MicroRNA 130a Regulates both Hepatitis C Virus and Hepatitis B Virus Replication through a Central Metabolic Pathway

ABSTRACT Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. IMPORTANCE: We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections

The Interplay between Immune and Metabolic Pathways in Kidney Disease

Kidney disease is a significant health problem worldwide, affecting an estimated 10% of the global population. Kidney disease encompasses a diverse group of disorders that vary in their underlying pathophysiology, clinical presentation, and outcomes. These disorders include acute kidney injury (AKI), chronic kidney disease (CKD), glomerulonephritis, nephrotic syndrome, polycystic kidney disease, diabetic kidney disease, and many others. Despite their distinct etiologies, these disorders share a common feature of immune system dysregulation and metabolic disturbances. The immune system and metabolic pathways are intimately connected and interact to modulate the pathogenesis of kidney diseases. The dysregulation of immune responses in kidney diseases includes a complex interplay between various immune cell types, including resident and infiltrating immune cells, cytokines, chemokines, and complement factors. These immune factors can trigger and perpetuate kidney inflammation, causing renal tissue injury and progressive fibrosis. In addition, metabolic pathways play critical roles in the pathogenesis of kidney diseases, including glucose and lipid metabolism, oxidative stress, mitochondrial dysfunction, and altered nutrient sensing. Dysregulation of these metabolic pathways contributes to the progression of kidney disease by inducing renal tubular injury, apoptosis, and fibrosis. Recent studies have provided insights into the intricate interplay between immune and metabolic pathways in kidney diseases, revealing novel therapeutic targets for the prevention and treatment of kidney diseases. Potential therapeutic strategies include modulating immune responses through targeting key immune factors or inhibiting pro-inflammatory signaling pathways, improving mitochondrial function, and targeting nutrient-sensing pathways, such as mTOR, AMPK, and SIRT1. This review highlights the importance of the interplay between immune and metabolic pathways in kidney diseases and the potential therapeutic implications of targeting these pathways

Pharmacological Inhibition of S100A4 Attenuates Fibroblast Activation and Renal Fibrosis

The TGF-β/Smad3 signaling pathway is an important process in the pathogenesis of kidney fibrosis. However, the molecular mechanisms are not completely elucidated. The current study examined the functional role of S100A4 in regulating TGF-β/Smad3 signaling in fibroblast activation and kidney fibrosis development. S100A4 was upregulated in the kidney in a murine model of renal fibrosis induced by folic acid nephropathy. Further, S100A4 was predominant in the tubulointerstitial cells of the kidney. Pharmacological inhibition of S100A4 with niclosamide significantly attenuated fibroblast activation, decreased collagen content, and reduced extracellular matrix protein expression in folic acid nephropathy. Overexpression of S100A4 in cultured renal fibroblasts significantly facilitated TGF-β1-induced activation of fibroblasts by increasing the expression of α-SMA, collagen-1 and fibronectin. In contrast, S100A4 knockdown prevented TGF-β1-induced activation of fibroblast and transcriptional activity of Smad3. Mechanistically, S100A4 interacts with Smad3 to stabilize the Smad3/Smad4 complex and promotes their translocation to the nucleus. In conclusion, S100A4 facilitates TGF-β signaling via interaction with Smad3 and promotes kidney fibrosis development. Manipulating S100A4 may provide a beneficial therapeutic strategy for chronic kidney disease

STAT6 Deficiency Attenuates Myeloid Fibroblast Activation and Macrophage Polarization in Experimental Folic Acid Nephropathy

Renal fibrosis is a pathologic feature of chronic kidney disease, which can lead to end-stage kidney disease. Myeloid fibroblasts play a central role in the pathogenesis of renal fibrosis. However, the molecular mechanisms pertaining to myeloid fibroblast activation remain to be elucidated. In the present study, we examine the role of signal transducer and activator of transcription 6 (STAT6) in myeloid fibroblast activation, macrophage polarization, and renal fibrosis development in a mouse model of folic acid nephropathy. STAT6 is activated in the kidney with folic acid nephropathy. Compared with folic-acid-treated wild-type mice, STAT6 knockout mice had markedly reduced myeloid fibroblasts and myofibroblasts in the kidney with folic acid nephropathy. Furthermore, STAT6 knockout mice exhibited significantly less CD206 and PDGFR-β dual-positive fibroblast accumulation and M2 macrophage polarization in the kidney with folic acid nephropathy. Consistent with these findings, STAT6 knockout mice produced less extracellular matrix protein, exhibited less severe interstitial fibrosis, and preserved kidney function in folic acid nephropathy. Taken together, these results have shown that STAT6 plays a critical role in myeloid fibroblasts activation, M2 macrophage polarization, extracellular matrix protein production, and renal fibrosis development in folic acid nephropathy. Therefore, targeting STAT6 may provide a novel therapeutic strategy for fibrotic kidney disease

Severe fever with thrombocytopenia syndrome virus inhibits exogenous Type I IFN signaling pathway through its NSs invitro.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a novel bunyavirus (SFTS virus, SFTSV). At present there is still no specific antiviral treatment for SFTSV; To understand which cells support SFTSV life cycle and whether SFTSV infection activates host innate immunity, four different cell lines (Vero, Hela, Huh7.5.1, and Huh7.0) were infected with SFTSV. Intracellular/extracellular viral RNA and expression of IFNα, and IFNß were detected by real-time RT- PCR following infection. To confirm the role of non-structural protein (NSs) of SFTSV in exogenous IFNα-induced Jak/STAT signaling, p-STAT1 (Western Blot), ISRE activity (Luciferase assay) and ISG expression (real-time PCR) were examined following IFNα stimulation in the presence or absence of over-expression of NSs in Hela cells. Our study showed that all the four cell lines supported SFTSV life cycle and SFTSV activated host innate immunity to produce type I IFNs in Hela cells but not in Huh7.0, Huh7.5.1 or Vero cells. NSs inhibited exogenous IFNα-induced Jak/STAT signaling as shown by decreased p-STAT1 level, suppressed ISRE activity and down-regulated ISG expression. Suppression of the exogenous Type I IFN-induced Jak/STAT signaling by NSs might be one of the mechanisms of SFTSV to evade host immune surveillance

Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

Hepatitis B virus (HBV) is an important account of infectious hepatitis and interferon (IFN) remains one of the best treatment options. Activation of type I IFN signaling pathway leads to expressions of IFN-stimulated genes (ISGs) which play important roles in antiviral and immunomodulatory responses to HBV or hepatitis C virus (HCV) infection. Our previous studies indicated that ISG15 and its conjugation (ISGylation) were exploited by HCV to benefit its replication and persistent infection. This study was designed to assess the role of ISG15 and ISGylation in HBV infection in vitro. The levels of ISG15 and ISGylation were upregulated by ISG15 plasmid transfection into HepG2.2.15 cells. Decreased ISGylation was achieved by siRNA targeting UBE1L, the only E1 activating enzyme for ISGylation. Overexpression of ISG15 and subsequent ISGylation significantly increased the levels of HBV DNA in the culture supernatants although the intracellular viral replication remained unaffected. Silencing UBE1L, with decreased ISGylation achieved, abrogated this ISGylation-mediated promoting effect. Our data indicated that overexpression of ISG15 stimulated HBV production in an ISGylation-dependent manner. Identification of ISG15-conjugated proteins (either HBV viral or host proteins) may reveal promising candidates for further antiviral drug development.Peer Reviewe

Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

Hepatitis B virus (HBV) is an important account of infectious hepatitis and interferon (IFN) remains one of the best treatment options. Activation of type I IFN signaling pathway leads to expressions of IFN-stimulated genes (ISGs) which play important roles in antiviral and immunomodulatory responses to HBV or hepatitis C virus (HCV) infection. Our previous studies indicated that ISG15 and its conjugation (ISGylation) were exploited by HCV to benefit its replication and persistent infection. This study was designed to assess the role of ISG15 and ISGylation in HBV infection in vitro. The levels of ISG15 and ISGylation were upregulated by ISG15 plasmid transfection into HepG2.2.15 cells. Decreased ISGylation was achieved by siRNA targeting UBE1L, the only E1 activating enzyme for ISGylation. Overexpression of ISG15 and subsequent ISGylation significantly increased the levels of HBV DNA in the culture supernatants although the intracellular viral replication remained unaffected. Silencing UBE1L, with decreased ISGylation achieved, abrogated this ISGylation-mediated promoting effect. Our data indicated that overexpression of ISG15 stimulated HBV production in an ISGylation-dependent manner. Identification of ISG15-conjugated proteins (either HBV viral or host proteins) may reveal promising candidates for further antiviral drug development

SFTSV inhibits exogenous IFNα-induced Jak/STAT signaling pathway through NSs.

<p>(A)<b>NSs of SFTSV expresses in Hela cells successfully.</b> Hela cells were transfected with empty vector pcDNA3.1 plasmid or NSs plasmid for 48hours, and total RNA were prepared with Trizol reagent, cDNA were harvested with reverse transcriptase for real time quantitative PCR. Protein samples were harvested for WB analyzed. (B) <b>Expression of GFP protein in Hela cells.</b> GFP-expressing plasmid DNA was transfected into Hela cells for 48 hours and GFP expression was observed under immunofluorescence microscope (200×). (C)<b>NSs inhibits the phosphorylation of STAT1.</b>Hela cells were transfected with NSs plasmid or empty vector pcDNA3.1 for 36 hours before IFNα was added to stimulate the cells for 15minutes. Total proteins were collected with protein lysis buffer for WB and densitometry analysis of western blot datafor p-STAT1. (D)<b>NSs suppresses the ISRE activity</b>. Hela cells were co-transfected with pISRE-luc plasmid and pRL-TK plasmid together with NSs plasmid, GFP-expressing plasmid or empty vector pcDNA3.1 plasmid for 24hours, and IFNα (100IU/mL)were added to the cells for 24hours, then samples were collected for dual-luciferase reporter assay. (E) <b>NSs down-regulates the expression of several ISGs.</b> Hela cells were transfectd with NSs plasmid or empty vectorpcDNA3.1 plasmid for 36hours, then we added IFNα (100IU/ml) to the cells for 12hours,RNA were collected with Trizol reagent, cDNA were used for real time quantitative PCR. Data were presented as mean± SD, n = 3.*p<0.05, **p<0.01, ***p<0.001.Data were representative of at least 3 repeated experiments.</p

SFTSV inhibits exogenous Type I IFN signaling pathway.

<p>(A)<b>SFTSV inhibits the phosphorylation of STAT1</b>. Hela cells were infected with SFTSV(MOI = 1.0) for2 hours and cultured for 36 hours before 100IU/ml IFNα was added for 15 minutes,and total protein were collected for WB and densitometry analysis for p-STAT1. (B)<b>SFTSV suppresses ISRE activity.</b> Hela cells were co-transfected with ISRE-luc plasmid 2μg per well and pRL-TK plasmid 2ng per well, then Hela cells were infected with SFTSV (MOI = 1.0) for 2 hours and cultured for 24hours, then we used 100IU/ml IFNα to stimulated the Hela cells for 24 hours, and samples were collected for dual-luciferase reporter assay. (C)<b>SFTSV down –regulates the expression of ISGs.</b> Hela cells were infected with 1.0MOI SFTSV for2 hours and culturedfor24hours, then treated with 100IU/ml IFNα for 12 hours, and total RNAs were isolated with Trizolreagent, The levels of ISG15, MxA, OAS3, GAPDH mRNA were measured by quantitative RT-PCR. Data were presented as mean± SD, n = 3. **p<0.01, ***p<0.001. Data are representative of at least 3 repeated experiments.</p