Integrin β1 Mediates Vaccinia Virus Entry through Activation of PI3K/Akt Signaling

Abstract

Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin β1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin β1 in lipid rafts on the cell surface, and the knockdown of integrin β1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin β1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin β1-dependent manner, suggesting that integrin β1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin β1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.

Vaccinia Virus Infection Requires Maturation of Macropinosomes

“…Our RNAi screen indicated that PI(3)K, PI(4)K, PI(5)K and PIKfyve were needed for MV infection (Figure 2). Previous studies have demonstrated a role for PI(3)K during multiple stages of VACV infection 68, 69 including entry 34, 39, 70. Consistent with this, PtdIns(3)P was detected on early MV‐containing macropinosomes until 30 min suggesting that these compartments undergo a lipid switch during their maturation (Figure 4A,B).…”

“…PIKfyve facilitates Rab7a‐independent fusion of LEs with each other and with LYs 30. In addition, the PIKfyve‐activating kinase, Akt, previously implicated in VACV entry 70, 71, was a hit in our screen. Interestingly, Akt has been reported to phosphorylate PIKfyve to facilitate lysosomal trafficking and degradation of EGFR 72.…”

Vascular Endothelial Growth Factor A Promotes Vaccinia Virus Entry into Host Cells via Activation of the Akt Pathway

“…In our study, silencing VEGF-A or inhibiting Akt activation did not prevent viral infection completely, supporting the concept that there are likely to be multiple pathways by which VV may enter cells, which may differ across cell types (43). Integrin ␤1-mediated phosphatidylinositol 3-kinase (PI3K)/Akt activation has very recently been reported to be essential for vaccinia virus entry (44). Integrin ␤1 is important for both vaccinia virus attachment and penetration.…”

Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

“…To facilitate the determination of the binding residues, we applied three-dimensional heteronuclear correlation NMR experiments (47,48) to the A17 fragments through uniform 13 Tables 1-3 and Fig. 2, B and C).…”

“…The viral L1 protein also mediates nonglycosaminoglycan binding to cells (11). Subsequently, the MV particles cluster at membrane lipid rafts (12), interact with integrin (13) and CD98 (14), and then undergo endocytosis (15)(16)(17). After uptake into cellular vesicles, the acidic environment triggers membrane fusion mediated by the viral entry fusion complex (18).…”