Biology subjects

Imamichi, T.

Publications and source records attributed to Imamichi, T..

11 recordsLinked to original sources

bioRxiv2026

IL-27 Reduces Cell-Surface LDLR in Human Macrophages and Restricts VSV-G-Pseudotyped HIV infection

The low-density lipoprotein receptor (LDLR) is a major cellular receptor for vesicular stomatitis virus glycoprotein (VSV-G), which is widely used to pseudotyped virus vectors. A replication incompetent VSV-G-pseudotyped HIV (VSV-G-HIVLuc) has been used in HIV infection studies. Although IL-27-polarized macrophages induce multiple antiviral genes and resist replication-competent HIV infection, their impact on VSV-GHIVLuc infection has not been studied. Here, we evaluated the role of IL-27 polarization on VSV-G-HIVLuc infection. Infection was monitored by luciferase activity. Cell-surface LDLR was analyzed by flow cytometry. LDLR transcripts and protein expression were quantified by qRT-PCR and Western blotting, respectively. Viral binding was assessed by measuring cell-associated p24 antigen, while post-entry events were evaluated by quantifying late reverse-transcription products. VSV-G-HIVLuc infection in IL-27-polarized M-CSF-induced macrophages (27M-Mac) and IL-27-polarized GM-CSFinduced macrophages (27GM-Mac) was reduced to 5.8{+/-}0.4% and 5.9{+/-}2.1%, respectively (both p<0.0001), compared to unpolarized macrophages. 27M-Mac and 27GM-Mac reduced cell-surface LDLR to 38.3{+/-}2.6% (p<0.01) and 31.4{+/-}3.2% (p<0.01). Western blotting showed that total LDLR protein decreased to 48{+/-}0.8% in 27M-Mac (p<0.01), whereas 27GM-Mac showed no significant change. However, LDLR transcripts increased approximately 1.6-1.9-fold following IL-27 treatment in both macrophage types (p<0.01). Viral binding to 27M-Mac decreased modestly, while binding to 27GM-Mac decreased significantly to 67.5 {+/-} 7.5% of that to GM-Mac (p<0.05, n=3). In contrast, late reverse-transcription products decreased to 23{+/-}2.1% (p<0.0001) and 44{+/-}1.5% (p<0.0001). These findings demonstrate that IL-27 downregulates cell-surface LDLR expression in macrophages despite increasing LDLR transcript abundance, suggesting that IL-27 restricts VSV-G-pseudotyped Lentiviral infection through both receptor-associated and additional post-entry mechanisms.

immunology↗

bioRxiv2026

A Metabolic Enzyme, Pyruvate Carboxylase, Functions as a Sequence-Selective Small RNA Sensor for Antiviral Immunity

Interleukin-27 (IL-27) is an anti-HIV cytokine that induces 14 novel microRNAs (miRNAs) in primary CD4(+) T cells. We previously reported that transfection of two of these miRNA mimics, miRTC10 and miRTC14 into human primary macrophages, differentially induced interferon (IFN)-2, -8, -13, and -{lambda}1 expression. However, the mechanism underlying this activation remains unclear. Here, we demonstrate that miRTC14 does not directly target canonical IFN-regulatory genes but instead engages with pyruvate carboxylase (PC) and laboratory of genetics and physiology-2 (LGP2/DHX58) as direct binding partner proteins. Functional analysis revealed that miRTC14 transfection induces IFN expression by more than 100-fold (p<0.001), whereas PC or LGP2 depletion by siRNAs markedly attenuated this response (50-100-fold reduction, p<0.01). Reconstitution of PC and LGP2 in deficient HEK293 cells restored miRTC14-driven IFN induction. Notably, IFN activation depended on sequence features at the duplex termini. However, PC-miRTC14-LGP2 axis activates TBK1-dependent phosphorylation of IRF3/7, similarly to canonical RNA sensors (RIG-I/MDA5), but this process induced differential IFN subtype. These findings establish PC as a miRNA-binding protein and define a previously unrecognized RNA-sensing mechanism, linking metabolic enzymes to RNA sequence-dependent innate immunity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/735367v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@ea70d3org.highwire.dtl.DTLVardef@1953a73org.highwire.dtl.DTLVardef@305f16org.highwire.dtl.DTLVardef@1b2daff_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO microRNA sequence-dependent activation of innate immunity by pyruvate carboxylase (PC). PC-miRTC14-LGP2 axis induces TBK1 phosphorylation via an as-yet-unidentified adapter(s). The activated TBK1 (p-TBK1) subsequently phosphorylates IRF3 and/or IRF7 leading to selective IFN subtypes (IFN-2, IFN-8, IFN-13, and IFN-{lambda}1). (Created in BioRender. Kariyawasam, U. (2027) https://BioRender.com/u82s04p) C_FIG

immunology↗

bioRxiv2025

Manganese mediates antiviral effects by driving an ATM -TBK1 phosphorylation signaling pathway

We previously reported that manganese (Mn) enhances innate immune responses to viral infection by inducing phosphorylation of TANK-binding kinase 1 (TBK1) in an Ataxia-telangiectasia mutated (ATM)-dependent manner. However, the underlying mechanism by which how Mn induces TBK1 phosphorylation remained unclear. Here, we show that Mn dose-dependently induced TBK1 phosphorylation in the presence of ATM across multiple cell lines, as well as in primary human macrophages and T cells. This phosphorylation was abolished in ATM-deficient cells, and we identified cytoplasmic ATM as a key mediator. Immunoprecipitation assays revealed that Mn promoted ATM phosphorylation at Ser1893, Ser1981, and Ser2996. TBK1 interacted with phosphorylated ATM at early stages, but upon phosphorylation, TBK1 dissociated from the ATM-TBK1 complex. This dissociation coincided with enhanced antiviral cytokine production. Furthermore, Mn dose-dependently suppressed HIV replication by inducing multiple antiviral host factors and cytokines. Together, these findings identify a cytoplasmic ATM-TBK1 phosphorylation cycle as a critical regulator of antiviral innate immunity and suggest Mn supplementation as a potential therapeutic approach against HIV and other viral infections. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/671272v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1c72f62org.highwire.dtl.DTLVardef@1f7e789org.highwire.dtl.DTLVardef@820fe0org.highwire.dtl.DTLVardef@e06fb3_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Mn-dependent activation of the ATM-TBK1 phosphorylation signaling pathway. At early time points, Mn phosphorylated ATM at multiple site. And then TBK1 bound to p-ATM and became phosphorylated. Phosphorylated TBK1 then dissociated from the complex at later stages (right panel). P-TBK1 participated in activating downstream TBK1-IRF signaling, thereby enhancing antiviral cytokines induction to DNA or RNA virus infection (left panel). C_FIG

immunology↗

bioRxiv2025

Delayed forebrain excitatory and inhibitory neurogenesis inSTRADA-related megalencephaly via mTOR hyperactivity

Biallelic pathogenic variants in STRADA, an upstream regulator of the mechanistic target of rapamycin (mTOR) pathway, result in megalencephaly, drug-resistant epilepsy, and severe intellectual disability. This study explores how mTOR pathway hyperactivity alters cell fate specification in dorsal and ventral forebrain development using STRADA knock-out human stem cell derived brain organoids. In both dorsal and ventral forebrain STRADA knock-out organoids, neurogenesis is delayed, with a predilection for progenitor renewal and proliferation and an increase in outer radial glia. Ventrally, interneuron subtypes shift to an increase in neuropeptide-Y expressing cells. Inhibition of the mTOR pathway with rapamycin results in rescue for most phenotypes. When mTOR pathway variants are present in all cells of the developing brain, overproduction of interneurons and altered interneuron cell fate may underlie mechanisms of megalencephaly, epilepsy, and cognitive impairment. Our findings suggest mTOR inhibition during fetal brain development as a potential therapeutic strategy in STRADA deficiency.

neuroscience↗

bioRxiv2024

Escherichia coli grown in cost-effective conical tubes produces more plasmid DNA

Before the COVID-19 pandemic, we grew plasmid-transformed Escherichia coli in Falcon round-bottom-polypropylene tubes (F-Round-PP) and isolated plasmid using a Miniprep kit. When obtaining sufficient quantities of F-Round-PP became problematic during the pandemic and the inflation, we grew them using any available tubes. Notably, we observed that plasmid yield from cells grown in a cost-effective Oxford conical-polypropylene tubes (O-Conical-PP) was higher than that from F-Round-PP. We assessed the impact using O-Conical-PP and other conical brand tubes. As a result, the plasmid yield from O-Conical-PP (the list price is nearly 1/3 of F-Round-PP) was 1.5-fold higher than other PP tubes (p<0.001). We propose that researchers may need to re-assess the effectiveness of their laboratory supplies to optimize the budget during this inflationary period.

microbiology↗

bioRxiv2023

Interleukin-27-induced HIV-resistant dendritic cells suppress reveres transcription following virus entry in an SPTBN1, Autophagy, and YB-1 independent manner

Interleukin (IL)-27, a member of the IL-12 family of cytokines, induces human immunodeficiency virus (HIV)-resistant monocyte-derived macrophages and T cells. This resistance is mediated via the downregulation of spectrin beta, non-erythrocytic 1 (SPTBN1), induction of autophagy, or suppression of the acetylation of Y-box binding protein-1 (YB-1); however, the role of IL-27 administration during the induction of immature monocyte-derived dendritic cells (iDC) is poorly investigated. In the current study, we investigated the function of IL-27-induced iDC (27DC) on HIV infection. 27DC inhibited HIV infection by 95 {+/-} 3 % without significant changes in the expression of CD4, CCR5, and SPTBN1 expression, autophagy induction and acetylation of YB-1 compared to iDC. An HIV proviral DNA copy number assay displayed that 27DC suppressed reverse transcriptase (RT) reaction without influencing the virus entry. A DNA microarray analysis was performed to identify the differentially expressed genes between 27DC and iDC. Compared to iDC, 51 genes were differentially expressed in 27DC, with more than 3-fold changes in four independent donors. Cross-reference analysis with the reported 2,214 HIV regulatory host genes identified nine genes as potential interests: Ankyrin repeat domain 22, Guanylate binding protein (GBP)-1, -2, -4, -5, Stabilin 1, Serpin family G member 1 (SERPING1), Interferon alpha inducible protein 6, and Interferon-induced protein with tetratricopeptide repeats 3. A knock-down study using si-RNA failed to determine a key factor associated with the anti-HIV activity due to the induction of robust amounts of off-target effects. Overexpression of each protein in cells had no impact on HIV infection. Thus, we could not define the mechanism of the anti-HIV effect in 27DC. However, our findings indicated that IL-27 differentiates monocytes into HIV-resistant DC, and the inhibitory mechanism differs from IL-27-induced HIV-resistant macrophages and T cells.

immunology↗

bioRxiv2022

The C-terminal domain of RNase H and the C-terminus amino acid residue regulate virus release and autoprocessing of a defective HIV-1 possessing M50I and V151I changes in integrase

Previously, we reported that an HIV-1 variant containing Met-to-Ile change at codon 50 and Val-to-Ile mutation at codon 151 of integrase (IN), HIV(IN:M50I/V151I), was an impaired virus. Despite the mutations being in IN, the virus release was significantly suppressed (p < 0.0001) and the initiation of autoprocessing was inhibited; the mechanism of the defect remains unknown. In the current study, we attempted to identify the critical domains or amino acid (aa) residue(s) that promote defects in HIV(IN:M50I/V151I), using a series of variants, including truncated or aa-substituted RNase H (RH) or IN. The results demonstrated that virus release and the initiation of autoprocessing were regulated by the C-terminal domains (CTDs) of RH and IN. Further studies illustrated that Asp at codon 109 of RH CTD and Asp at the C terminus of IN induces the defect. This result indicated that the CTD of RH and IN in GagPol and particular aa positions in RH and IN regulated the virus release and the initiation of autoprocessing, and these sites could be potential targets for the developing new therapies.

microbiology↗

bioRxiv2022

Interleukin-27 skews TNF-alpha-induced inflammatory microenvironment in keratinocytes

Inflammaging has received considerable attention because aging is characterized by low-grade, chronic and asymptomatic inflammation, concomitant with increased blood levels of senescence-associated secretory phenotype (SASP) factors, including IL-1, IL-6, IL-8, IL-18 and tumor necrosis factor-alpha (TNF-alpha). On the other hand, IL-27 is not categorized as SASP factors though it is known that IL-27 has pleiotropic roles in inflammation. Here, we evaluated the interaction between TNF-alpha and IL-27 in the context of low-grade inflammation by using in HaCaT cells. TNF-alpha induced significant upregulation of IL-6 and IL-8 through the experimental concentrations (~10 ng/ml) of TNF-alpha, while the mRNA expression levels of IL-1RA, IL-10 and IL-18BP were unchanged. After confirming the expression of IL-27 receptor in HaCaT cells, we examined the effects of IL-27 alone on the cytokine expression. IL-27 alone significantly enhanced mRNA expression levels of IL-10 and IL-18BP by 1.61-fold and 1.46-fold, respectively, and also enhanced mRNA expression levels of IL-6 by 2.32-fold. In the presence of 100 ng/ml IL-27, the expression levels of anti-inflammatory cytokines, IL-1RA, IL-10 and IL-18BP, were significantly upregulated with the treatment of a physiological concentration (1 ng/ml) TNF-alpha. Taken together, a high concentration of IL-27 exhibits anti-inflammatory effects in the presence of a low concentration of TNF-alpha when keratinocytes are the recipient of IL-27 signaling, suggesting the anti-inflammatory roles of IL-27 in inflammaging may be regulated by TNF-alpha concentration.

immunology↗

bioRxiv2021

S100A8 and S100A9, biomarkers of SARS-Cov-2-infected patients, suppress HIV replication in primary macrophages

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils and form a heterodimer complex. Recently, both proteins were identified as novel biomarkers of SARS-CoV-2 infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibits HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads in SARS-CoV2 co-infection.

microbiology↗

bioRxiv2021

Natural Occurring Non-Synonymous Single Nucleotide Polymorphisms in Integrase and RNase H Regulate Assembly and Autoprocessing of HIV-1

Recently, a genome-wide association study using plasma HIV RNA reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus naive patients were associated with virus load (VL). However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP using site-directed mutagenesis and examined their replicative abilities and biological properties. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found an impaired virus. Despite the mutation being in integrase, a quantification assay demonstrated that the virus release was significantly suppressed (P<0.001). Transmission electron microscopy analyses revealed that the accumulation of abnormal shapes of buds on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins in the HIV(IN:M50I) particles. Forster Resonance Energy Transfer (FRET) assay displayed that GagPol containing IN:M50I (GagPol(IN:M50I)) significantly increased the efficiency of homodimerization (P<0.05) and heterodimerization with Gag (P<0.001), compared to GagPol(WT). HIV replication assay using a series of variants of HIV(IN:M50I) elucidated that the C-terminus residues, Asn at codon 288, plays a key role in the defect and the impaired maturation and replication capability was rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser change at codon 79 in RNase H. These data demonstrate that Gag and GagPol assembly, virus release and autoprocessing are not only regulated by integrase but also RNase H. ImportanceA nascent HIV-1 is noninfectious. To become an infectious virus, Gag and GagPol polyproteins in the particles need to be cleaved by mature HIV protease (PR). PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by a self-cleavage is called autoprocessing. Here, during the evaluation of roles of naturally emerging non-synonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase in GagPol which increases the efficiency of heterodimerization with Gag. This defect was recovered by co-existing of other SNPs: Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

microbiology↗

bioRxiv2019

siRNA containing a unique 5-nucleotide motif acts as a quencher of IFI16-mediated innate immune response

We previously reported that small interfering RNA (siRNA) enhances DNA or DNA virus mediated-interferon (IFN)-{lambda}1 induction through retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) crosstalk-signalling pathway. Here we provide further evidence of a new role for siRNA. siRNA containing a 5-nucleotide (nt) motif sequence suppresses DNA-mediated IFNs and inflammatory cytokines. We determined that motif siRNA inhibits the induction when the motif is located at the 3 or 5-terminus of siRNA. Using THP1-Lucia ISG cells with various DNA stimulants, it is demonstrated that motif siRNA inhibits DNA or DNA virus but not RNA virus-mediated signaling. Motif siRNA specifically interrupts IFI16 binding to DNA and has 2.5-fold higher affinity to IFI16 than that of siRNA without the motif. Collectively, these findings may shed lights on a novel function of siRNA with the 5-nt motif as a quencher of innate immunity and facilitate the development of potential therapeutics to treat diseases in which this pathway is dysregulated.

immunology↗