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1.
EMBO J ; 41(11): e111210, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35471636

ABSTRACT

Recent work reported the existence of a mammalian cell-autonomous antiviral defence based on RNA interference (RNAi), which relies on the accumulation of virus-derived small interfering RNAs (vsiRNAs) to guide the degradation of complementary viral RNAs. In a new study, Zhang et al (2022) find that, in infected mice, vsiRNAs can enter the bloodstream via their incorporation into extracellular vesicles (EVs) and confer sequence-specific antiviral activity to recipient cells, thus indicating that mammalian antiviral RNAi participates in both cell-autonomous and non-cell-autonomous host defence.


Subject(s)
Antiviral Agents , Viruses , Animals , Mammals/genetics , Mice , RNA Interference , RNA, Double-Stranded , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Viral/genetics
2.
Immunity ; 54(10): 2180-2182, 2021 10 12.
Article in English | MEDLINE | ID: mdl-34644551

ABSTRACT

RNA interference (RNAi) provides antiviral defense in many organisms, including plants, insects, and nematodes. In this issue of Immunity, Fang et al. (2021) utilize designer peptides targeting viral suppressors of RNAi to provide evidence for the relevance of RNAi to antiviral immunity in mammals, also revealing the potential of this approach toward antiviral therapy.


Subject(s)
Antiviral Agents , Animals , Antiviral Agents/therapeutic use , RNA Interference
3.
Nat Commun ; 11(1): 5387, 2020 11 03.
Article in English | MEDLINE | ID: mdl-33144593

ABSTRACT

The Human Silencing Hub (HUSH) complex is necessary for epigenetic repression of LINE-1 elements. We show that HUSH-depletion in human cell lines and primary fibroblasts leads to induction of interferon-stimulated genes (ISGs) through JAK/STAT signaling. This effect is mainly attributed to MDA5 and RIG-I sensing of double-stranded RNAs (dsRNAs). This coincides with upregulation of primate-conserved LINE-1s, as well as increased expression of full-length hominid-specific LINE-1s that produce bidirectional RNAs, which may form dsRNA. Notably, LTRs nearby ISGs are derepressed likely rendering these genes more responsive to interferon. LINE-1 shRNAs can abrogate the HUSH-dependent response, while overexpression of an engineered LINE-1 construct activates interferon signaling. Finally, we show that the HUSH component, MPP8 is frequently downregulated in diverse cancers and that its depletion leads to DNA damage. These results suggest that LINE-1s may drive physiological or autoinflammatory responses through dsRNA sensing and gene-regulatory roles and are controlled by the HUSH complex.


Subject(s)
Epigenesis, Genetic/physiology , Gene Expression Regulation, Neoplastic , Gene Silencing/physiology , Interferon Type I/metabolism , Long Interspersed Nucleotide Elements/physiology , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , DNA Damage , Down-Regulation , Gene Knockout Techniques , HEK293 Cells , HeLa Cells , Humans , Inflammation , Interferon-Induced Helicase, IFIH1/metabolism , Long Interspersed Nucleotide Elements/genetics , Phosphoproteins/metabolism , RNA, Double-Stranded , Receptors, Immunologic , Sequence Analysis, RNA , Signal Transduction
4.
EMBO J ; 38(8)2019 04 15.
Article in English | MEDLINE | ID: mdl-30872283

ABSTRACT

To protect against the harmful consequences of viral infections, organisms are equipped with sophisticated antiviral mechanisms, including cell-intrinsic means to restrict viral replication and propagation. Plant and invertebrate cells utilise mostly RNA interference (RNAi), an RNA-based mechanism, for cell-intrinsic immunity to viruses while vertebrates rely on the protein-based interferon (IFN)-driven innate immune system for the same purpose. The RNAi machinery is conserved in vertebrate cells, yet whether antiviral RNAi is still active in mammals and functionally relevant to mammalian antiviral defence is intensely debated. Here, we discuss cellular and viral factors that impact on antiviral RNAi and the contexts in which this system might be at play in mammalian resistance to viral infection.


Subject(s)
Host-Pathogen Interactions/immunology , Mammals/immunology , RNA Interference , RNA, Viral/genetics , Virus Diseases/immunology , Viruses/immunology , Animals , Antiviral Agents/administration & dosage , Host-Pathogen Interactions/genetics , Mammals/genetics , Mammals/virology , Virus Diseases/genetics , Virus Diseases/virology , Virus Replication , Viruses/isolation & purification
5.
EMBO J ; 37(4)2018 02 15.
Article in English | MEDLINE | ID: mdl-29351913

ABSTRACT

In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG-I-like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon-stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG-I, MDA5 and, the least-studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus-derived double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi-dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA-mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2-mediated antagonism of dsRNA-mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.


Subject(s)
DEAD-box RNA Helicases/metabolism , Interferon Type I/metabolism , RNA Helicases/metabolism , RNA Interference , RNA Viruses/physiology , RNA, Double-Stranded/metabolism , RNA, Small Interfering/genetics , Ribonuclease III/metabolism , DEAD-box RNA Helicases/genetics , Gene Expression Regulation , HeLa Cells , Humans , RNA Helicases/genetics , RNA, Double-Stranded/genetics , RNA, Viral/genetics , Ribonuclease III/genetics , Signal Transduction
6.
EMBO J ; 35(23): 2505-2518, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27815315

ABSTRACT

RNA interference (RNAi) elicited by long double-stranded (ds) or base-paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence-specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN-responsive cells with type I IFN Notably, transfection with long dsRNA specifically vaccinates IFN-deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system.


Subject(s)
Gene Expression Regulation , RNA Interference , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , Animals , Cells, Cultured , Immunity, Innate , Interferon Type I/antagonists & inhibitors , Mice , RNA Viruses/immunology
8.
Elife ; 3: e01535, 2014 Feb 18.
Article in English | MEDLINE | ID: mdl-24550253

ABSTRACT

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells. DOI: http://dx.doi.org/10.7554/eLife.01535.001.


Subject(s)
DEAD-box RNA Helicases/metabolism , Encephalomyocarditis virus/metabolism , RNA Helicases/metabolism , RNA, Antisense/metabolism , RNA, Viral/metabolism , Animals , Antiviral Agents/pharmacology , Chlorocebus aethiops , DEAD-box RNA Helicases/genetics , Encephalomyocarditis virus/drug effects , Encephalomyocarditis virus/genetics , Encephalomyocarditis virus/immunology , Gene Expression Regulation, Viral , HEK293 Cells , HeLa Cells , Host-Pathogen Interactions , Humans , Immunity, Innate , Influenza A virus/genetics , Influenza A virus/metabolism , Interferon-Induced Helicase, IFIH1 , Interferons/genetics , Interferons/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , RNA Helicases/genetics , RNA, Antisense/genetics , RNA, Viral/genetics , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/genetics , Signal Transduction , Transfection , Vero Cells , Virus Replication
9.
J Biol Chem ; 286(10): 8128-8140, 2011 Mar 11.
Article in English | MEDLINE | ID: mdl-21169362

ABSTRACT

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.


Subject(s)
Capsid Proteins/metabolism , Carrier Proteins/metabolism , Evolution, Molecular , HIV-1/physiology , Leukemia Virus, Murine/physiology , Virus Replication/physiology , Animals , Antiviral Restriction Factors , Capsid Proteins/genetics , Carrier Proteins/genetics , Cells, Cultured , Humans , Murinae , Mutation , Protein Structure, Secondary , Sequence Homology, Amino Acid , Tripartite Motif Proteins , Ubiquitin-Protein Ligases
10.
J Virol ; 84(11): 5790-801, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20219908

ABSTRACT

Retroviruses are both powerful evolutionary forces and dangerous threats to genome integrity. As such, they have imposed strong selective pressure on their hosts, notably triggering the emergence of restriction factors, such as TRIM5 alpha, that act as potent barriers to their cross-species transmission. TRIM5 alpha orthologues from different primates have distinct retroviral restriction patterns, largely dictated by the sequence of their C-terminal PRYSPRY domain, which binds the capsid protein of incoming virions. Here, by combining genetic and functional analyses of human and squirrel monkey TRIM5 alpha, we demonstrate that the coiled-coil domain of this protein, thus far essentially known for mediating oligomerization, also conditions the spectrum of antiretroviral activity. Furthermore, we identify three coiled-coil residues responsible for this effect, one of which has been under positive selection during primate evolution, notably in New World monkeys. These results indicate that the PRYSPRY and coiled-coil domains cooperate to determine the specificity of TRIM5 alpha-mediated capture of retroviral capsids, shedding new light on this complex event.


Subject(s)
Carrier Proteins/metabolism , Proteins/metabolism , Animals , Antiviral Restriction Factors , Base Sequence , Binding Sites , Capsid Proteins/metabolism , Carrier Proteins/chemistry , Host-Pathogen Interactions , Humans , Macaca , Protein Structure, Tertiary , Proteins/chemistry , Retroviridae , Saimiri , Species Specificity , Tripartite Motif Proteins , Ubiquitin-Protein Ligases
11.
Nature ; 463(7278): 237-40, 2010 Jan 14.
Article in English | MEDLINE | ID: mdl-20075919

ABSTRACT

More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.


Subject(s)
Embryonic Stem Cells/metabolism , Endogenous Retroviruses/genetics , Gene Silencing , Genes, Intracisternal A-Particle/genetics , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , 5' Untranslated Regions/genetics , Acetylation , Animals , DNA Methylation , Embryo, Mammalian/metabolism , Embryo, Mammalian/virology , Embryonic Stem Cells/virology , Fibroblasts , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Histones/metabolism , Leukemia Virus, Murine/genetics , Leukemia Virus, Murine/physiology , Lysine/metabolism , Methylation , Mice , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Promoter Regions, Genetic/genetics , Repressor Proteins/deficiency , Repressor Proteins/genetics , Tripartite Motif-Containing Protein 28
12.
Virology ; 378(2): 233-42, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18586294

ABSTRACT

Human TRIM5alpha restricts N-tropic murine leukemia virus (N-MLV) but not B-tropic MLV (B-MLV) infection. Here we study B30.2/SPRY domain mutants of human TRIM5alpha that acquire the ability to inhibit B-MLV infection prior to reverse transcription without losing the ability to restrict N-MLV infection. Remarkably, these mutants gain the ability to decrease the amount of particulate B-MLV capsids in the cytosol of infected cells. In addition, these mutants gain the ability to restrict SIV(mac) and HIV-2 infection. B-MLV and SIV(mac) infections were blocked by the mutant TRIM5alpha proteins prior to reverse transcription. Thus, the range of retroviruses restricted by human TRIM5alpha can be increased by changes in the B30.2/SPRY domain, which also result in the ability to cause premature uncoating of the restricted retroviral capsid.


Subject(s)
Carrier Proteins/physiology , Leukemia Virus, Murine/immunology , Mutation, Missense , Antiviral Restriction Factors , Capsid/metabolism , Carrier Proteins/genetics , Cell Line , Cytoplasm/virology , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , HIV-2/immunology , Humans , Leukemia Virus, Murine/physiology , Protein Structure, Tertiary , Simian Immunodeficiency Virus/immunology , Tripartite Motif Proteins , Ubiquitin-Protein Ligases
13.
PLoS Pathog ; 3(12): e200, 2007 Dec 28.
Article in English | MEDLINE | ID: mdl-18166079

ABSTRACT

TRIM5alpha is a restriction factor that limits infection of human cells by so-called N- but not B- or NB-tropic strains of murine leukemia virus (MLV). Here, we performed a mutation-based functional analysis of TRIM5alpha-mediated MLV restriction. Our results reveal that changes at tyrosine(336) of human TRIM5alpha, within the variable region 1 of its C-terminal PRYSPRY domain, can expand its activity to B-MLV and to the NB-tropic Moloney MLV. Conversely, we demonstrate that the escape of MLV from restriction by wild-type or mutant forms of huTRIM5alpha can be achieved through interdependent changes at positions 82, 109, 110, and 117 of the viral capsid. Together, our results support a model in which TRIM5alpha-mediated retroviral restriction results from the direct binding of the antiviral PRYSPRY domain to the viral capsid, and can be prevented by interferences exerted by critical residues on either one of these two partners.


Subject(s)
Carrier Proteins/metabolism , Fibroblasts/virology , Leukemia Virus, Murine/growth & development , Retroviridae Infections/virology , Tumor Virus Infections/virology , Amino Acid Substitution , Animals , Antiviral Restriction Factors , Capsid/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cells , Fibroblasts/cytology , Fibroblasts/immunology , HIV Infections/immunology , HIV Infections/virology , HIV-1/genetics , HIV-1/growth & development , Humans , Kidney/cytology , Leukemia Virus, Murine/genetics , Leukemia Virus, Murine/immunology , Mice , Molecular Sequence Data , Protein Structure, Tertiary , Retroviridae Infections/immunology , Tripartite Motif Proteins , Tumor Virus Infections/immunology , Ubiquitin-Protein Ligases
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