Nucleolin Promotes IRES-Driven Translation of Foot-and-Mouth Disease Virus by Supporting the Assembly of Translation Initiation Complexes.

Nucleolin (NCL), a stress-responsive RNA-binding protein, has been implicated in the translation of internal ribosome entry site (IRES)-containing mRNAs, which encode proteins involved in cell proliferation, carcinogenesis, and viral infection (type I IRESs). However, the details of the mechanisms by which NCL participates in IRES-driven translation have not hitherto been described. Here, we identified NCL as a protein that interacts with the IRES of foot-and-mouth disease virus (FMDV), which is a type II IRES. We also mapped the interactive regions within FMDV IRES and NCL in vitro. We found that NCL serves as a substantial regulator of FMDV IRES-driven translation but not of bulk cellular or vesicular stomatitis virus cap-dependent translation. NCL also modulates the translation of and infection by Seneca Valley virus (type III-like IRES) and classical swine fever virus (type III IRES), which suggests that its function is conserved in unrelated IRES-containing viruses. We also show that NCL affects viral replication by directly regulating the production of viral proteins and indirectly regulating FMDV RNA synthesis. Importantly, we observed that the cytoplasmic relocalization of NCL during FMDV infection is a substantial step for viral IRES-driven translation and that NCL specifically promotes the initiation phase of the translation process by recruiting translation initiation complexes to viral IRES. Finally, the functional importance of NCL in FMDV pathogenicity was confirmed in vivo. Taken together, our findings demonstrate a specific function for NCL in selective mRNA translation and identify a target for the development of a broad-spectrum class of antiviral interventions. IMPORTANCE FMDV usurps the cellular translation machinery to initiate viral protein synthesis via a mechanism driven by IRES elements. It allows the virus to shut down bulk cellular translation, while providing an advantage for its own gene expression. With limited coding capacity in its own genome, FMDV has evolved a mechanism to hijack host proteins to promote the recruitment of the host translation machinery, a process that is still not well understood. Here, we identified nucleolin (NCL) as a positive regulator of the IRES-driven translation of FMDV. Our study supports a model in which NCL relocalizes from the nucleus to the cytoplasm during the course of FMDV infection, where the cytoplasmic NCL promotes FMDV IRES-driven translation by bridging the translation initiation complexes with viral IRES. Our study demonstrates a previously uncharacterized role of NCL in the translation initiation of IRES-containing viruses, with important implications for the development of broad antiviral interventions.

Ribosomal Protein L13 Promotes IRES-Driven Translation of Foot-and-Mouth Disease Virus in a Helicase DDX3-Dependent Manner.

Internal ribosome entry site (IRES)-driven translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. In the current study, we identified the ribosomal protein L13 (RPL13) as a critical regulator of IRES-driven translation of foot-and-mouth disease virus (FMDV) but found that it is not essential for cellular global translation. RPL13 is also a determinant for translation and infection of Seneca Valley virus (SVV) and classical swine fever virus (CSFV), and this suggests that its function may also be conserved in unrelated IRES-containing viruses. We further showed that depletion of DEAD box helicase DDX3 disrupts binding of RPL13 to the FMDV IRES, whereas the reduction in RPL13 expression impairs the ability of DDX3 to promote IRES-driven translation directly. DDX3 cooperates with RPL13 to support the assembly of 80S ribosomes for optimal translation initiation of viral mRNA. Finally, we demonstrated that DDX3 affects the recruitment of the eukaryotic initiation factor eIF3 subunits e and j to the viral IRES. This work provides the first connection between DDX3 and eIF3e/j and recognition of the role of RPL13 in modulating viral IRES-dependent translation. This previously uncharacterized process may be involved in selective mRNA translation.IMPORTANCE Accumulating evidence has unveiled the roles of ribosomal proteins (RPs) belonging to the large 60S subunit in regulating selective translation of specific mRNAs. The translation specificity of the large-subunit RPs in this process is thought provoking, given the role they play canonically in catalyzing peptide bond formation. Here, we have identified the ribosomal protein L13 (RPL13) as a critical regulator of IRES-driven translation during FMDV infection. Our study supports a model whereby the FMDV IRESs recruit helicase DDX3 recognizing RPL13 to facilitate IRES-driven translation, with the assistance of eIF3e and eIF3j. A better understanding of these specific interactions surrounding IRES-mediated translation initiation could have important implications for the selective translation of viral mRNA and thus for the development of effective prevention of viral infection.

Sec62 Suppresses Foot-and-Mouth Disease Virus Proliferation by Promotion of IRE1α-RIG-I Antiviral Signaling.

Foot-and-mouth disease virus (FMDV) is highly infectious and causes a major plague in animal farming. Unfolded protein response is one of the major cellular responses to pathogenic infections, which performs a crucial role in cell survival, apoptosis, and antiviral innate immune response. In this study, we showed that FMDV infection activated two unfolded protein response branches (PERK-eIF2α and ATF6 signaling) in both baby hamster kidney cells (BHK-21) and porcine kidney (PK-15) cells, whereas it suppressed the IRE1α-XBP1 signaling by decreasing IRE1α level. Further study revealed IRE1α signaling as an important antiviral innate immune mechanism against FMDV. Sec62, the transport protein, was greatly decreased at the late stages of FMDV infection. By overexpression and knockdown study, we also found that the expression of Sec62 was positively involved in the levels of IRE1α and RIG-I and subsequent activation of downstream antiviral signaling pathways in FMDV-infected PK-15 cells. Taken together, our study demonstrates that Sec62 is an important antiviral factor that upregulates IRE1α-RIG-I-dependent antiviral innate immune responses, and FMDV evades antiviral host defense mechanism by downregulating Sec62-IRE1α/RIG-I.

Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs.

Porcine vesicular disease caused by Senecavirus A (SVA) is a newly emerging disease in many countries. Based on clinical signs only, it is very challenging to distinguish SVA infection from other similar diseases, such as foot and mouth disease, swine vesicular disease, and vesicular stomatitis. Therefore, it is crucial to establish a detection assay for the clinical diagnosis of SVA infection. In this study, a pair of specific primers were designed based on the highly conserved L/VP4 gene sequence of SVA. The established SYBR green I-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect SVA nucleic acids in clinical samples. The limit of detection SVA nucleic acids by qRT-PCR was 6.4 × 101 copies/µL, which was significantly more sensitive than that by gel electrophoresis of 6.4 × 103 copes/µL. This assay was specific and had no cross-reaction with other seven swine viruses. Using SYBR green I-based qRT-PCR, the SVA positive rates in experimental animal samples and field samples were 67.60% (96/142) and 80% (24/30) respectively. The results demonstrate that SYBR green I-based qRT-PCR is a rapid and specific method for the clinical diagnosis and epidemiological investigation of related vesicular diseases caused by SVA.

Three-dimensional structure of foot-and-mouth disease virus and its biological functions.

Foot-and-mouth disease (FMD), an acute, violent, infectious disease of cloven-hoofed animals, remains widespread in most parts of the world. It can lead to a major plague of livestock and an economical catastrophe. Structural studies of FMD virus (FMDV) have greatly contributed to our understanding of the virus life cycle and provided new horizons for the control and eradication of FMDV. To examine host-FMDV interactions and viral pathogenesis from a structural perspective, the structures of viral structural and non-structural proteins are reviewed in the context of their relevance for virus assembly and dissociation, formation of capsid-like particles and virus-receptor complexes, and viral penetration and uncoating. Moreover, possibilities for devising novel antiviral treatments are discussed.

Ribosomal Protein L13 Participates in Innate Immune Response Induced by Foot-and-Mouth Disease Virus.

In addition to ribosomal protein synthesis and protein translation, ribosomal proteins also participate in tumorigenesis and tumor progression, immune responses, and viral replication. Here, we show that ribosomal protein L13 (RPL13) participates in the antiviral immune response induced by foot-and-mouth disease virus (FMDV), inhibiting FMDV replication. The overexpression of RPL13 promoted the induction and activation of the promoters of the nuclear factor-κB (NF-κB) and interferon-ß (IFN-ß) genes, and the expression and protein secretion of the antiviral factor IFN-ß and proinflammatory cytokine interleukin-6 (IL-6). The knockdown of RPL13 had the opposite effects. We also found that the FMDV 3Cpro protease interacts with RPL13, and that its activity reduces the expression of RPL13, thus antagonizing the RPL13-mediated antiviral activity. This study extends our knowledge of the extraribosomal functions of ribosomal proteins and provides new scientific information on cellular antiviral defenses and virus-antagonizing mechanisms.

The DDX23 Negatively Regulates Translation and Replication of Foot-and-Mouth Disease Virus and Is Degraded by 3C Proteinase.

DEAD-box helicase 23 (DDX23) is a host nuclear helicase, which is a part of the spliceosomal complex and involved in pre-mRNA splicing. To investigate whether DDX23, an internal ribosomal entry sites transacting factor (ITAF) affects foot-and-mouth disease virus (FMDV) replication and translation through internal ribosome entry site (IRES)-dependent manner. For this, we utilized a pull-down assay, Western blotting, quantitative real-time PCR, confocal microscopy, overexpression and small interfering RNA knockdown, as well as the median tissue culture infective dose. Our findings showed that FMDV infection inhibited DDX23 expression and the overexpression of DDX23 reduced viral replication, however, CRISPR Cas9 knockout/small interfering RNA knockdown increased FMDV replication. FMDV IRES domain III and IV interacted with DDX23, whereas DDX23 interacted with FMDV 3C proteinase and significantly degraded. The enzymatic activity of FMDV 3C proteinase degraded DDX23, whereas FMDV degraded DDX23 via the lysosomal pathway. Additionally, IRES-driven translation was suppressed in DDX23-overexpressing cells, and was enhanced in DDX23 knocked down. Collectively, our results demonstrated that DDX23 negatively affects FMDV IRES-dependent translation, which could be a useful target for the design of antiviral drugs.

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.