NLRP3 inflammasome activation by Foot-and-mouth disease virus infection mainly induced by viral RNA and non-structural protein 2B.

Foot-and-mouth disease virus (FMDV) is a positive-strand RNA virus of the family Picornaviridae. Early studies show that some viruses of Picornaviridae, such as EMCV and EV71, induce NLRP3 inflammasome activation. Our current study demonstrates that FMDV induces the secretion of caspase-1 and interleukin 1 beta (IL-1ß), as well as activates the NLRP3 inflammasome in a dose- and time-dependent manner. Meanwhile, NLRP3 inflammasome can suppress FMDV replication during virus infection. Both FMDV RNA and viroporin 2B stimulate NLRP3 inflammasome activation. FMDV RNA triggers NLRP3 inflammasome through p-NF-κB/p65 pathway not dependent on RIG-I inflammasome. FMDV 2B activates NLRP3 inflammasome through elevation of intracellular ion, but not dependent on mitochondrial reactive oxygen species (ROS) and lysosomal cathepsin B. It further demonstrates that 2B viroporin activates NLRP3 inflammasome and induces IL-1ß in mice, which enhances the specific immune response against FMDV as an ideal self-adjuvant for FMD VLPs vaccine in guinea pigs. The results reveal a series of regulations between NLRP3 inflammasome complex and FMDV. Amino acids 140-145 of 2B is essential for forming an ion channel. By mutating the amino acid and changing the hydrophobic properties, the helical transmembrane region of the viroporin 2B is altered, so that the 2B is insufficient to trigger the activation of NLRP3 inflammasome. This study demonstrates the functions of FMDV RNA and 2B viroporin activate NLRP3 inflammasome and provides some useful information for the development of FMD vaccine self-adjuvant, which is also helpful for the establishment of effective prevention strategies by targeting NLRP3 inflammasome.

Foot-and-mouth disease virus infection stimulates innate immune signaling in the mouse macrophage RAW 264.7 cells.

The innate immune system acts as the first line of defense against invasion by bacterial and viral pathogens. The role of macrophages in innate immune responses to foot-and-mouth disease virus (FMDV) is poorly understood. To determine the mechanism underlying activation of innate immunity after FMDV infection in macrophages, we performed FMDV infection in mouse macrophage RAW 264.7 cells and found that FMDV serotype O infection induced a cytopathic effect. We then evaluated the gene expression profile in macrophage RAW 264.7 cells after FMDV infection using systematic microarray analysis. Gene ontology annotation and enrichment analysis revealed that FMDV promoted expression in a group of genes that are enriched in innate immune response and inflammatory response processes. Further research demonstrated that FMDV serotype O infection enhanced NF-κB, Toll-like, and RIG-I-like receptor signaling pathways and proteins expression and increased transcription and expression of a series of cytokines and interferons, as proved by qRT-PCR, Western blot, ELISA, and dual-luciferase reporter assay. Our study concluded that FMDV infection triggers the innate immune response in macrophages after activation of multiple innate immune pathway receptors and proteins by FMDV serotype O, resulting in activation and secretion of a series of cytokines and interferons.

Foot-and-mouth disease virus-like particles as integrin-based drug delivery system achieve targeting anti-tumor efficacy.

The surface of foot-and-mouth disease virus (FMDV)-like particles (VLPs) contains a conserved arginine-glycine-aspartic acid (RGD) motif. Natural FMDV specifically attaches to overexpressed integrin receptors in several cancer cells. The FMDV VLPs produced in Escherichia coli were used for the first time as a delivery system of anti-tumor drug doxorubicin (DOX). The DOX-loaded VLPs exhibited a distinct release profile in different physiological conditions. The effects of FMDV-VLPs-DOX on cellular internalization and viability were evaluated in vitro by cell imaging, MTT assay and apoptosis, respectively. The anti-tumor efficacy in vivo was also determined in a nude mouse xenograft model based on tumor volume/weight and histological changes. The FMDV-VLPs-DOX complex significantly inhibited the proliferation of tumor and improved the pathological damage of DOX to non-targeting tissues. All results supported the potential of FMDV VLPs as a platform for specific targeted delivery of drugs or chemical reagents.

The application of virus-like particles as vaccines and biological vehicles.

Virus-like particles (VLPs) can be spontaneously self-assembled by viral structural proteins under appropriate conditions in vitro while excluding the genetic material and potential replication probability. In addition, VLPs possess several features including can be rapidly produced in large quantities through existing expression systems, highly resembling native viruses in terms of conformation and appearance, and displaying repeated cluster of epitopes. Their capsids can be modified via genetic insertion or chemical conjugation which facilitating the multivalent display of a homologous or heterogeneous epitope antigen. Therefore, VLPs are considered as a safe and effective candidate of prophylactic and therapeutic vaccines. VLPs, with a diameter of approximately 20 to 150 nm, also have the characteristics of nanometer materials, such as large surface area, surface-accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), inerratic spatial structure, and good biocompatibility. Therefore, assembled VLPs have great potential as a delivery system for specifically carrying a variety of materials. This review summarized recent researches on VLP development as vaccines and biological vehicles, which demonstrated the advantages and potential of VLPs in disease control and prevention and diagnosis. Then, the prospect of VLP biology application in the future is discussed as well.

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

MiR-361 and miR-34a suppress foot-and-mouth disease virus proliferation by activating immune response signaling in PK-15 cells.

Foot-and-mouth disease (FMD) severely impacts cloven-hoofed live-stock production, leading to serious economic losses and international restriction on the trade of animals and animal products worldwide. MiRNAs serve key roles in viral immunity and regulation. However, the knowledge about miRNAs regulation in FMDV infection is still limited. In this study, we found that FMDV infection caused rapid cytopathic in PK-15 cell. To investigate the miRNAs' function in FMDV infection, we performed knockdown of endogenous Dgcr8 using its specific siRNA and found that interference of Dgcr8 inhibited cellular miRNA expression and increased FMDV production, including viral capsid proteins expression, viral genome copies and virus titer, suggesting that miRNAs play an important role in FMDV infection. To obtain a full perspective on miRNA expression profiling after FMDV infection, we performed miRNA sequencing and found that FMDV infection caused inhibition of miRNA expression in PK-15 cells. Together with the target prediction result, miR-34a and miR-361 were screened for further study. Function study showed that no matter plasmid or mimics-mediated overexpression of miR-34a and miR-361 both suppressed FMDV replication, while inhibition of endogenous miR-34a and miR-361 expression using specific inhibitors significantly increased FMDV replication. Further study showed that miR-34a and miR-361 stimulated IFN-ß promoter activity and activated interferon-stimulated response element (ISRE). In addition, ELISA test found that miR-361 and miR-34a increased secretion level of IFN-ß and IFN-γ, which may contribute to repression of FMDV replication. This study preliminary revealed that miR-361 and miR-34a inhibited FMDV proliferation via stimulating immune response.

Foot-and-mouth disease virus infection suppresses autophagy and NF-кB antiviral responses via degradation of ATG5-ATG12 by 3Cpro.

Autophagy-related protein ATG5-ATG12 is an essential complex for the autophagophore elongation in autophagy, which has been reported to be involved in foot-and-mouth disease virus (FMDV) replication. Previous reports show that ATG5-ATG12 positively or negatively regulates type I interferon (IFN-α/ß) pathway during virus infection. In this study, we found that FMDV infection rapidly induced LC3 lipidation and GFP-LC3 subcellular redistribution at the early infection stage in PK-15 cells. Along with infection time course to 2-5 h.p.i., the levels of LC3II and ATG5-ATG12 were gradually reduced. Further study showed that ATG5-ATG12 was degraded by viral protein 3Cpro, demonstrating that FMDV suppresses autophagy along with viral protein production. Depletion of ATG5-ATG12 by siRNA knock down significantly increased the FMDV yields, whereas overexpression of ATG5-ATG12 had the opposite effects, suggesting that degradation of ATG5-ATG12 benefits virus growth. Further experiment showed that overexpression of ATG5-ATG12 positively regulated NF-кB pathway during FMDV infection, marked with promotion of IKKα/ß phosphorylation and IκBα degradation, inhibition of p65 degradation, and facilitation of p65 nuclear translocation. Meanwhile, ATG5-ATG12 also promoted the phosphorylation of TBK1 and activation of IRF3 via preventing TRAF3 degradation. The positive regulation of NF-кB and IRF3 pathway by ATG5-ATG12 resulted in enhanced expression of IFN-ß, chemokines/cytokines, and IFN stimulated genes, including anti-viral protein PKR. Altogether, above findings suggest that ATG5-ATG12 positively regulate anti-viral NF-κB and IRF3 signaling during FMDV infection, thereby limiting FMDV proliferation. FMDV has evolved mechanisms to counteract the antiviral function of ATG5-ATG12, via degradation of them by viral protein 3Cpro.

Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase.

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.

Quantitative Proteomic Analysis of BHK-21 Cells Infected with Foot-and-Mouth Disease Virus Serotype Asia 1.

Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantitatively study the host cell gene expression profile, in order to achieve an unbiased overview of the protein expression changes in BHK-21 cells infected with FMDV serotype Asia 1. The SILAC-based approach identified overall 2,141 proteins, 153 of which showed significant alteration in the expression level 6 h post FMDV infection (57 up-regulated and 96 down-regulated). Among these proteins, six cellular proteins, including three down-regulated (VPS28, PKR, EVI5) and three up-regulated (LYPLA1, SEC62 and DARs), were selected according to the significance of the changes and/or the relationship with PKR. The expression level and pattern of the selected proteins were validated by immunoblotting and confocal microscopy. Furthermore, the functions of these cellular proteins were assessed by small interfering RNA-mediated depletion, and their functional importance in the replication of FMDV was demonstrated by western blot, reverse transcript PCR (RT-PCR) and 50% Tissue Culture Infective Dose (TCID50). The results suggest that FMDV infection may have effects on the expression of specific cellular proteins to create more favorable conditions for FMDV infection. This study provides novel data that can be utilized to understand the interactions between FMDV and the host cell.

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein.

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca(2+) concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.