The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins

Abstract

Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVβ1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins.

Fig 1

A fluorescence-based assay to quantify HMPV binding. (A) Schematic of HMPV binding assay. R18-MPV was bound to cells on ice to prevent virus fusion. Adding detergent chemically dequenched the R18 dye associated with virus membranes, resulting in measurable fluorescence proportional to the amount of virus bound to cells. (B) R18-MPV binding to human bronchial epithelial (BEAS-2B) cells results in a linear increase in fluorescence (slope = 144.1 ± 8.8; r² = 0.9817). Binding was measured as fluorescence of cell-bound virus after addition of 1% Triton X-100. Results are means ± standard errors of the means (SEM) for three independent experiments performed in triplicate. (C) R18-MPV binding correlates with infectivity (slope = 0.009 ± 0.0004; r² = 0.9910). R18-MPV binding was assessed as for panel B; identical wells were incubated at 37°C in culture medium, and HMPV-infected cells were enumerated at 24 h postbinding as described in Materials and Methods. Results are means ± SEM for three independent experiments performed in triplicate.

Fig 2

Integrin expression on human bronchial epithelial (BEAS-2B) cells. BEAS-2B cells were stained for surface expression of specific integrins and analyzed by flow cytometry. Expression of α2 (A), αV (B), α5 (C), β1 (D), and β3 (E) integrin subunits or the αVβ3 heterodimer (F) is shown in shaded histograms, relative to an isotype control antibody (open histograms). The number in the upper right corner of each panel represents mean fluorescence intensity of integrin expression.

Fig 3

HMPV binding and subsequent infection depend upon RGD-binding integrins. (A to E) HMPV binding (black circles) or infection at 24 h (gray squares) was determined in the absence or presence of α2 (A), β3 (B), α5 (C), β1 (D), or αV (E) integrin function-blocking antibodies. Results from at least three independent experiments performed in triplicate are expressed as mean percent inhibition relative to untreated control; error bars indicate SEM. *, P ≤ 0.05 from Student's t test, comparing antibody treatment to no treatment. (F) The highest concentrations of αV, β1, or α5 function-blocking antibodies were used alone or in combination to target multiple integrins, and HMPV binding (black bars) or infectivity (gray bars) was determined. Asterisks indicate an additive effect of combination blockade on HMPV infectivity (gray bars); Student's t test comparing two treatments, P ≤ 0.05.

Fig 4

HMPV infection depends upon α5β1 and αV integrins. HMPV infection at 24 h was determined in the absence or presence of αV, α5, or a combination of the two integrin function-blocking antibodies. Infected cells were enumerated as described in Materials and Methods. Results from at least three independent experiments performed in triplicate are expressed as mean percent inhibition relative to untreated control (no MAb); error bars indicate SEM. *, additive effect of combination blockade on HMPV infectivity; Student's t test comparing two treatments, P ≤ 0.05.

Fig 5

HMPV F binds to RGD-binding integrins in the absence of G. (A) Electron micrographs of HMPV and F-only and F+G virus-like particles (VLPs). Sucrose-purified particles were stained with uranyl formate and imaged on an FEI Morgagni electron microscope (magnification, ×28,000). Protein spikes are visible projecting from the surfaces of both virus and VLPs. (B) Ten to 30 particles were chosen for morphology analysis; diameters and glycoprotein spike lengths were measured using AMT Image Capture Engine software. (C) Viral matrix (M), fusion (F), and attachment (G) protein incorporation into VLPs was confirmed by Western blotting. Virus and VLPs were immunoprecipitated with an anti-F monoclonal antibody (2 μg) and analyzed by Western blotting. Viral proteins were detected with HMPV M-specific or HMPV polyclonal A2 virus-specific (F and G) antibodies and fluorescent secondary antibodies using the Li-Cor Odyssey infrared imaging system. Lanes: 1, mock; 2, HMPV; 3, F-VLP; 4, F+G-VLP. Uncleaved protein (F0) and the large subunit from the cleaved form (F1) of the fusion protein were detected. M bands were detected in a different channel than F and G bands on the same blot and thus appear on a different image. (D) R18-MPV or R18-VLP binding was measured in the absence or presence of integrin function-blocking antibodies (α2 or a combination of αV, α5, and β1 to block all available RGD-binding integrins). Results from three independent experiments are expressed as mean percent inhibition relative to an untreated control (no MAb). Error bars indicate SEM; *, P ≤ 0.05.

Fig 6

The HMPV F RGD motif is required for HMPV infection. (A) Titers from reverse-engineered viruses recovered with either the native RGD motif (F-wt) or F-RAE are shown as mean titer for two independently recovered viruses; error bars indicate SEM. The limit of detection is shown as a dotted line. (B and C) Light microscopy images depicting typical plaque size and morphology for HMPV F-wt and F-RAE. LLC-MK2 cells were infected with either HMPV F-wt or F-RAE and incubated for 4 days under a semisolid medium, permitting only cell-to-cell virus spread. HMPV-infected cells are stained black. Magnifications, 2.5× (B) and 10× (C).

Fig 7

HMPV fusion occurs slowly over the course of several hours. (A) R18-MPV fusion with live, adherent human bronchial epithelial (BEAS-2B) cells. R18 fluorescence increases over time as R18 dye self-quenched in the virus membrane dilutes into cellular membranes during virus-mediated membrane fusion. The zero time point was imaged before incubating cells at 37°C to initiate virus fusion. Live cell images were captured with a Zeiss Axiovert 200 microscope using a 40× objective with 359/461-nm (DAPI) and 556/573-nm (R18) filters at the indicated time points. (B) R18-MPV fusion was measured with a plate reader. Curves represent fluorescence of cells (gray diamonds), cells plus R18-MPV at 4°C (blue squares), or cells plus R18-MPV at 37°C (red circles). Results for triplicate wells (mean ± standard deviation [SD]) from a representative experiment are shown. (C) R18-MPV fusion was measured in the absence (red circles) or presence (green triangles) of neutralizing HMPV antiserum (dilution, 1:40) or for heat-inactivated virus (black squares). Percent R18 dequenching was calculated as described in Materials and Methods. Curves represent mean percent R18 dequenching for three independent experiments, monitored for triplicate wells. Error bars indicate SEM.

Fig 8

HMPV fusion is not triggered by HMPV G or RGD-binding integrins. (A) R18 F-VLP fusion was measured in the absence (circles) or presence (triangles) of neutralizing HMPV antiserum or for heat-inactivated particles (squares). R18-G-VLP (diamonds) dequenching was monitored as a background control. Percent R18 dequenching was calculated as described in Materials and Methods, and curves represent means for three independent experiments monitored for triplicate wells. Error bars indicate SEM. (B and C) HMPV G does not alter HMPV F-mediated fusion kinetics or the extent of fusion at 4 h. R18-labeled HMPV (black circles), F-VLPs (gray squares), or F+G-VLPs (open squares) were bound to the surface of BEAS-2B cells, and R18 fluorescence was monitored for 4 h. Triton X-100 was added after 4 h to determine the extent of virus or VLP fusion. Curves in panel B represent mean percent R18 dequenching for three independent experiments, monitored for duplicate wells. Bars in panel C represent the extent of fusion observed after 4 h for three independent experiments; error bars indicate SEM. *, P < 0.05; N.S., P > 0.05. (D to F) HMPV F binding to RGD-binding integrins does not alter HMPV fusion kinetics. R18-labeled HMPV (D), F-VLP (E), or F+G-VLP (F) fusion was assessed in the absence of antibodies (black lines) or in the presence of integrin function-blocking antibodies against α2 integrins (gray lines) or all RGD-binding integrins (αV plus α5 plus β1) (dotted lines). Curves represent mean percent R18 dequenching for three independent experiments monitored for duplicate wells. Error bars are not shown for figure clarity. Dequenching rates were not significantly altered in the presence of integrin antibodies; however, significantly less HMPV and VLPs bound during RGD-binding integrin blockade (data not shown). (G) F-RGD (wt) VLPs (10 μg), F-RAE VLPs (10 μg), and producer cell lysates (50 μg) were analyzed by Western blotting. Uncleaved (F0) and cleaved (F1) HMPV F were detected with an F-specific MAb and fluorescent secondary antibody using the Li-Cor Odyssey infrared imaging system. C, untransfected 293-F cell lysate. (H) The RGD integrin-binding motif is not required for efficient F-mediated hemifusion. R18 VLP fusion was measured for F-RGD (circles), F-RAE (triangles), or G-only (squares) particles as described for panel A.

Fig 9

Productive HMPV transcription depends upon RGD-binding integrin-mediated virus entry. HMPV (MOI = 0.25 PFU/cell) was bound to the surface of BEAS-2B cells in the absence or presence of α2 integrin or RGD-binding integrin (αV plus α5 plus β1) function-blocking antibodies. Levels of HMPV N transcript were determined at 8 h postinfection by real-time RT-PCR, relative to the GAPDH cellular gene. The 2^−ΔΔCT method was used to correct for input genome and compare transcript levels in treated and untreated samples. Data are presented as the average level of HMPV N transcript detected relative to an untreated (no-MAb) control; error bars indicate SEM for six biological replicates from two independent experiments.