Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH

Department of Molecular & Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, United States
Journal of Virology (Impact Factor: 4.44). 12/2006; 80(22):10931-41. DOI: 10.1128/JVI.01287-06
Source: PubMed


Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.

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    • "Contrary to this, NDV fusion was enhanced in acidic environments [47]. The pH-dependent HMPV strain CAN97-83 requires trypsin treatment to mediate proteolysis processing and also low-pH pulses for its fusion activity [22]. "
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    ABSTRACT: To understand the molecular mechanisms of Avian metapneumovirus (aMPV) and the requirements involved in the infection and fusion, trypsin treatment was done in the different stages of virus; before infection, during entry and after virus infection followed by aMPV infection. The growth kinetics of aMPV was compared in time dependent manner. The effect of trypsin was found in the later stage of aMPV infection increasing the numbers of infected cells with the significant higher titer of infectious virions to that of trypsin treated before infection, during entry and aMPV. A serine protease inhibitor reduced aMPV replication in a significant way, whereas cysteine peptidase (E-64), aspartic protease (pepstatin A), and metalloprotease (phosphoramidon) inhibitors had no effect on aMPV replication. Inoculation of aMPV on Vero cells expressing the membrane-associated protease TMPRSS2 resulted in higher virus titers than that inoculated on normal Vero cells and is statistically significant (p<0.05). Also, an inhibitor of clathrin/caveolae-mediated endocytosis had no effect on virus progeny, indicating that aMPV does not use the endocytic pathway for entry but undergoes direct fusion. The effect of lysosomotropic agents was not significant, suggesting that aMPV does not require low-pH environment in endosomes to fuse its envelope with the plasma membrane. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · May 2015 · Molecular and Cellular Probes
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    • "The attachment (G) protein plays a role in virus attachment and is expressed as a single polypeptide chain, which subsequently undergoes extensive N- and O-linked glycosylation [7]. The fusion (F) protein mediates fusion of the virus and host-cell membranes, and is initially synthesised as a single polypeptide chain (F0) that undergoes proteolytic cleavage to generate the mature and active form of the protein, consisting of F1 and F2 protein subunits [8]. The virus also expresses a third membrane-associated protein called the matrix (M) protein, which is analogous to the M protein of RSV and is a major determinant of virus morphology [9]. "
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    ABSTRACT: Human metapneumovirus (HMPV) is now a major cause of lower respiratory infection in children. Although primary isolation of HMPV has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious HMPV have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material that can be achieved following HMPV infection. In this study we demonstrate that expression of the HMPV F, G and M proteins in mammalian cells leads to HMPV virus-like particles (VLP) formation. This experimental strategy will serve as a model system to allow the process of HMPV virus assembly to be examined. The HMPV F, G and M proteins were expressed in mammalian cell lines. Protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. VLP formation was examined using sucrose density gradient centrifugation and electron microscopy analysis. Analysis of cells co-expressing of the F, G and M proteins demonstrated that these proteins interacted. Furthermore, in cells co-expression the three HMPV proteins the formation VLPs was observed. Image analysis revealed the VLPs had a similar morphology to the filamentous virus morphology that we observed on HMPV-infected cells. The capacity of each protein to initiate VLP formation was examined using a VLP formation assay. Individual expression of each virus protein showed that the G protein was able to form VLPs in the absence of the other virus proteins. Furthermore, co-expression of the G protein with either the M or F proteins facilitated their incorporation into the VLP fraction. Co-expression of the F, G and M proteins lead to the formation of VLPs, and that incorporation of the F and M proteins into VLPs is facilitated by their interaction with the G protein. Our data suggests that the G protein plays a central role in VLP formation, and further suggests that the G protein may also play a role in the recruitment of the F and M proteins to sites of virus particle formation during HMPV infection.
    Full-text · Article · Sep 2013 · Virology Journal
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    • "followed by low pH [55] [56]. Moreover, the fusion protein of the human metapneumovirus is activated by proteolysis and by acidic pH [20] [25]. "
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    ABSTRACT: Most paramyxoviruses enter the cell by direct fusion of the viral envelope with the plasma membrane. Our previous studies have shown the colocalization of NDV with the early endosome marker EEA1 and the inhibition of NDV fusion by the caveolin-phosphorylating drug PMA prompted us to propose that NDV enters the cells via endocytosis. Here we show that the virus-cell fusion and cell-cell fusion promoted by NDV-F are increased by about 30% after brief exposure to low pH in HeLa and ELL-0 cells but not in NDV receptor-deficient cell lines such as GM95 or Lec1. After a brief low-pH exposure, the percentage of NDV fusion at 29°C was similar to that at 37°C without acid-pH stimulation, meaning that acid pH would decrease the energetic barrier to enhance fusion. Furthermore, preincubation of cells with the PKC inhibitor bisindolylmaleimide led to the inhibition of about 30% of NDV infectivity, suggesting that a population of virus enters cells through receptor-mediated endocytosis. Moreover, the involvement of the GTPase dynamin in NDV entry is shown as its specific inhibitor, dynasore, also impaired NDV fusion and infectivity. Optimal infection of the host cells was significantly affected by drugs that inhibit endosomal acidification such as concanamycin A, monensin and chloroquine. These results support our hypothesis that entry of NDV into ELL-0 and HeLa cells occurs through the plasma membrane as well as by dynamin-, low pH- and receptor-dependent endocytosis.
    Preview · Article · Aug 2013 · Biochimica et Biophysica Acta
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