m6A reader proteins: the executive factors in modulating viral replication and host immune response.

N6-Methyladenosine (m6A) modification is the most abundant covalent modification of RNA. It is a reversible and dynamic process induced by various cellular stresses including viral infection. Many m6A methylations have been discovered, including on the genome of RNA viruses and on RNA transcripts of DNA viruses, and these methylations play a positive or negative role on the viral life cycle depending on the viral species. The m6A machinery, including the writer, eraser, and reader proteins, achieves its gene regulatory role by functioning in an orchestrated manner. Notably, data suggest that the biological effects of m6A on target mRNAs predominantly depend on the recognition and binding of different m6A readers. These readers include, but are not limited to, the YT521-B homology (YTH) domain family, heterogeneous nuclear ribonucleoproteins (HNRNPs), insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs), and many others discovered recently. Indeed, m6A readers have been recognized not only as regulators of RNA metabolism but also as participants in a variety of biological processes, although some of these reported roles are still controversial. Here, we will summarize the recent advances in the discovery, classification, and functional characterization of m6A reader proteins, particularly focusing on their roles and mechanisms of action in RNA metabolism, gene expression, and viral replication. In addition, we also briefly discuss the m6A-associated host immune responses in viral infection.

Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication.

Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.

Hsp70-1: upregulation via selective phosphorylation of heat shock factor 1 during coxsackieviral infection and promotion of viral replication via the AU-rich element.

Coxsackievirus B3 (CVB3) is the primary pathogen of viral myocarditis. Upon infection, CVB3 exploits the host cellular machineries, such as chaperone proteins, to benefit its own infection cycles. Inducible heat shock 70-kDa proteins (Hsp70s) are chaperone proteins induced by various cellular stress conditions. The internal ribosomal entry site (IRES) within Hsp70 mRNA allows Hsp70 to be translated cap-independently during CVB3 infection when global cap-dependent translation is compromised. The Hsp70 protein family contains two major members, Hsp70-1 and Hsp70-2. This study showed that Hsp70-1, but not Hsp70-2, was upregulated during CVB3 infection both in vitro and in vivo. Then a novel mechanism of Hsp70-1 induction was revealed in which CaMKIIγ is activated by CVB3 replication and leads to phosphorylation of heat shock factor 1 (HSF1) specifically at Serine 230, which enhances Hsp70-1 transcription. Meanwhile, phosphorylation of Ser230 induces translocation of HSF1 from the cytoplasm to nucleus, thus blocking the ERK1/2-mediated phosphorylation of HSF1 at Ser307, a negative regulatory process of Hsp70 transcription, further contributing to Hsp70-1 upregulation. Finally, we demonstrated that Hsp70-1 upregulation, in turn, stabilizes CVB3 genome via the AU-rich element (ARE) harbored in the 3' untranslated region of CVB3 genomic RNA.

Coxsackievirus-induced miR-21 disrupts cardiomyocyte interactions via the downregulation of intercalated disk components.

Intercalated disks (ICDs) are substantial connections maintaining cardiac structures and mediating signal communications among cardiomyocytes. Deficiency in ICD components such as desmosomes, fascia adherens and gap junctions leads to heart dysfunction. Coxsackievirus B3 (CVB3) infection induces cardiac failure but its pathogenic effect on ICDs is unclear. Here we show that CVB3-induced miR-21 expression affects ICD structure, i.e., upregulated miR-21 targets YOD1, a deubiquitinating enzyme, to enhance the K48-linked ubiquitination and degradation of desmin, resulting in disruption of desmosomes. Inhibition of miR-21 preserves desmin during CVB3 infection. Treatment with proteasome inhibitors blocks miR-21-mediated desmin degradation. Transfection of miR-21 or knockdown of YOD1 triggers co-localization of desmin with proteasomes. We also identified K108 and K406 as important sites for desmin ubiquintination and degradation. In addition, miR-21 directly targets vinculin, leading to disturbed fascia adherens evidenced by the suppression and disorientation of pan-cadherin and α-E-catenin proteins, two fascia adherens-components. Our findings suggest a new mechanism of miR-21 in modulating cell-cell interactions of cardiomyocytes during CVB3 infection.

MiR-126 promotes coxsackievirus replication by mediating cross-talk of ERK1/2 and Wnt/ß-catenin signal pathways.

Coxsackievirus B3 (CVB3) is one of the most prevalent causes of viral myocarditis and is associated with many other pathological conditions. CVB3 replication relies on host cellular machineries and causes direct damage to host cells. MicroRNAs have been found to regulate viral infections but their roles in CVB3 infection are still poorly understood. Here we describe a novel mechanism by which miR-126 regulates two signal pathways essential for CVB3 replication. We found that CVB3-induced ERK1/2 activation triggered the phosphorylation of ETS-1 and ETS-2 transcription factors, which induced miR-126 upregulation. By using both microRNA mimics and inhibitors, we proved that the upregulated miR-126 suppressed sprouty-related, EVH1 domain containing 1 (SPRED1) and in turn enhanced ERK1/2 activation. This positive feedback loop of ERK1/2-miR-126-ERK1/2 promoted CVB3 replication. Meanwhile, miR-126 expression stimulated GSK-3ß activity and induced degradation of ß-catenin through suppressing LRP6 and WRCH1, two newly identified targets in the Wnt/ß-catenin pathway, which sensitized the cells to virus-induced cell death and increased viral progeny release to initiate new infections. Our results demonstrate that upregulated miR-126 upon CVB3 infection targets SPRED1, LRP6, and WRCH1 genes, mediating cross-talk between ERK1/2 and Wnt/ß-catenin pathways, and thus promoting viral replication and contributes to the viral cytopathogenicity.

Antisense DNA and RNA agents against picornaviruses.

Anti-picornaviral antisense agents are part of a broader group of nucleic acid-based molecules developed for sequence-specific inhibition of translation and/or transcription of the target sequence through induced nuclease activity or physical hindrance. Three types of nucleic acid-based gene silencing molecules can be distinguished, including DNA-base antisense oligonucleotides (ASO), nucleic acid enzymes (ribozyme and DNAzyme) and double-stranded small interfering RNA (siRNA or microRNA). These antisense DNA and RNA molecules have been widely studied for gene functional studies and therapeutic purposes. In this review, we focus on drug development using ASO and siRNA strategies to inhibit picornavirus infections. The picornavirus genome organization and life cycle is described, followed by discussion of design considerations, chemical modifications and drug delivery approaches. Recent studies using antisense against picornavirus are reviewed. Finally, we compare the advantages and disadvantages of the antisense agents with those of other therapeutics, taking into consideration their limitations which need to be overcome to achieve the final goal of clinical application.