Pathogenic hantaviruses bind plexin-semaphorin-integrin domains present at the apex of inactive, bent alphavbeta3 integrin conformers

Abstract

The alphavbeta3 integrins are linked to human bleeding disorders, and pathogenic hantaviruses regulate the function of alphavbeta3 integrins and cause acute vascular diseases. alphavbeta3 integrins are present in either extended (active) or dramatically bent (inactive) structures, and interconversion of alphavbeta3 conformers dynamically regulates integrin functions. Here, we show that hantaviruses bind human alphavbeta3 integrins and that binding maps to the plexin-semaphorin-integrin (PSI) domain present at the apex of inactive, bent, alphavbeta3-integrin structures. Pathogenic hantaviruses [New York-1 virus (NY-1V) and Hantaan virus (HTNV)] bind immobilized beta3 polypeptides containing the PSI domain, and human (but not murine) beta3 polypeptides inhibit hantavirus infectivity. Substitution of human beta3 residues 1-39 for murine beta3 residues directed pathogenic hantavirus infection of nonpermissive CHO cells expressing chimeric alphavbeta3 receptors. Mutation of murine beta3 Asn-39 to Asp-39 present in human beta3 homologues (N39D) permitted hantavirus infection of cells and specified PSI domain residue interactions with pathogenic hantaviruses. In addition, cell-surface expression of alphavbeta3 locked in an inactive bent conformation conferred hantavirus infectivity of CHO cells. Our findings indicate that hantaviruses bind to a unique domain exposed on inactive integrins and, together with prior findings, suggest that this interaction restricts alphavbeta3 functions that regulate vascular permeability. Our findings suggest mechanisms for viruses to direct hemorrhagic or vascular diseases and provide a distinct target for modulating alphavbeta3-integrin functions.

Fig. 1.

β3-Integrin hantavirus interactions. (A) αvβ3 heterodimers were secreted from baculovirus infected Sf-9 cells as described (25). Secreted αvβ3 was purified by using ConA, followed by RGD-affinity chromatography (25), and detected by silver stain or Western blot analysis with rabbit anti-αvβ3. (B) Purified αvβ3 was incubated with NY-1V, HTNV, and PHV (DMEM/2% FCS) before adsorption to VeroE6 cells. Infected cells were methanol-fixed at 24 h after infection, immunoperoxidase-stained for hantavirus N-protein, and quantitated (17). ConA- and RGD-purified material from Bac-Rec-VP5-infected (26) Sf-9 cell supernatants was used as a control. Rabbit anti-αvβ3 (10 μg) binding to αvβ3 prevented αvβ3 from inhibiting infection. Results are presented as a percentage of infected cells (n ≈ 100) in the absence of αvβ3 and represent three independent experiments. (C) mAb LM-142 was bound to protein A/G resin and coated with secreted purified αvβ3 (500 ng). Vitronectin, fibronectin, RGD peptides (20 μg/μl), or BSA were adsorbed to αvβ3 resin in DMEM/2% FCS. NY-1V (5000 FFU) was adsorbed to untreated or protein-treated resin (2 h at 25°C). After washing (five times with DMEM/2% FCS), resin and bound virus were applied to VeroE6 cells, monolayers were washed, and at 48 h after infection, hantavirus-infected cells were quantitated, as shown in B. In two samples, NY-1V was pretreated with either secreted αvβ3 (200 ng) or control Sf-9 supernatent (Fig. 1B) before adsorption to resin containing αvβ3. Data are expressed as the percentage of control (resin without αvβ3). At least three independent experiments were performed with similar results. (D) β3-Integrin homologue swaps CHO cells expressing full-length Huαv. Indicated human or murine β3 integrins or β3-integrin chimeras were analyzed by FACS for αvβ3 and infected with pathogenic NY-1V, HTNV, or PHV (≈1,000 FFU) as described above, and cells were quantitated as in B at 24 h after infection. Chimeric β3 integrins (1–762) contain indicated human β3 residues inserted into a murine β3-integrin background. Data are expressed as percentage of mock-transfected CHO cells, and experiments were performed at least three times with similar results.

Fig. 2.

Binding to residues 1–136. Ni²⁺–NTA resin was incubated with 4 μg of a human or murine β3 polypeptides (residues 1–136; Fig. 7). NY-1V, HTNV, or PHV (≈5,000 FFU) were incubated with resin in DMEM/2% FCS (2 h, 25C). Resin was washed (five times, DMEM/2% FCS), and resin and bound virus were applied to VeroE6 cells and analyzed as described for Fig. 1C. The mean number of infected cells in the control (resin alone) was ≈100, and experiments were performed at least three times with similar results.

Fig. 3.

β3 polypeptides inhibit infection. Increasing amounts of human or murine β3 polypeptides (residues 1–53, Fig. 7) were incubated (2 h at 4°C in DMEM/2% FCS) with NY-1V, HTNV, and PHV, and they were subsequently adsorbed to VeroE6 cells. Polypeptide quantities are indicated as follows: none, black; 0.75 μM, white; 1.5 μM, dark gray; 3.0 μM, hatched; and 6 μM, light gray). Cells were washed, and infected cells at 24 h after infection were quantitated as described for Fig. 1B. The mean number of infected cells in controls (no added polypeptides) was ≈60, and experiments were performed at least three times with similar results.

Fig. 4.

PSI-domain residue interactions. (A) Human and murine β3-integrin homologues (residues 1–43) are aligned. Residue differences are shown in bold. Only three residues differ between human and bovine β3 (underlined). (B) Indicated murine β3-subunit residues were mutated to homologous human residues and expressed in 6-His-tagged 1–53 polypeptides. Mutant polypeptides (6 μM) were added during adsorption and assayed for their ability to inhibit infection as in Fig. 3. The mean number of infected cells in the controls (media alone) was ≈60, and experiments were repeated at least three times with similar results. (C) CHO cells (expressing Huαv and WT or mutated murine β3 integrins substituted with homologous human β3 residues at indicated positions) were analyzed by FACS for αvβ3 and assayed for their ability to confer hantavirus infection as in Fig. 1D. The mean number of infected cells in control CHO cells was ≈15, and results are representative of three independent experiments.

Fig. 5.

Bent and extended αvβ3 integrins. (A) VeroE6 cells were pretreated with Ca⁺² (1 mM) or Mn⁺² (40 μM) for 1 h before infection with NY-1V or HTNV. Virus was adsorbed (DMEM/2% FCS, 1 h), and the number of infected cells was quantitated at 24 h after infection, as described in Fig. 1B.(B) CHO cells transfected with Huαv and WT and locked-bent (αv-G307C and β3-R563C) (13) or locked-extended (β3-C5A) (21) β3 were analyzed by FACS for αvβ3; infected with NY-1V, HTNV, or PHV; and assayed as described for Fig. 1D. The mean number of infected cells in controls was ≈15, and results are representative of three independent experiments. (C) Model of pathogenic hantavirus interaction with bent αvβ3. For integrin, the α-subunit (gold), β-subunit (blue), and PSI domain (black) are indicated. For hantavirus proteins, G₁/G₂ (red and blue), N protein (green), and polymerase (yellow) are indicated.