Polybasic KKR motif in the cytoplasmic tail of Nipah virus fusion protein modulates membrane fusion by inside-out signaling.
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The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where approximately 20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r(2) = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms. (+info)
Foodborne transmission of Nipah virus, Bangladesh.
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We investigated an outbreak of encephalitis in Tangail District, Bangladesh. We defined case-patients as persons from the outbreak area in whom fever developed with new onset of seizures or altered mental status from December 15, 2004, through January 31, 2005. Twelve persons met the definition; 11 (92%) died. Serum specimens were available from 3; 2 had immunoglobulin M antibodies against Nipah virus by capture enzyme immunoassay. We enrolled 11 case-patients and 33 neighborhood controls in a case-control study. The only exposure significantly associated with illness was drinking raw date palm sap (64% among case-patients vs. 18% among controls, odds ratio [OR] 7.9, p = 0.01). Fruit bats (Pteropus giganteus) are a nuisance to date palm sap collectors because the bats drink from the clay pots used to collect the sap at night. This investigation suggests that Nipah virus was transmitted from P. giganteus to persons through drinking fresh date palm sap. (+info)
Genetic analysis of J-virus and Beilong virus using minireplicons.
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J-virus (JPV), isolated from wild mice in Australia, and Beilong virus (BeiPV), originally isolated from human mesangial cells in China and subsequently detected in rat mesangial cells, represent a new group of paramyxoviruses which have exceptionally large genomes (>19 kb) and contain more than six transcriptional units. In this study, minireplicons were employed to assess the taxonomic status of JPV and BeiPV. Our results demonstrated that, whilst the genome replication machineries of JPV and BeiPV can be interchanged, they were not functional when exchanged with that of Nipah virus. These studies indicate that JPV and BeiPV are closely related to each other and support the classification of these two viruses into a separate genus. In addition, the minireplicons were also used to demonstrate that these large-genome viruses also comply with the 'rule of six' and that over-expression of the C protein has a detrimental effect on minigenome replication. (+info)
Human neuronal cell protein responses to Nipah virus infection.
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BACKGROUND: Nipah virus (NiV), a recently discovered zoonotic virus infects and replicates in several human cell types. Its replication in human neuronal cells, however, is less efficient in comparison to other fully susceptible cells. In the present study, the SK-N-MC human neuronal cell protein response to NiV infection is examined using proteomic approaches. RESULTS: Method for separation of the NiV-infected human neuronal cell proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was established. At least 800 protein spots were resolved of which seven were unique, six were significantly up-regulated and eight were significantly down-regulated. Six of these altered proteins were identified using mass spectrometry (MS) and confirmed using MS/MS. The heterogenous nuclear ribonucleoprotein (hnRNP) F, guanine nucleotide binding protein (G protein), voltage-dependent anion channel 2 (VDAC2) and cytochrome bc1 were present in abundance in the NiV-infected SK-N-MC cells in contrast to hnRNPs H and H2 that were significantly down-regulated. CONCLUSION: Several human neuronal cell proteins that are differentially expressed following NiV infection are identified. The proteins are associated with various cellular functions and their abundance reflects their significance in the cytopathologic responses to the infection and the regulation of NiV replication. The potential importance of the ratio of hnRNP F, and hnRNPs H and H2 in regulation of NiV replication, the association of the mitochondrial protein with the cytopathologic responses to the infection and induction of apoptosis are highlighted. (+info)
Molecular determinants of antiviral potency of paramyxovirus entry inhibitors.
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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. (+info)
Single amino acid changes in the Nipah and Hendra virus attachment glycoproteins distinguish ephrinB2 from ephrinB3 usage.
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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. (+info)
Cis-acting elements in the antigenomic promoter of Nipah virus.
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Genome synthesis in paramyxoviruses, including Nipah virus (NiV), is controlled by sequence elements that reside in the non-coding nucleotides at the 5'-trailer (3'-antigenomic) end that make up the antigenomic promoter (AGP). Using a chloramphenicol acetyl transferase-based plasmid-driven minigenome system, the terminal 96 nt of NiV AGP were first mutagenized in blocks of three hexamers to enable broad mapping of the minigenome functional regions. This was followed by further dissection of these functional regions to define the cis-acting elements contained therein. Results based on RNA analysis and reporter gene activity identified a bipartite promoter structure similar to that seen in related viruses, but with some distinct differences: in NiV, each of the two discrete replication control elements was bimodal, characterized by a critical conserved region (nt 1-12 and 79-91) and a contiguous non-conserved region (nt 13-36 and 73-78), which appeared less important. The regulatory role of these less critical regions was underscored by the use of a two-step mutation strategy, which revealed the additive detrimental effect of substitutions in this part of the terminal element. The structure and sequence characteristics of the internal control element was also different: it involved four contiguous hexamers, and the region encompassing three of these (nt 79-96, corresponding to hexamers 14, 15 and 16), although analogous in position to the equivalent element in the Sendai virus AGP, was characterized by the distinct 5'-(GNNNUG)(14-15)(GNNNNN)(16) motif. (+info)
Vertical transmission and fetal replication of Nipah virus in an experimentally infected cat.
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A female adult cat developed clinical disease 13 days after subcutaneous inoculation with Nipah virus (NiV) and was discovered to be pregnant at necropsy. Viral genome was detected in a variety of specimens, including blood, serum, tonsil swabs, and urine, up to 3 days before the onset of disease. Samples collected postmortem, including placenta, uterine fluid, and fetal tissues, were also positive for NiV genome, and the placenta and uterine fluid contained high levels of recoverable virus. The high levels of viral shedding in the adult combined with fetal viral replication suggests that both vertical and horizontal transmission of NiV could play a role in spillover events, an essential element in the epidemiology of Henipavirus infection. (+info)