binding, interfere with F activation or fusion, or interfere with the protease processing of F. However, the assay is safe, because these particles can only produce infectious progeny in cells expressing HeV G/F. These features allow experimentation and antiviral assessment for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. We report the use of this screen to discover effective inhibitors of henipavirus replication and the evaluation of a well-known compound with previously unidentified properties that may allow its immediate use for henipaviruses.     

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal.