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Comparative Study
, 11 (5), 515-21

Integrin alphavbeta3 Is a Coreceptor for Human Cytomegalovirus

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Comparative Study

Integrin alphavbeta3 Is a Coreceptor for Human Cytomegalovirus

Xin Wang et al. Nat Med.

Abstract

Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that causes birth defects in newborns and severe disease in immunocompromised individuals. The broad tropism of HCMV infection suggests that it uses multiple receptors. We recently showed that the epidermal growth factor receptor (EGFR) serves as a receptor for HCMV. Here we show that HCMV also uses integrin alphavbeta3 as a coreceptor. Upon infection, HCMV glycoproteins gB and gH independently bind to EGFR and alphavbeta3, respectively, to initiate viral entry and signaling. Alphavbeta3 then translocates to lipid rafts where it interacts with EGFR to induce coordinated signaling. The coordination between EGFR and alphavbeta3 is essential for the early events of HCMV infection, including viral entry, RhoA downregulation, stress-fiber disassembly and viral nuclear trafficking. Our findings support a model in which EGFR and alphavbeta3 work together as coreceptors for HCMV entry and signaling. This discovery is fundamental to understanding HCMV pathogenesis and developing treatment strategies targeted to viral receptors.

Figures

Figure 1
Figure 1
Both αvβ3 and EGFR are required for HCMV infection, and HCMV binds both EGFR and αvβ3 independently. (a, b) HCMV infection blocking assays revealed that inhibition occurred when EGFR or αvβ3 was blocked (a), and the effects were dose-dependent (b). (c) IE gene products were detected in cells expressing both EGFR and αvβ3. (d) IE gene products decreased when HCMV-EGFR or HCMV-αvβ3 binding was inhibited, (e) EGFR- and αvβ3-specific antibodies inhibited HCMV binding, as determined by real-time PCR. (f) Immunofluorescence photomicrographs localizing UL94 show that binding of HCMV to MB468 cells was inhibited by blocking HCMV-EGFR or HCMV-αvβ3 interaction.
Figure 2
Figure 2
Coordinated signaling between EGFR and αvβ3 is generated when both αvβ3 and EGFR are activated via gH and gB, respectively. (a) Both αvβ3 and EGFR were activated upon HCMV infection, concurrently with the activation of downstream Src and PI3-K signaling. (b,c) HCMV activated EGFR-mediated PI3-K and αvβ3-mediated Src signaling. Coordination between EGFR-and αvβ3-mediated signal occurred when both receptors were activated, (d1) HCMV interacted with αvβ3 via gH. (d2) HCMV-induced phosphorylation of β3 and Src were inhibited by blocking gH. (e) Crosstalk between EGFR and αvβ3 was inhibited by Ly29 or PP2 but not by U0126. PO4: phosphorylated; Constru: construct.
Figure 3
Figure 3
HCMV induces the formation of an EGFR-αvβ3 complex when both αvβ3 and EGFR are activated. (a) EGFR co-precipitated with αvβ3 but not with αvβ5 in lysates from HCMV-infected HEL cells at 5 and 10 min p.i. (b) The interaction between EGFR and αvβ3 was disrupted by inhibiting the activation of EGFR, αvβ3, PI3-K, or Src. IP: immunoprecipitation; WB: western blot.
Figure 4
Figure 4
The lipid raft microdomain is involved in regulating HCMV-induced formation of EGFR-αvβ3 complex and coordination of signaling. (a1, a2) Activated αvβ3 moved from non-raft fractions (a2) into lipid raft fractions (a1), where it colocalized with activated EGFR at 5 and 10 min p.i. (b1, b2) EGFR and αvβ3, together with p85 subunit of PI3-K and Src, formed multimeric complexes only when both receptors were co-localized in lipid raft microdomains. (c) Disruption of lipid rafts with MβCD reduced HCMV-induced signaling coordination. The effect of MβCD was reversed by cholesterol (Chol). TfR: transferrin receptor.
Figure 5
Figure 5
Coordination of PI3-K signaling and Src signaling within lipid rafts is required for HCMV entry. (a) Ly29, PP2, and MβCD inhibited HCMV entry (as determined by real-time PCR) into HEL cells. Results are shown as percentages of inhibition, (b) Immunofluorescence photomicrographs localizing UL94 show that Ly29, PP2, and MβCD inhibited the internalization of HCMV into MB468 cells.
Figure 6
Figure 6
Coordination between EGFR- and αvβ3-mediated signaling is required for HCMV-induced downregulation of RhoA activity, disruption of stress fibers, and virus nuclear translocation. (a) HCMV induced downregulation of the RhoA/cofilin pathway, (b) RhoA/cofilin downregulation was inhibited by blocking EGFR- or αvβ3-mediated signaling, (c) Ly29 or PP2 inhibited HCMV nuclear translocation (determined by real-time PCR), which was relieved by Y-27632. (d) HCMV induced stress fiber disassembly, which correlated with UL94 nuclear translocation. (e) Stress fiber disassembly and UL94 nuclear translocation were inhibited by blocking PI3-K or Src signaling. The inhibition was relieved by Y-27632. Cytochalasin B (Cyto B) facilitated HCMV nuclear translocation.

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