The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3' trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of ...tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The persistence of HIV despite suppressive antiretroviral therapy is a major roadblock to HIV eradication. Current strategies focused on inducing the expression of latent HIV fail to clear the ...persistent reservoir, prompting the development of new approaches for killing HIV-positive cells. Recently, acitretin was proposed as a pharmacological enhancer of the innate cellular defense network that led to virus reactivation and preferential death of infected cells. We evaluated the capacity of acitretin to reactivate and/or to facilitate immune-mediated clearance of HIV-positive cells. Acitretin did not induce HIV reactivation in latently infected cell lines (J-Lat and ACH-2). We could observe only modest induction of HIV reactivation by acitretin in latently green fluorescent protein-HIV-infected Jurkat cells, comparable to suboptimal concentrations of vorinostat, a known latency-reversing agent (LRA). Acitretin induction was insignificant, however, compared to optimal concentrations of LRAs. Acitretin failed to reactivate HIV in a model of latently infected primary CD4
T cells but induced retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral signaling (MAVS) expression in infected and uninfected cells, confirming the role of acitretin as an innate immune modulator. However, this effect was not associated with selective killing of HIV-positive cells. In conclusion, acitretin-mediated stimulation of the RIG-I pathway for HIV reactivation is modest and thus may not meaningfully affect the HIV reservoir. Stimulation of the RIG-I-dependent interferon (IFN) cascade by acitretin may not significantly affect the selective destruction of latently infected HIV-positive cells.
Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) has been shown to restrict retroviruses and DNA viruses by decreasing the pool of intracellular deoxynucleotides. In turn, ...SAMHD1 is controlled by cyclin-dependent kinases (CDK) that regulate the cell cycle and cell proliferation. Here, we explore the effect of CDK6 inhibitors on the replication of herpes simplex virus type 1 (HSV-1) in primary monocyte-derived macrophages (MDM).
MDM were treated with palbociclib, a selective CDK4/6 inhibitor, and then infected with a GFP-expressing HSV-1. Intracellular deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method.
CDK6 inhibitor palbociclib blocked SAMHD1 phosphorylation, intracellular dNTP levels and HSV-1 replication in MDM at subtoxic concentrations. Treatment of MDM with palbociclib reduced CDK2 activation, measured as the phosphorylation of the T-loop at Thr160. The antiviral activity of palbociclib was lost when SAMHD1 was degraded by viral protein X. Similarly, palbociclib did not block HSV-1 replication in SAMHD1-negative Vero cells at subtoxic concentrations, providing further evidence for a role of SAMHD1 in mediating the antiviral effect.
SAMHD1-mediated HSV-1 restriction is controlled by CDK and points to a preferential role for CDK6 and CDK2 as mediators of SAMHD1 activation. Similarly, the restricting activity of SAMHD1 against DNA viruses suggests that control of dNTP availability is the major determinant of its antiviral activity. This is the first study describing the anti-HSV-1 activity of palbociclib.
Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing ...number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3′ ends of mitochondrial pre‐tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile‐onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism
Although natural killer (NK) cells are recognized for their modulation of immune responses, the mechanisms by which human NK cells mediate immune regulation are unclear. Here, we report that ...expression of human leukocyte antigen (HLA)-DP, a ligand for the activating NK cell receptor NKp44, is significantly upregulated on CD8+ effector T cells, in particular in human cytomegalovirus (HCMV)+ individuals. HLA-DP+ CD8+ T cells expressing NKp44-binding HLA-DP antigens activate NKp44+ NK cells, while HLA-DP+ CD8+ T cells not expressing NKp44-binding HLA-DP antigens do not. In line with this, frequencies of HLA-DP+ CD8+ T cells are increased in individuals not encoding for NKp44-binding HLA-DP haplotypes, and contain hyper-expanded CD8+ T cell clones, compared to individuals expressing NKp44-binding HLA-DP molecules. These findings identify a molecular interaction facilitating the HLA-DP haplotype-specific editing of HLA-DP+ CD8+ T cell effector populations by NKp44+ NK cells and preventing the generation of hyper-expanded T cell clones, which have been suggested to have increased potential for autoimmunity.
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•Effector CD8+ T cells express on their surface the HLA class II heterodimer HLA-DP•Expression of HLA-DP on effector CD8+ T cells is upregulated upon HCMV infection•NKp44+ NK cells interact with CD8+ T cells carrying NKp44-binding HLA-DP molecules•CD8+ T cells expressing NKp44-binding HLA-DPs show reduced clonal expansion
Padoan et al. show that HLA-DP expression on CD8+ T cells is upregulated upon HCMV infection, in particular on effector CD8+ T cells. NKp44+ NK cells can regulate HLA-DP+ CD8+ T cells in individuals expressing NKp44-binding HLA-DP haplotypes, with consequences for CD8+ T cell clonality.
Midostaurin is a multi-kinase inhibitor with antineoplastic activity. We assessed the capacity of midostaurin to affect early and late steps of HIV-1 infection and to reactivate HIV-1 latently ...infected cells, alone or in combination with histone deacetylase inhibitors (HDACi) known to act as latency-reversing agents (LRA). Acute HIV-1 infection was assessed by flow cytometry in three cell types treated with midostaurin in the presence or absence of SAMHD1. Non-infected cells were treated with midostaurin and harvested for Western blot analysis. Macrophage infections were also measured by quantitative RT-PCR. HIV-1 latency reactivation was assessed in several latency models. Midostaurin induced G2/M arrest and inhibited CDK2, preventing the phosphorylation of SAMHD1 associated to inhibition of its dNTPase activity. In the presence of SAMHD1, midostaurin blocked HIV-1 DNA formation and viral replication. However, following Vpx-mediated SAMHD1 degradation, midostaurin increased viral transcripts and virus replication. In three out of four HIV-1 latency models, including primary CD4+ T cells, midostaurin effectively reversed HIV-1 latency and was synergistic in combination with LRA vorinostat and panobinostat. Our study describes a dual effect for midostaurin in HIV-1 infection, antiviral or proviral depending on SAMHD1 activation, and highlights a role for active SAMHD1 in regulating the activity of potential HIV-1 latency reversal agents.
•The multikinase inhibitor midostaurin blocked acute HIV-1 infection but reactivates latent virus.•Midostaurin's proviral activity is masked by its antiviral effect on SAMHD1.•Midostaurin's latency reversal was synergistic with known latency reversal agents.•SAMHD1 may regulate the activity of potential latency reversal agents.
SAMHD1 is a triphosphohydrolase that restricts HIV-1 by limiting the intracellular dNTP pool required for reverse transcription. Although SAMHD1 is expressed and active/unphosphorylated in most cell ...lines, its restriction activity is thought to be relevant only in non-cycling cells. However, an in depth evaluation of SAMHD1 function and relevance in cycling cells is required. Here, we show that SAMHD1-induced degradation by HIV-2 Vpx affects the dNTP pool and HIV-1 replication capacity in the presence of the 3′-azido-3′-deoxythymidine (AZT) in cycling cells. Similarly, in SAMHD1 knockout cells, HIV-1 showed increased replicative capacity in the presence of nucleoside inhibitors, especially AZT, that was reverted by re-expression of wild type SAMHD1. Sensitivity to non-nucleoside inhibitors (nevirapine and efavirenz) or the integrase inhibitor raltegravir was not affected by SAMHD1. Combination of three mutations (S18A, T21A, T25A) significantly prevented SAMHD1 phosphorylation but did not significantly affect HIV-1 replication in the presence of AZT. Our results demonstrate that SAMHD1 is active in HIV-1 permissive cells, does not modify susceptibility to HIV-1 infection but strongly affects sensitivity to nucleoside inhibitors.
•HIV-1 showed increased replicative capacity in the presence of AZT in SAMHD1 knockout cells.•Multiple mutations prevented SAMHD1 phosphorylation but did not affect HIV-1 replication in the presence of AZT.•The activity of SAMHD1 became apparent by addition of a competitive thymidine analogue.•Results demonstrate that SAMHD1 is active in apparently HIV-1 permissive cells.
HIV-1 infection is thought to impair type I interferon (IFN-I) production in macrophages, a cell type that is also relatively resistant to HIV-1 cytotoxic effects. Here, we show that monocyte ...differentiation into macrophages by M-CSF led to cell proliferation and susceptibility to HIV-1 infection that induced cell cycle arrest and increased cell death. Established HIV-1 infection of monocyte-derived macrophages induced the upregulation of the pattern recognition receptors MDA5 and Rig-I that serve as virus sensors; production of interferon-β, and transcription of interferon-stimulated genes including CXCL10. Infected macrophages showed increased expression of p21 and subsequent inactivation of cyclin-CDK2 activity leading to a hypo-phosphorylated active retinoblastoma protein (pRb) and deactivation of E2F1-dependent transcription and CDK1 downregulation. Additionally, HIV-1 infection limited deoxynucleotide pool by downregulation of the ribonucleotide reductase subunit R2 (RNR2) and reactivation of the HIV-1 restriction factor SAMHD1 together with increased cell death. In conclusion, HIV-1 induced an innate antiviral mechanism associated to IFN-I production, interferon stimulated gene activation, and p21-mediated G2/M arrest leading to elevated levels of cell death in monocyte derived macrophages. Upregulation of MDA5 and Rig-I may serve as targets for the development of antiviral strategies leading to the elimination of HIV-1 infected cells.
•Productive HIV-1 infection of macrophages triggers an innate antiviral response.•HIV-1 infected macrophages activate interferon stimulated genes and cell death.•HIV-1 infection increases Rig-I and MDA5 expression and p21-mediated cell cycle arrest.•Infected macrophages become refractory to over infection through SAMHD1 activation.•Innate immune responses may serve as targets for eradicating HIV-1 infection.
The enzyme tRNase Z, a member of the metallo-beta-lactamase family, endonucleolytically removes 3' trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of ...tRNase Z, tRNase Z.sup.S, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase Z.sup.L, related to tRNase Z.sup.S through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase Z.sup.S . tRNase Z.sup.L consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: N.sub.dom T.sub.prox, located at the carboxy end of the amino domain proximal to the linker, and T.sub.flex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F.sub.329 and L.sub.332) at the carboxy end of N.sub.dom T.sub.prox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the N.sub.dom - linker boundary. The Ala substitution for N.sub.378 in the T.sub.flex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R.sub.382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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