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Tyrosine residues in the cytoplasmic domains affect sorting and fusion activity of the Nipah virus glycoproteins in polarized epithelial cells.

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.

SUBMITTER: Weise C

PROVIDER: S-EPMC2897613 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Tyrosine residues in the cytoplasmic domains affect sorting and fusion activity of the Nipah virus glycoproteins in polarized epithelial cells.

Weise Carolin C Erbar Stephanie S Lamp Boris B Vogt Carola C Diederich Sandra S Maisner Andrea A

Journal of virology 20100519 15

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 tha ...[more]

PMID: 20484517

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Project description:Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (hemagglutinin [HA] tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly, however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F-G interactions, supporting the F/G dissociation model of henipavirus-induced membrane fusion, even in the context of heterologous glycoprotein pairs.IMPORTANCE The NiV and HeV henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytium formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential coinfections of these or subsequent hosts may alter the outcome of disease.

Project description:Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in β-strand c (V114M) of domain II. We have previously shown that the analogous β-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E β-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo. IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

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Tyrosine residues in the cytoplasmic domains affect sorting and fusion activity of the Nipah virus glycoproteins in polarized epithelial cells.

Publications

Tyrosine residues in the cytoplasmic domains affect sorting and fusion activity of the Nipah virus glycoproteins in polarized epithelial cells.

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