Abstract
Virus entry is an attractive target for therapeutic intervention. Here, using a combination of electron microscopy, immunofluorescence assay, siRNA interference, specific pharmacological inhibitors, and dominant negative mutation, we demonstrated that the entry of foot-and-mouth disease virus (FMDV) triggered a substantial amount of plasma membrane ruffling. We also found that the internalization of FMDV induced a robust increase in fluid-phase uptake, and virions internalized within macropinosomes colocalized with phase uptake marker dextran. During this stage, the Rac1-Pak1 signaling pathway was activated. After specific inhibition on actin, Na+/H+ exchanger, receptor tyrosine kinase, Rac1, Pak1, myosin II, and protein kinase C, the entry and infection of FMDV significantly decreased. However, inhibition of phosphatidylinositol 3-kinase (PI3K) did not reduce FMDV internalization but increased the viral entry and infection to a certain extent, implying that FMDV entry did not require PI3K activity. Results showed that internalization of FMDV exhibited the main hallmarks of macropinocytosis. Moreover, intracellular trafficking of FMDV involves EEA1/Rab5-positive vesicles. The present study demonstrated macropinocytosis as another endocytic pathway apart from the clathrin-mediated pathway. The findings greatly expand our understanding of the molecular mechanisms of FMDV entry into cells, as well as provide potential insights into the entry mechanisms of other picornaviruses.
Highlights
foot-and-mouth disease virus (FMDV) is non-enveloped and consists of a densely packed icosahedral protein shell, which is approximately 30 nm in diameter surrounding a single-stranded, positive-sense RNA genome of about 8400 nt in length[3,4]
Optical microscope observations revealed that after the BHK-21 cells were infected by FMDV (MOI 1) for 4 h, the cells presented obvious cytopathic effect (CPE)
Studies showed that the field isolates of FMDV invade the cells via a clathrin-mediated endocytosis (CME) pathway involving the integrin receptor[27,28,29,30]
Summary
FMDV is non-enveloped and consists of a densely packed icosahedral protein shell, which is approximately 30 nm in diameter surrounding a single-stranded, positive-sense RNA genome of about 8400 nt in length[3,4]. Clathrin-coated vesicles containing the endocytic cargo are formed with a diameter of about 120 nm, which deliver the cargo into endosomes This process depends on adaptor protein 2, GTPase dynamin, and other important accessory proteins, such as AP180 and Eps[157,8]. Viruses using this pathway include Semliki forest virus[9], vesicular stomatitis virus[10], and adenovirus 2/58 Another known endocytic pathway is mediated by caveolin with the formation of caveolae, which are flask-shaped invaginations in the plasma membrane. These ruffles collapse toward the cell membrane, enveloping the virus-receptor complex together with the dissociated virus and other fluid-phase macromolecules[13,14,15,16] They pinch off from the cell membrane and enter the cytoplasm as large (0.5–10 μ m) and irregularly shaped endocytic vesicles known as macropinosomes[14,15,16]. This study demonstrated a novel pathway for FMDV entry and may facilitate understanding of the endocytic mechanisms of other picornaviruses
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