[show abstract][hide abstract] ABSTRACT: Proteolytic activation of the fusion protein of the highly pathogenic Nipah virus (NiV F) is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion. Unlike other paramyxoviral fusion proteins, functional NiV F activation requires endocytosis and pH-dependent cleavage at a monobasic cleavage site by endosomal proteases. Using prototype Vero cells, cathepsin L was previously identified to be a cleavage enzyme. Compared to Vero cells, MDCK cells showed substantially higher F cleavage rates in both NiV-infected and NiV F-transfected cells. Surprisingly, this could not be explained either by an increased F endocytosis rate or by elevated cathepsin L activities. On the contrary, MDCK cells did not display any detectable cathepsin L activity. Though we could confirm cathepsin L to be responsible for F activation in Vero cells, inhibitor studies revealed that in MDCK cells, cathepsin B was required for F-protein cleavage and productive replication of pathogenic NiV. Supporting the idea of an efficient F cleavage in early and recycling endosomes of MDCK cells, endocytosed F proteins and cathepsin B colocalized markedly with the endosomal marker proteins early endosomal antigen 1 (EEA-1), Rab4, and Rab11, while NiV F trafficking through late endosomal compartments was not needed for F activation. In summary, this study shows for the first time that endosomal cathepsin B can play a functional role in the activation of highly pathogenic NiV.
Journal of Virology 01/2012; 86(7):3736-45. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract Background: In spring 2009, a new swine-origin influenza A (H1N1) virus emerged in Mexico. During the following weeks the virus spread worldwide, prompting the World Health Organization to declare the first influenza pandemic of the 21st century. Sustained human-to-human transmission and severe disease progression observed in some patients urged public health authorities to respond rapidly to the disease outbreak and vaccine manufacturers to develop pandemic influenza vaccines for mass distribution. With the onset of the pandemic we began to explore the potential of academic/industrial collaboration to accelerate the production of vaccines during an outbreak of an emerging virus by combining the use of an academic BSL-4 laboratory with the expertise of a commercial vaccine manufacturer. Methods and results: To obtain virus seed stocks used for the production of a vaccine to combat the pandemic H1N1 2009 influenza virus (H1N1pdm), we followed various strategies: (i) optimization of cell culture conditions for growth of wild-type H1N1pdm isolates; (ii) classical reassortment of H1N1pdm and standard influenza vaccine donor strain PR8; and (iii) generation of corresponding reassortant viruses using reverse genetics. To ensure a rapid transition to production, the entire potential seed stock development process was carried out in a certified canine kidney suspension cell line (MDCK 33016-PF) under Good Manufacturing Practice (GMP) conditions. Conclusions: The outcome of this study indicates that a combination of different experimental strategies is the best way to cope with the need to develop vaccines rapidly in the midst of an emerging pandemic.
Clinical Chemistry and Laboratory Medicine 01/2011; 50(7):1275-9. · 3.01 Impact Factor
[show abstract][hide abstract] ABSTRACT: The highly pathogenic Nipah virus (NiV) is aerially transmitted and causes a systemic infection after entering the respiratory tract. Airway epithelia are thus important targets in primary infection. Furthermore, virus replication in the mucosal surfaces of the respiratory or urinary tract in later phases of infection is essential for virus shedding and transmission. So far, the mechanisms of NiV replication in epithelial cells are poorly elucidated. In the present study, we provide evidence that bipolar targeting of the two NiV surface glycoproteins G and F is of biological importance for fusion in polarized epithelia. We demonstrate that infection of polarized cells induces focus formation, with both glycoproteins located at lateral membranes of infected cells adjacent to uninfected cells. Supporting the idea of a direct spread of infection via lateral cell-to-cell fusion, we could identify basolateral targeting signals in the cytoplasmic domains of both NiV glycoproteins. Tyrosine 525 in the F protein is part of an endocytosis signal and is also responsible for basolateral sorting. Surprisingly, we identified a dityrosine motif at position 28/29 in the G protein, which mediates polarized targeting. A dileucine motif predicted to function as sorting signal is not involved. Mutation of the targeting signal in one of the NiV glycoproteins prevented the fusion of polarized cells, suggesting that basolateral or bipolar F and G expression facilitates the spread of NiV within epithelial cell monolayers, thereby contributing to efficient virus spread in mucosal surfaces in early and late phases of infection.
Journal of Virology 08/2010; 84(15):7634-41. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nipah virus (NiV), a highly pathogenic paramyxovirus, causes respiratory disease in pigs and severe febrile encephalitis in humans with high mortality rates. On the basis of the structural similarity of viral fusion (F) proteins within the family Paramyxoviridae, we designed and tested 18 quinolone derivatives in a NiV and measles virus (MV) envelope protein-based fusion assay beside evaluation of cytotoxicity. We found five compounds successfully inhibiting NiV envelope protein-induced cell fusion. The most active molecules (19 and 20), which also inhibit the syncytium formation induced by infectious NiV and show a low cytotoxicity in Vero cells, represent a promising lead quinolone-type compound structure. Molecular modeling indicated that compound 19 fits well into a particular protein cavity present on the NiV F protein that is important for the fusion process.
Journal of Medicinal Chemistry 07/2009; 52(14):4257-65. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae which originated from bats, encodes for a fusion (F) protein which is proteolytically processed within endosomes by cathepsin L. We show here that sequence requirements for NiV F activation differ markedly from other para- or orthomyxoviral fusion proteins. In contrast to other viral fusion proteins with monobasic cleavage sites, processing of NiV F proteins with one single basic amino acid in the cleavage peptide by exogenous trypsin is very inefficient, and introduction of a consensus sequence for furin does not result in cleavage by this ubiquitous protease. In contrast, a multibasic cleavage peptide in the NiV F protein completely impairs proteolytic processing and the generation of biological activity.
[show abstract][hide abstract] ABSTRACT: Cell entry and cell-to-cell spread of the highly pathogenic Nipah virus (NiV) requires binding of the NiV G protein to cellular ephrin receptors and subsequent NiV F-mediated fusion. Since expression levels of the main NiV entry receptor ephrin-B2 (EB2) are highly regulated in vivo to fulfill the physiological functions in axon guidance and angiogenesis, the goal of this study was to determine if changes in the EB2 expression influence NiV infection.
Surprisingly, transfection of increasing EB2 plasmid concentrations reduced cell-to-cell fusion both in cells expressing the NiV glycoproteins and in cells infected with NiV. This effect was attributed to the downregulation of the NiV glycoproteins from the cell surface. In addition to the influence on cell-to-cell fusion, increased EB2 expression significantly reduced the total amount of NiV-infected cells, thus interfered with virus entry. To determine if the negative effect of elevated EB2 expression on virus entry is a result of an increased EB2 signaling, receptor function of a tail-truncated and therefore signaling-defective DeltacEB2 was tested. Interestingly, DeltacEB2 fully functioned as NiV entry and fusion receptor, and overexpression also interfered with virus replication.
Our findings clearly show that EB2 signaling does not account for the striking negative impact of elevated receptor expression on NiV infection, but rather that the ratio between the NiV envelope glycoproteins and surface receptors critically influence cell-to-cell fusion and virus entry.
[show abstract][hide abstract] ABSTRACT: Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes severe diseases in animals and humans. Endothelial cell (EC) infection is an established hallmark of NiV infection in vivo. Despite systemic virus spread via the vascular system, EC in brain and lung are preferentially infected whereas EC in other organs are less affected. As in vivo, we found differences in the infection of EC in cell culture. Only brain-derived primary or immortalized EC were found to be permissive to NiV infection. Using a replication-independent fusion assay, we could show that the lack of infection in non-brain EC was due to a lack of receptor expression. The NiV entry receptors ephrinB2 (EB2) or ephrinB3 were only expressed in brain endothelia. The finding that EB2 expression in previously non-permissive aortic EC rendered the cells permissive to infection then demonstrated that EB2 is not only necessary but also sufficient to allow the establishment of a productive NiV infection. This strongly suggests that limitations in receptor expression restrict virus entry in certain EC subsets in vivo, and are thus responsible for the differences in EC tropism observed in human and animal NiV infections.
[show abstract][hide abstract] ABSTRACT: The recent discovery that the Nipah virus (NiV) fusion protein (F) is activated by endosomal cathepsin L raised the question if NiV utilize pH- and protease-dependent mechanisms of entry. We show here that the NiV receptor ephrin B2, virus-like particles and infectious NiV are internalized from the cell surface. However, endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of infection of new host cells. Our data clearly demonstrate that proteolytic activation of the NiV F protein is required before incorporation into budding virions but not after virus entry.
[show abstract][hide abstract] ABSTRACT: Nipah virus (NiV) is a highly pathogenic paramyxovirus, which emerged in 1998 from fruit bats in Malaysia and caused an outbreak of severe respiratory disease in pigs and fatal encephalitis in humans with high mortality rates. In contrast to most paramyxoviruses, NiV can infect a large variety of mammalian species. Due to this broad host range, its zoonotic potential, its high pathogenicity for humans, and the lack of effective vaccines or therapeutics, NiV was classified as a biosafety level 4 pathogen. This article provides an overview of the molecular characteristics of NiV focusing on the structure, functions, and unique biological properties of the two NiV surface glycoproteins, the receptor-binding G protein, and the fusion protein F. Since viral glycoproteins are major determinants for cell tropism and virus spread, a detailed knowledge of these proteins can help to understand the molecular basis of viral pathogenicity.
Annals of the New York Academy of Sciences 05/2007; 1102:39-50. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nipah virus (NiV) is a recently emerged and highly pathogenic paramyxovirus that causes a systemic infection in animals and humans and can infect a wide range of cultured cells. Interestingly, the NiV fusion (F) protein has a single arginine at the cleavage site similar to paramyxoviruses that are activated by exogenous trypsin-like enzymes only present in specific cells and tissues and therefore only cause localized infections. We show here that NiV F activation is not mediated by an exogenous serum protease but by an endogenous ubiquitous cellular protease after endocytosis of the protein. In addition to endocytosis, acidification of the endosome is a prerequisite for F cleavage. These results show that activation of the NiV F protein depends on a type of proteolytic cleavage that is clearly different from what is known for other paramyxoviral and orthomyxoviral fusion proteins. To our knowledge, this is the first example of a viral class I fusion protein whose activation depends on clathrin-mediated constitutive endocytosis.
Journal of Biological Chemistry 09/2005; 280(33):29899-903. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nipah virus (NiV), a highly pathogenic member of the family Paramyxoviridae, encodes the surface glycoproteins F and G. Since internalization of the NiV envelope proteins from the cell surface might be of functional importance for viral pathogenesis either by regulating cytopathogenicity or by modulating recognition of infected cells by the immune system, we analyzed the endocytosis of the NiV F and G proteins. Interestingly, we found both glycoproteins to be internalized in infected and transfected cells. As endocytosis is normally mediated by tyrosine- or dileucine-dependent signals in the cytoplasmic tails of transmembrane proteins, all potential internalization signals in the NiV glycoproteins were mutated. Whereas the G protein appeared to be constitutively internalized with the bulk flow during membrane turnover, uptake of the F protein was found to be signal mediated. F endocytosis clearly depended on a membrane-proximal YXXPhi motif and was found to be of functional importance for the biological activity of the protein.
Journal of Virology 04/2005; 79(6):3865-72. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nipah virus (NiV), a highly pathogenic paramyxovirus, causes a systemic infection in vivo and is able to replicate in cultured cells of many species and organs. Such pantropic paramyxoviruses generally encode fusion (F) proteins with multibasic cleavage sites activated by furin or other ubiquitous intracellular host cell proteases. In contrast, NiV has an F protein with a single arginine (R109) at the cleavage site, as is the case with paramyxoviruses that are activated by trypsin-like proteases only present in specific cells or tissues and therefore only cause localized infections. Unlike these viruses, cleavage of the NiV F protein is ubiquitous and does not require the addition of exogenous proteases in cell culture. To determine the importance of the amino acid sequence at the NiV F protein cleavage site for ubiquitous activation, we generated NiV F proteins with mutations around R109. Surprisingly, neither the exchange of amino acids upstream of R109 nor replacement of the basic residue itself interfered with F cleavage. Thus, R109 is not essential for F cleavage and activation. Our data demonstrate that NiV F-protein activation depends on a novel type of proteolytic cleavage that has not yet been described for any other paramyxovirus F protein. NiV F activation is mediated by a ubiquitous protease that requires neither a monobasic nor a multibasic cleavage site and therefore differs from the furin- or trypsin-like proteases known to activate other ortho- and paramyxovirus fusion proteins.
Journal of Virology 10/2004; 78(18):9705-12. · 5.08 Impact Factor