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Coxsackieviruses possess two proteases that are engaged in cleaving viral polyprotein and hijacking host cell processes such as RNA biosynthesis. Integrator subunit 10 (INTS10), a subunit of the integrator complex, facilitates the processing of small nuclear RNAs (U1 and U2 snRNAs) to regulate cellular transcription. We found that INST10 can be cleaved by Coxsackievirus B (CVB). Hence, we hypothesized that INST10 may play a role in CVB infection. In this study, INTS10 is identified as the substrate of CVB3 protease 3C (3Cpro). The cleavage occurs at the residue Q221 and yields a fragment. Depletion of INTS10 enhanced CVB3 replication and blocked snRNA processing. Overexpression of U1 snRNA inhibited CVB3 infection, whereas its knockdown conversely enhanced it. Similarly, knockdown of U2 snRNA was found to promote CVB3 replication. Taken together, the 3Cpro-mediated cleavage of INTS10 disrupts U snRNA processing, which in turn counteracts the inhibitory effect of snRNA U1 and U2 on virus replication and subverts host defenses.

Group B Coxsackieviruses (CVB) are one of the causative pathogens of myocarditis, which may progress to cardiomyopathy. The pathogenesis of CVB is not fully understood, and effective antiviral therapy is not available. N-acetylcysteine (NAC), the classic antioxidant, has been used in clinical practice for several decades to treat various medical conditions. In this study, the anti-CVB effect of NAC was investigated. We show that NAC dramatically suppressed viral replication and alleviated cardiac injury induced by CVB3. To further study the antiviral mechanism of NAC, RNA-sequencing was performed for CVB3-infected cells with NAC treatment. We found that eukaryotic elongation factor 1 alpha 1 (EEF1A1) is one of the most upregulated genes in CVB3-infected cells. However, EEF1A2, the highly homologous isoform of EEF1A1, remains unchanged. EEF1A1 expression was significantly suppressed by NAC treatment in CVB3-infected cells, while EEF1A2 was not affected. eEF1A1 knockdown significantly inhibited CVB3 replication, implicating that eEF1A1 facilitates viral replication. Importantly, we show that eEF1A1, which was not expressed in the myocardia of newborn mice, was significantly upregulated by CVB3 infection. NAC markedly downregulated the expression of eEF1A1 but not eEF1A2 in the myocardia of CVB3-infected mice. Furthermore, NAC accelerated eEF1A1 degradation by promoting autophagy in CVB3-infected cells. We show that p62, one of the critical adaptors of autophagic targets, interacts with eEF1A1 and was downregulated in CVB3-infected cells upon NAC treatment. Taken together, this study demonstrated that NAC shows a potent anti-CVB effect through the downregulation of eEF1A1.

Coxsackievirus B (CVB) belongs to Enterovirus genus within the Picornaviridae family, and it is one of the most common causative pathogens of viral myocarditis in young adults. The pathogenesis of myocarditis caused by CVB has not been completely elucidated. In CVB infection, autophagy is manipulated to facilitate viral replication. Here we report that protein 2B, one of the non-structural proteins of CVB3, possesses autophagy-inducing capability. The autophagy-inducing motif of protein 2B was identified by the generation of truncated 2B and site-directed mutagenesis. The expression of 2B alone was sufficient to induce the formation of autophagosomes in HeLa cells, while truncated 2B containing the two hydrophobic regions of the protein also induced autophagy. In addition, we demonstrated that a single amino acid substitution (56V→A) in the stem loop in between the two hydrophobic regions of protein 2B abolished the formation of autophagosomes. Moreover, we found that 2B and truncated 2B with autophagy-inducting capability were co-localized with LC3-II. This study indicates that protein 2B relies on its transmembrane hydrophobic regions to induce the formation of autophagosomes, while 56 valine residue in the stem loop of protein 2B might exert critical structural influence on its two hydrophobic regions. These results may provide new insight for understanding the molecular mechanism of autophagy triggered by CVB infection.

AbstractGlycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane protein that can modulate osteoblasts and bone mineralization. Periodontal disease (PD) is characterized by gum inflammation, alveolar bone resorption, and tooth loss. In this study, we found that GPNMB is highly expressed in inflamed periodontal tissue through microarray and immunohistochemistry (IHC) assays. The role of GPNMB in the pathogenesis of PD was evaluated with primary human periodontal ligament cells (hPDLCs) treated with lipopolysaccharide (LPS) and a GPNMB-expressing lentivirus (lenti-GP). In the hPDLCs treated with LPS and lenti-GP, the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 was suppressed and that of IL-10 was upregulated. GPNMB significantly decreased apoptosis in the hPDLCs treated with LPS. GPNMB could upregulate the expression of Jumonji domain-containing protein 3 (Jmjd3), a histone 3 lysine 27 (H3K27) demethylase that is linked to the modulation of the inflammatory response and apoptosis. Taken together, our data find that GPNMB is highly expressed in gum tissue with PD and may be an anti-inflammatory player in the pathogenesis of PD.

Coxsackievirus B (CVB) is the major causative pathogen for severe diseases such as viral myocarditis, meningitis, and pancreatitis. There is no effective antiviral therapy currently available for CVB infection primarily due to that the pathogenesis of CVB has not been completely understood. Viruses are obligate intracellular pathogens which subvert cellular processes to ensure viral replication. Dysregulation of ubiquitination has been implicated in CVB infection. However, how ubiquitination is involved in CVB infection remains unclear. Here we found that the 3D protein of CVB3, the RNA-dependent RNA polymerase, was modified at K220 by K48-linked polyubiquitination which promoted its degradation through proteasome. Proteomic analysis showed that the E3 ligase TRIM56 was upregulated in CVB3-infected cells, while the majority of TRIMs remained unchanged. Pull-down and immunoprecipitation analyses showed that TRIM56 interacted with CVB3 3D. Immunofluorescence observation showed that viral 3D protein was colocalized with TRIM56. TRIM56 overexpression resulted in enhanced ubiquitination of CVB3 3D and decreased virus yield. Moreover, TRIM56 was cleaved by viral 3C protease in CVB3-infected cells. Taken together, this study demonstrated that TRIM56 mediates the ubiquitination and proteasomal degradation of the CVB3 3D protein. These findings demonstrate that TRIM56 is an intrinsic cellular restriction factor against CVB infection, and enhancing viral protein degradation could be a potential strategy to control CVB infection.

BACKGROUND: Stress granules (SGs) are granular aggregates in the cytoplasm that are formed under a variety of stress situations including viral infection. Previous studies indicate that poliovirus, a member of Picornaviridae, can induce SG formation. However, the exact mechanism by which the picornaviruses induce SG formation is unknown. METHOD: The localization of SG markers in cells infected with coxsackievirus B3 (CVB3) or enterovirus 71 (EV71) and in cells expressing each viral protein was determined via immunofluorescence assays or plasmid transfection. Eight plasmids expressing mutants of the 2A protease (2Aᵖʳᵒ) of CVB3 were generated using a site-directed mutagenesis strategy. The cleavage efficiencies of eIF4G by CVB3 2Aᵖʳᵒ and its mutants were determined via western blotting assays. RESULTS: In this study, we found that CVB3 infection induced SG formation, as evidenced by the co-localization of some accepted SG markers in viral infection-induced granules. Furthermore, we identified that 2Aᵖʳᵒ of CVB3 was the key viral component that triggered SG formation. A 2Aᵖʳᵒ mutant with the G122E mutation, which exhibited very low cleavage efficiency toward eIF4G, significantly attenuated its capacity for SG induction, indicating that the protease activity was required for 2Aᵖʳᵒ to initiate SG formation. Finally, we observed that SGs also formed in EV71-infected cells. Expression of EV71 2Aᵖʳᵒ alone was also sufficient to cause SG formation. CONCLUSION: Both CVB3 and EV71 infections can induce SG formation, and 2Aᵖʳᵒ plays a crucial role in the induction of SG formation during these infections. This finding may help us to better understand how picornaviruses initiate the SG response.

Coxsackievirus B3 (CVB3) is a common causative agent in the development of inflammatory cardiomyopathy. However, whether the expression of peripheral blood microRNAs (miRNAs) is altered in this process is unknown. The present study investigated changes to miRNA expression in the peripheral blood of CVB3-infected mice. Utilizing miRNA microarray technology, differential miRNA expression was examined between normal and CVB3-infected mice. The present results suggest that specific miRNAs were differentially expressed in the peripheral blood of mice infected with CVB3, varying with infection duration. Using miRNA microarray analysis, a total of 96 and 89 differentially expressed miRNAs were identified in the peripheral blood of mice infected with CVB3 for 3 and 6 days, respectively. Quantitative polymerase chain reaction was used to validate differentially expressed miRNAs, revealing a consistency of these results with the miRNA microarray analysis results. The biological functions of the differentially expressed miRNAs were then predicted by bioinformatics analysis. The potential biological roles of differentially expressed miRNAs included hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. These results may provide important insights into the mechanisms responsible for the progression of CVB3 infection.

Aims: To evaluate the stability of coxsackievirus B (CVB) genome integrated with the enhanced green fluorescent protein gene (egfp) and provide valuable information for the use of the recombinant CVB variant. Methods: A CVB3 variant expressing eGFP was constructed by insertion of the egfp open-reading frame (ORF) at the 5′ end of CVB3 ORF. The recombinant virus CVB3-eGFP was serially passaged in HeLa cells. The deletions in the CVB3-eGFP genome around egfp were examined by reverse transcription polymerase chain reaction and sequencing. Results: Genomic deletions of CVB3-eGFP could be observed as early as the 2nd passage. Sequencing showed that the genomic deletions caused either viral ORF shifts or partial deletions of the viral VP4 coding sequence. The 6th passage of CVB3-eGFP was checked by plaque assay for eGFP expression. All plaque-like foci showed eGFP expression. eGFP expression was also viewed in HeLa cells infected with plaque-forming viruses. Conclusions: The insertion of egfp destabilized the CVB3 genome. The genomic deletions led to lethal mutations because of the termination of viral protein synthesis due to viral ORF shift and loss of partial viral gene. These findings imply that experimental data based on CVB integrated with the reporter gene should be interpreted with caution.

Abstract Human papillomavirus (HPV) is a dsDNA virus and its high-risk subtypes increase cancer risks. Yet, the mechanism of HPV infection and pathogenesis still remain unclear. Therefore, understanding the molecular mechanisms, and the pathogenesis of HPV are crucial in the prevention of HPV related cancers. In this study, we analyzed cervix squamous cell carcinoma (CESC) and head and neck carcinoma (HNSC) combined data to investigate various HPV induced cancer common feature. We showed that epidermal growth factor receptor (EGFR) was downregulated in HPV positive (HPV+) cancer, and that HPV+ cancer patients exhibited better prognosis than HPV negative (HPV−) cancer patients. Our study also showed that TP53 mutation rate is lower in HPV+ cancer than in HPV− cancer and that TP53 can be modulated by HPV E7 protein. However, there was no significant difference in the expression of wildtype TP53 in both groups. Subsequently, we constructed HPV-human interaction network and found that EGFR is a critical factor. From the network, we also noticed that EGFR is regulated by HPV E7 protein and hsa-miR-944. Moreover, while phosphorylated EGFR is associated with a worse prognosis, EGFR total express level is not significantly correlated with prognosis. This indicates that EGFR activation will induce a worse outcome in HPV+ cancer patients. Further enrichment analysis showed that EGFR downstream pathway and cancer relative pathway are diversely activated in HPV+ cancer and HPV− cancer. In summary, HPV E7 protein downregulates EGFR that downregulates phosphorylated EGFR and inhibit EGFR related pathways which in turn and consequently induce better prognosis. Importance Although HPV infection has been studied in various cancer types, there are only limited studies that have focused on the common effect of HPV related cancer. Consequently, this study focused on CESC and HNSC, two cancer types with high HPV infection proportion in cohort, thereby, intending to dig out the common effects and mechanisms of HPV+ cancers. Unlike some virus-human interaction prediction studies, the P-HIPSter database provides virus-human protein interaction based on protein structure prediction. Through this data, our interaction network was able to uncover previously unnoticed protein interactions. Our finding revealed that HPV infection caused various gene expression differences, and a great amount of which interact with EGFR, a cancer related gene. Therefore, since EGFR is associated with HPV+ cancer patients’ survival, some FDA proved EGFR inhibitors would be potential anti-HPV drugs.

As ubiquitous innate immune cells, macrophages are crucial for tissue homeostasis and disease pathogenesis. Although our understanding of macrophage subsets and functions has advanced, no effective strategies are available for targeting macrophages to treat diseases in clinical settings due to their heterogeneity. Transcription factors that regulate macrophage function have received increasing attention. CCAAT/enhancer-binding protein delta (CEBPD), an inflammation-associated transcription factor characterized by low basal expression but rapid induction by stimuli, has emerged as a key regulator of macrophages. CEBPD governs diverse biological processes in macrophages through its target genes. Furthermore, macrophage CEBPD significantly contributes to various pathologies. Modulating CEBPD expression or activity in macrophages could regulate various molecular processes to improve disease progression and alleviate organ damage; therefore, novel CEBPD-based therapeutic methods for treating diseases have attracted attention. In this review, we describe the factors upstream and downstream of CEBPD in macrophages. We then summarize recent advances in the regulation of macrophage biological processes by CEBPD. Finally, we discuss the contribution of macrophage CEBPD to various diseases and highlight strategies for developing novel therapies to modulate macrophage function by targeting CEBPD.