Human metapneumovirus (HMPV) binding and infection are mediated by interactions between the HMPV fusion protein and heparan sulfate - PubMed (original) (raw)

Human metapneumovirus (HMPV) binding and infection are mediated by interactions between the HMPV fusion protein and heparan sulfate

Andres Chang et al. J Virol. 2012 Mar.

Abstract

Human metapneumovirus (HMPV) is a major worldwide respiratory pathogen that causes acute upper and lower respiratory tract disease. The mechanism by which this virus recognizes and gains access to its target cell is still largely unknown. In this study, we addressed the initial steps in virus binding and infection and found that the first binding partner for HMPV is heparan sulfate (HS). While wild-type CHO-K1 cells are permissive to HMPV infection, mutant cell lines lacking the ability to synthesize glycosaminoglycans (GAGs), specifically, heparan sulfate proteoglycans (HSPGs), were resistant to binding and infection by HMPV. The permissiveness to HMPV infection was also abolished when CHO-K1 cells were treated with heparinases. Importantly, using recombinant HMPV lacking both the G and small hydrophobic (SH) proteins, we report that this first virus-cell binding interaction is driven primarily by the fusion protein (HMPV F) and that this interaction is needed to establish a productive infection. Finally, HMPV binding to cells did not require β1 integrin expression, and RGD-mediated interactions were not essential in promoting HMPV F-mediated cell-to-cell membrane fusion. Cells lacking β1 integrin, however, were less permissive to HMPV infection, indicating that while β1 integrins play an important role in promoting HMPV infection, the interaction between integrins and HMPV occurs after the initial binding of HMPV F to heparan sulfate proteoglycans.

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Figures

Fig 1

HMPV F RGA mutant promotes cell-to-cell membrane fusion at levels similar to those for HMPV F WT. (A) Representative SDS-polyacrylamide gel of lysates from metabolically labeled and biotinylated Vero cells transfected with F WT versus F RGA. The total amount of F was immunoprecipitated with an F-specific antiserum, and the surface population was separated using streptavidin beads. (B) Quantification of SDS-PAGE bands (n = 3) corresponding to total and surface expression of HMPV F WT and RGA mutant. Data represented as percentage of surface or total WT F0 or F1 expression. (C) Representative pictures (n = 4) of syncytium assays in Vero cells transfected with F WT or the HMPV F RGA mutant are shown. (D) Luciferase reporter gene assay (n = 3) used to compare fusion activity between HMPV F WT and HMPV F RGA mutant. MCS, multiple-cloning site, empty vector pCAGGS control. Error bars, mean ± standard error of the mean.

Fig 2

Initial HMPV binding is independent of β1 integrin expression. (A) Expression levels of αv and β1 integrins in Vero, GD25, β1GD25, and CHO-K1 cells assessed by flow cytometry. Control (gray), secondary antibody only. (B) Different MOIs (0.5, 1, 2, 5, 10, 25, and 50) of HMPV were bound to GD25 (closed circles), β1GD25 (closed squares), and CHO-K1 (open circles) cells. HMPV binding was assessed by HRP activity following immunolabeling of the HMPV N protein. Data were normalized to those for virus bound to Vero cells at an MOI of 10. Results shown are averages of duplicate wells for 3 independent experiments. (C) Representative Western blots used for quantification in panel D. WT rgHMPV (lanes 2, 6, and 10), ΔG mutant HMPV (lanes 3, 7, and 11), and ΔG/ΔSH mutant HMPV (lanes 4, 8, and 12) at an MOI of 7 were bound to Vero (lanes 1 to 4), GD25 (lanes 5 to 8), and β1GD25 (lanes 9 to 12) cells. Lysates of virus bound to cells were collected, and a volume corresponding to an equal number of cells was analyzed with an anti-HMPV F antibody. The control cell lane without virus is shown in lanes 1, 5, and 9. (D) The signal intensity of specific bands for HMPV F protein on the Western blot was quantified for WT (black bars), ΔG (grey bars), and ΔG/ΔSH (white bars) HMPV bound to the different cell lines and normalized to WT Vero cell binding efficiency (n = 6). Error bars, mean ± standard error of the mean; brackets, P > 0.05, analysis of variance. (E) An equal number of WT, ΔG, and ΔG/ΔSH viral particles concentrated by a sucrose cushion were lysed and blotted for HMPV F using anti-HMPV F antisera. Quantification of the signal of the specific bands on the Western blot is shown (n = 10). Data were normalized to those for control WT GFP-expressing HMPV. Error bars, mean ± standard error of the mean.

Fig 3

HMPV infection is enhanced by the expression of β1 integrin. Vero, GD25, β1GD25, and CHO-K1 cells were infected with different amounts of WT rgHMPV. GFP expression of at least 10,000 cells was analyzed by FACS the following day. Results shown are averages for 3 independent experiments done in duplicate. No statistical significance between CHO-K1 and Vero cell infectivity was seen. Error bars, mean ± standard error of the mean.

Fig 4

Effective HMPV infection requires the expression of a trypsin- and proteinase K-sensitive cellular surface protein. Vero or CHO-K1 cells were treated with 0%, 0.0625%, or 0.125% trypsin (A, C, and E) or 0 μg/ml or 300 μg/ml of proteinase K (B, D, and F) prior to either infection by control rgPIV5 (A and B) or rgHMPV (C and D) or analysis of αv and β1 integrin expression (E and F; n = 3). GFP expression or FITC fluorescence intensity of at least 10,000 cells was analyzed by FACS the following day (n = 5 for trypsin and n = 6 for proteinase K treatment). Data in panels A and B are expressed as percentage of total infected cells, whereas data in C and D are expressed as percentage of cells infected normalized to the results for Vero cells not treated with proteinase K. Error bars, mean ± standard error of the mean.

Fig 5

Initial HMPV binding is dependent on GAG expression and is mediated by the F protein. (A to C) CHO-K1, pgsA745, Lec1, and ldlDLec1 cells were exposed to different MOIs (0, 1, 10, and 50) of HMPV WT (A), ΔG (B), or ΔG/ΔSH (C) at 4°C. Results shown are averages of 6 independent experiments done in duplicate. (D) Representative Western blot used for quantification in panel E. WT rgHMPV (lanes 2, 6, 10, and 14), HMPV ΔG (lanes 3, 7, 11, and 15), and HMPV ΔG/ΔSH (lanes 4, 8, 12, and 16) at an MOI of 7 were bound to Lec1 (lanes 1 to 4), ldlDLec1 (lanes 5 to 8), pgsA745 (lanes 9 to 12), and control CHO-K1 (lanes 13 to 16) cells. Lysates of virus bound to cells were collected, and a volume corresponding to an equal number of cells was analyzed with an anti-HMPV antibody. The control cell line without virus is shown in lanes 1, 5, 9, and 13. (E) Quantification of Western blot signals showing binding activity of the WT, ΔG mutant, and ΔG/ΔSH mutant HMPV. Data were normalized to those for WT Vero binding efficiency (n = 10). Error bars, mean ± standard error of the mean; asterisks, P < 0.001.

Fig 6

HMPV infection is greatly reduced in the absence of GAGs. CHO-K1, pgsA745, Lec1, and ldlDLec1 cells were infected with different MOIs (0, 1, 10, and 50) of rgHMPV, and GFP expression of at least 10,000 cells was analyzed by FACS the following day. Results shown are averages of 3 independent experiments done in duplicate. Error bars, mean ± standard error of the mean.

Fig 7

HMPV binding and infection are decreased in the absence of heparan sulfate. (A and B) HMPV ΔG/ΔSH binding to CHO-K1 (lanes 1 to 4), ldlDLec1 (lanes 5 to 8), pgsD677 (lanes 9 to 12), and pgsA745 (lanes 13 to 16) cells was assessed after a 2-h treatment with heparinases 1 and 3 (lanes 4, 8, 12, and 16) or chondroitinase ABC (lanes 3, 7, 11, and 15) or mock treatment (lanes 2, 6, 10, and 14). The control cell line without virus is shown in lanes 1, 5, 9, and 13. The signal for the specific bands on the Western blots represented in panel A (n = 3) were quantified and shown in panel B. (C) pgsD677 cells were exposed to HMPV or PIV5 at an MOI of 10. Data shown are normalized to the infectivity of CHO-K1 cells and represent the averages of 3 independent experiments done in duplicate (n = 3). (D) CHO-K1 cells treated for 1 h at 37°C with 2 mIU/ml of heparinase 1 or heparinase 3 or 20 mIU/ml of chondroitinase ABC (mock treatment as a control) were exposed to 1 MOI of either HMPV or PIV5 at 4°C, followed by normal incubation to allow infection to occur. pgsA745 cell infection is shown as a control. Data shown are normalized to the infectivity of CHO-K1 cells in the absence of any treatments and represent the averages of 3 independent experiments done in duplicate (n = 3). Error bars, mean ± standard error of the mean.

Fig 8

Homology model of the prefusion and postfusion form of HMPV F. (A) The amino acid sequence of HMPV F was threaded onto the crystal structure of the prefusion form of PIV5 F (70) using DeepView Swiss-PdvViewer (version 4.0.1) software. The RGD sequence of HMPV (red) is covered by two stretches of amino acid (yellow and green in inset). (B) Individual sequences of the F1 and F2 subunits of HMPV F were threaded onto the crystal structure of the postfusion form of Newcastle disease virus F (61).

Fig 9

Potential mechanisms for the initial steps of HMPV infection via interactions with HS. (A) HMPV could bind to any heparan sulfate proteoglycan or any specific modification in the HS moiety through HMPV F, concentrating the virus and allowing HMPV F to interact with a more specific viral receptor or coreceptor, which could also be an HSPG. This interaction, in turn, could allow the virus to interact with a potential fusion receptor that will activate HMPV F. (B) HMPV could bind to a specific HSPG or any specific modification in the HS moiety through HMPV F, and that interaction could be sufficient for HMPV F triggering and viral entry. (C) HMPV could bind to any HSPG or any specific modification in the HS moiety through HMPV F, and that interaction could be sufficient for HMPV F triggering and viral entry. Integrin αvβ1 expression renders the cell more permissive to HMPV infection.

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Human metapneumovirus (HMPV) binding and infection are mediated by interactions between the HMPV fusion protein and heparan sulfate - PubMed (original) (raw)