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Artikel ; Online: Epitranscriptomic regulation of HIV-1 full-length RNA packaging.

Pereira-Montecinos, Camila / Toro-Ascuy, Daniela / Ananías-Sáez, Catarina / Gaete-Argel, Aracelly / Rojas-Fuentes, Cecilia / Riquelme-Barrios, Sebastián / Rojas-Araya, Bárbara / García-de-Gracia, Francisco / Aguilera-Cortés, Paulina / Chnaiderman, Jonás / Acevedo, Mónica L / Valiente-Echeverría, Fernando / Soto-Rifo, Ricardo

Nucleic acids research

2022 Band 50, Heft 4, Seite(n) 2302–2318

Abstract: During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we ... ...

Abstract	During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.
Mesh-Begriff(e)	5' Untranslated Regions ; Adenosine/genetics ; Adenosine/metabolism ; Gene Products, gag/genetics ; HIV-1/metabolism ; Methylation ; RNA, Viral/genetics ; RNA, Viral/metabolism ; Virion/metabolism
Chemische Substanzen	5' Untranslated Regions ; Gene Products, gag ; RNA, Viral ; Adenosine (K72T3FS567)
Sprache	Englisch
Erscheinungsdatum	2022-02-07
Erscheinungsland	England
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkac062
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: Differences in the internalization of self-inactivating VSVG-pseudotyped murine leukemia virus-based vectors in human and murine cells.

Acevedo, Mónica Loreto / García-de Gracia, Francisco / Miranda-Cárdenas, Camila / Soto-Rifo, Ricardo / Aguayo, Francisco / León, Oscar

Journal of virological methods

2018 Band 255, Seite(n) 14–22

Abstract: Self-inactivating VSVG-pseudotyped murine leukemia virus (SIN-VSVG-MLV) has been widely used to generate stable cell lines and produce gene delivery vectors. Despite the broad cellular tropism of the VSVG-pseudotyped MLV, we observed differential viral ... ...

Abstract	Self-inactivating VSVG-pseudotyped murine leukemia virus (SIN-VSVG-MLV) has been widely used to generate stable cell lines and produce gene delivery vectors. Despite the broad cellular tropism of the VSVG-pseudotyped MLV, we observed differential viral transduction efficiency depending on the host cell type used. In order to determine the mechanism underlying these differences, we used a GFP-expressing SIN-VSVG-MLV and analyzed the major steps of viral transduction in different cell lines including human epithelial, T-lymphocytes, monocytes and murine fibroblast cells. We observed the better transduction efficiency in HeLa cells, which was 20-fold higher than THP-1 and NIH/3T3 cells. To quantify viral internalization, we determined genomic RNA content by quantifying the early reverse transcription product. Genomic RNA and transduction levels were correlated with HeLa cells showing the higher amount of early RT product followed by tsA201 cells, while NIH/3T3, Jurkat and THP-1 had the lowest amounts. Similar results were observed when the late reverse transcription product was analyzed. Reverse transcription efficiency was 66-85% in HeLa cells and about 30% in tsA201, NIH/3T3, Jurkat and THP-1 cells. Viral integration, determined by Alu-Nested-qPCR, was higher for HeLa and lowerst for Jurkat and THP-1 cells. Interestingly, we observed that viral entry was correlated with the cellular availability of clathrin-mediated endocytosis, which was higher in HeLa and tsA201 cells, potentially explaining the higher rates of SIN-VSVG-MLV transduction and early RT synthesis observed in these cell lines. In conclusion, the SIN-VSVG-MLV vector showed significantly different rates of infectivity depending on the host cell type, possibly due to differential rates of viral internalization.
Mesh-Begriff(e)	Animals ; Cell Line ; Genetic Vectors/genetics ; Humans ; Leukemia Virus, Murine/genetics ; Leukemia Virus, Murine/metabolism ; Leukemia Virus, Murine/physiology ; Membrane Glycoproteins/genetics ; Membrane Glycoproteins/metabolism ; Mice ; Transduction, Genetic ; Viral Envelope Proteins/genetics ; Viral Envelope Proteins/metabolism ; Virus Internalization
Chemische Substanzen	G protein, vesicular stomatitis virus ; Membrane Glycoproteins ; Viral Envelope Proteins
Sprache	Englisch
Erscheinungsdatum	2018-02-06
Erscheinungsland	Netherlands
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2018.02.005
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev

García-de-Gracia, Francisco / Gaete-Argel, Aracelly / Riquelme-Barrios, Sebastián / Pereira-Montecinos, Camila / Rojas-Araya, Bárbara / Aguilera, Paulina / Oyarzún-Arrau, Aarón / Rojas-Fuentes, Cecilia / Acevedo, Mónica L / Chnaiderman, Jonás / Valiente-Echeverría, Fernando / Toro-Ascuy, Daniela / Soto-Rifo, Ricardo

RNA biology. 2021 May 04, v. 18, no. 5

2021

Abstract: Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E ... ...

Abstract	Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.
Schlagwörter	Human immunodeficiency virus 1 ; physiological transport ; ribonucleoproteins ; ribosomes
Sprache	Englisch
Erscheinungsverlauf	2021-0504
Umfang	p. 745-758.
Erscheinungsort	Taylor & Francis
Dokumenttyp	Artikel
Anmerkung	NAL-AP-2-clean
ISSN	1555-8584
DOI	10.1080/15476286.2020.1832375
Datenquelle	NAL Katalog (AGRICOLA)

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Artikel ; Online: CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev.

RNA biology

2020 Band 18, Heft 5, Seite(n) 745–758

Abstract	Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.
Mesh-Begriff(e)	Cells, Cultured ; Down-Regulation ; Eukaryotic Initiation Factors/physiology ; HEK293 Cells ; HIV-1/genetics ; HIV-1/metabolism ; HeLa Cells ; Humans ; Jurkat Cells ; Protein Biosynthesis/genetics ; gag Gene Products, Human Immunodeficiency Virus/biosynthesis ; rev Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors ; rev Gene Products, Human Immunodeficiency Virus/physiology
Chemische Substanzen	CTIF protein, human ; Eukaryotic Initiation Factors ; gag Gene Products, Human Immunodeficiency Virus ; rev Gene Products, Human Immunodeficiency Virus
Sprache	Englisch
Erscheinungsdatum	2020-10-25
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1555-8584
ISSN (online)	1555-8584
DOI	10.1080/15476286.2020.1832375
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses.

bioRxiv : the preprint server for biology

2021

Abstract: Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the ... ...

Abstract	Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.
Sprache	Englisch
Erscheinungsdatum	2021-05-21
Erscheinungsland	United States
Dokumenttyp	Preprint
DOI	10.1101/2021.05.19.444823
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

Research square

2021

Abstract	Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.
Sprache	Englisch
Erscheinungsdatum	2021-05-27
Erscheinungsland	United States
Dokumenttyp	Preprint
DOI	10.21203/rs.3.rs-555275/v1
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA.

Toro-Ascuy, Daniela / Rojas-Araya, Bárbara / García-de-Gracia, Francisco / Rojas-Fuentes, Cecilia / Pereira-Montecinos, Camila / Gaete-Argel, Aracelly / Valiente-Echeverría, Fernando / Ohlmann, Théophile / Soto-Rifo, Ricardo

Nucleic acids research

2018 Band 46, Heft 21, Seite(n) 11539–11552

Abstract: Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the ... ...

Abstract	Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.
Mesh-Begriff(e)	Cell Line ; Eukaryotic Initiation Factor-4A/genetics ; Eukaryotic Initiation Factor-4A/metabolism ; HIV-1/genetics ; HIV-1/metabolism ; HeLa Cells ; Humans ; Multiprotein Complexes/genetics ; Multiprotein Complexes/metabolism ; Nuclear Cap-Binding Protein Complex/genetics ; Nuclear Cap-Binding Protein Complex/metabolism ; Protein Binding ; RNA Splicing ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Viral/genetics ; RNA, Viral/metabolism ; Virus Replication/genetics ; gag Gene Products, Human Immunodeficiency Virus/biosynthesis ; gag Gene Products, Human Immunodeficiency Virus/genetics ; rev Gene Products, Human Immunodeficiency Virus/genetics ; rev Gene Products, Human Immunodeficiency Virus/metabolism
Chemische Substanzen	Multiprotein Complexes ; Nuclear Cap-Binding Protein Complex ; RNA, Messenger ; RNA, Viral ; gag Gene Products, Human Immunodeficiency Virus ; rev Gene Products, Human Immunodeficiency Virus ; rev protein, Human Immunodeficiency Virus-1 ; Eukaryotic Initiation Factor-4A (EC 2.7.7.-)
Sprache	Englisch
Erscheinungsdatum	2018-09-18
Erscheinungsland	England
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gky851
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses.

Bonaventure, Boris / Rebendenne, Antoine / Chaves Valadão, Ana Luiza / Arnaud-Arnould, Mary / Gracias, Ségolène / Garcia de Gracia, Francisco / McKellar, Joe / Labaronne, Emmanuel / Tauziet, Marine / Vivet-Boudou, Valérie / Bernard, Eric / Briant, Laurence / Gros, Nathalie / Djilli, Wassila / Courgnaud, Valérie / Parrinello, Hugues / Rialle, Stéphanie / Blaise, Mickaël / Lacroix, Laurent /

Lavigne, Marc / Paillart, Jean-Christophe / Ricci, Emiliano P / Schulz, Reiner / Jouvenet, Nolwenn / Moncorgé, Olivier / Goujon, Caroline

EMBO reports

2022 Band 23, Heft 11, Seite(n) e54061

Abstract: Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation ... ...

Abstract	Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.
Mesh-Begriff(e)	Humans ; DEAD-box RNA Helicases/genetics ; DEAD-box RNA Helicases/metabolism ; HIV-1/physiology ; Positive-Strand RNA Viruses/physiology ; SARS-CoV-2/physiology ; Virus Replication
Chemische Substanzen	DEAD-box RNA Helicases (EC 3.6.4.13) ; DDX42 protein, human (EC 3.6.1-)
Sprache	Englisch
Erscheinungsdatum	2022-09-26
Erscheinungsland	England
Dokumenttyp	Journal Article
ZDB-ID	2020896-0
ISSN	1469-3178 ; 1469-221X
ISSN (online)	1469-3178
ISSN	1469-221X
DOI	10.15252/embr.202154061
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor

Bonaventure, Boris / Rebendenne, Antoine / Garcia de Gracia, Francisco / Tauziet, Marine / McKellar, Joe / Chaves Valadão, Ana Luiza / Courgnaud, Valérie / Bernard, Eric / Briant, Laurence / Gros, Nathalie / Djilli, Wassila / Arnaud-Arnould, Mary / Parrinello, Hugues / Rialle, Stéphanie / Moncorgé, Olivier / Goujon, Caroline

bioRxiv

Abstract: Genome-wide CRISPR/Cas9 knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. With the aim of identifying new cellular inhibitors of HIV-1, we have developed a strategy in which we took advantage of the ability ... ...

Abstract	Genome-wide CRISPR/Cas9 knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. With the aim of identifying new cellular inhibitors of HIV-1, we have developed a strategy in which we took advantage of the ability of type 1 interferon (IFN) to potently inhibit HIV-1 infection, in order to create a cellular environment hostile to viral replication. This approach led to the identification of the DEAD-box RNA helicase DDX42 as an intrinsic inhibitor of HIV-1. Depletion of endogenous DDX42 using siRNA or CRISPR/Cas9 knock-out increased HIV-1 infection, both in model cell lines and in physiological targets of HIV-1, primary CD4+ T cells and monocyte-derived macrophages (MDMs), and irrespectively of the IFN treatment. Similarly, the overexpression of a dominant-negative mutant of DDX42 positively impacted HIV-1 infection, whereas wild-type DDX42 overexpression potently inhibited HIV-1 infection. The positive impact of endogenous DDX42 depletion on HIV-1 infection was directly correlated to an increase in viral DNA accumulation. Interestingly, proximity ligation assays showed that DDX42, which can be mainly found in the nucleus but is also present in the cytoplasm, was in the close vicinity of HIV-1 Capsid during infection of primary monocyte-derived macrophages. Moreover, we show that DDX42 is also able to substantially decrease infection with other retroviruses and retrotransposition of long interspersed elements-1 (LINE-1). Finally, we reveal that DDX42 potently inhibits other pathogenic viruses, including Chikungunya virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Schlagwörter	covid19
Sprache	Englisch
Erscheinungsdatum	2020-10-28
Verlag	Cold Spring Harbor Laboratory
Dokumenttyp	Artikel ; Online
DOI	10.1101/2020.10.28.359356
Datenquelle	COVID19

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Artikel ; Online: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses

bioRxiv

Abstract	Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.
Schlagwörter	covid19
Sprache	Englisch
Erscheinungsdatum	2021-05-19
Verlag	Cold Spring Harbor Laboratory
Dokumenttyp	Artikel ; Online
DOI	10.1101/2021.05.19.444823
Datenquelle	COVID19

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