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1.
J Thromb Haemost ; 2021 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-34538015

RESUMO

OBJECTIVE: Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID-19 patients. Given that platelets are key regulators of thrombosis, inflammation, and immunity they represent prime candidates as mediators of COVID-19-associated pathogenesis. The objective of this study was to understand the contribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the platelet phenotype via phenotypic (activation, aggregation) and transcriptomic characterization. APPROACH AND RESULTS: In a cohort of 3915 hospitalized COVID-19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication, and biomarkers of inflammation and thrombosis, we find platelet count, size, and immaturity are associated with increased critical illness and all-cause mortality. Bone marrow, lung tissue, and blood from COVID-19 patients revealed the presence of SARS-CoV-2 virions in megakaryocytes and platelets. Characterization of COVID-19 platelets found them to be hyperreactive (increased aggregation, and expression of P-selectin and CD40) and to have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. In vitro mechanistic studies highlight that the interaction of SARS-CoV-2 with megakaryocytes alters the platelet transcriptome, and its effects are distinct from the coronavirus responsible for the common cold (CoV-OC43). CONCLUSIONS: Platelet count, size, and maturity associate with increased critical illness and all-cause mortality among hospitalized COVID-19 patients. Profiling tissues and blood from COVID-19 patients revealed that SARS-CoV-2 virions enter megakaryocytes and platelets and associate with alterations to the platelet transcriptome and activation profile.

2.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168039

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos
3.
Mol Ther Oncolytics ; 8: 71-81, 2018 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-29888320

RESUMO

Through the action of two virus-encoded decapping enzymes (D9 and D10) that remove protective caps from mRNA 5'-termini, Vaccinia virus (VACV) accelerates mRNA decay and limits activation of host defenses. D9- or D10-deficient VACV are markedly attenuated in mice and fail to counter cellular double-stranded RNA-responsive innate immune effectors, including PKR. Here, we capitalize upon this phenotype and demonstrate that VACV deficient in either decapping enzyme are effective oncolytic viruses. Significantly, D9- or D10-deficient VACV displayed anti-tumor activity against syngeneic mouse tumors of different genetic backgrounds and human hepatocellular carcinoma xenografts. Furthermore, D9- and D10-deficient VACV hyperactivated the host anti-viral enzyme PKR in non-tumorigenic cells compared to wild-type virus. This establishes a new genetic platform for oncolytic VACV development that is deficient for a major pathogenesis determinant while retaining viral genes that support robust productive replication like those required for nucleotide metabolism. It further demonstrates how VACV mutants unable to execute a fundamental step in virus-induced mRNA decay can be unexpectedly translated into a powerful anti-tumor therapy.

4.
J Virol ; 92(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29793959

RESUMO

In response to virus-induced shutoff host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), a substrate of the double-stranded RNA (dsRNA)-activated kinase protein kinase R (PKR). The detection of innate immune sensors and effectors like PKR at SGs suggests a role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus 1 (HSV-1), the virus-encoded virion host shutoff (VHS) endoribonuclease is required to restrict interferon production, PKR activation, and SG formation, although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (ΔVHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for beta interferon mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING signaling downstream of cGMP-AMP synthase (cGAS)-dependent DNA sensing is required for beta interferon induction. In contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2α-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation, and beta interferon to address this question. We show that dsRNA is localized to SGs and that SGs can function to promote PKR activation in the context of a DNA virus infection, but we find no evidence to support their importance for interferon induction during HSV-1 infection.


Assuntos
Grânulos Citoplasmáticos/imunologia , Fibroblastos/imunologia , Herpesvirus Humano 1/imunologia , Imunidade Inata , Ribonucleases/imunologia , Transdução de Sinais/imunologia , Proteínas Virais/imunologia , Células Cultivadas , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/genética , Fibroblastos/metabolismo , Fibroblastos/virologia , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/imunologia , RNA Viral/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Transdução de Sinais/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
6.
Cell Host Microbe ; 17(3): 332-344, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25766294

RESUMO

By accelerating global mRNA decay, many viruses impair host protein synthesis, limiting host defenses and stimulating virus mRNA translation. Vaccinia virus (VacV) encodes two decapping enzymes (D9, D10) that remove protective 5' caps on mRNAs, presumably generating substrates for degradation by the host exonuclease Xrn1. Surprisingly, we find VacV infection of Xrn1-depleted cells inhibits protein synthesis, compromising virus growth. These effects are aggravated by D9 deficiency and dependent upon a virus transcription factor required for intermediate and late mRNA biogenesis. Considerable double-stranded RNA (dsRNA) accumulation in Xrn1-depleted cells is accompanied by activation of host dsRNA-responsive defenses controlled by PKR and 2'-5' oligoadenylate synthetase (OAS), which respectively inactivate the translation initiation factor eIF2 and stimulate RNA cleavage by RNase L. This proceeds despite VacV-encoded PKR and RNase L antagonists being present. Moreover, Xrn1 depletion sensitizes uninfected cells to dsRNA treatment. Thus, Xrn1 is a cellular factor regulating dsRNA accumulation and dsRNA-responsive innate immune effectors.


Assuntos
Exorribonucleases/metabolismo , Interações Hospedeiro-Patógeno , Imunidade Inata , Proteínas Associadas aos Microtúbulos/metabolismo , RNA de Cadeia Dupla/metabolismo , Vírus Vaccinia/imunologia , Vírus Vaccinia/fisiologia , Replicação Viral , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos
7.
Commun Integr Biol ; 5(3): 243-7, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22896784

RESUMO

Cytoplasmic poly(A)-binding proteins (PABPs) regulate mRNA stability and translation. Although predominantly localized in the cytoplasm, PABP proteins also cycle through the nucleus. Recent work has established that their steady-state localization can be altered by cellular stresses such as ultraviolet (UV) radiation, and infection by several viruses, resulting in nuclear accumulation of PABPs. Here, we present further evidence that their interaction with and release from mRNA and translation complexes are important in determining their sub-cellular distribution and propose an integrated model for regulated nucleo-cytoplasmic transport of PABPs.

8.
J Cell Sci ; 124(Pt 19): 3344-55, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21940797

RESUMO

Poly(A)-binding protein 1 (PABP1) has a fundamental role in the regulation of mRNA translation and stability, both of which are crucial for a wide variety of cellular processes. Although generally a diffuse cytoplasmic protein, it can be found in discrete foci such as stress and neuronal granules. Mammals encode several additional cytoplasmic PABPs that remain poorly characterised, and with the exception of PABP4, appear to be restricted in their expression to a small number of cell types. We have found that PABP4, similarly to PABP1, is a diffusely cytoplasmic protein that can be localised to stress granules. However, UV exposure unexpectedly relocalised both proteins to the nucleus. Nuclear relocalisation of PABPs was accompanied by a reduction in protein synthesis but was not linked to apoptosis. In examining the mechanism of PABP relocalisation, we found that it was related to a change in the distribution of poly(A) RNA within cells. Further investigation revealed that this change in RNA distribution was not affected by PABP knockdown but that perturbations that block mRNA export recapitulate PABP relocalisation. Our results support a model in which nuclear export of PABPs is dependent on ongoing mRNA export, and that a block in this process following UV exposure leads to accumulation of cytoplasmic PABPs in the nucleus. These data also provide mechanistic insight into reports that transcriptional inhibitors and expression of certain viral proteins cause relocation of PABP to the nucleus.


Assuntos
Proteínas Sanguíneas/metabolismo , Núcleo Celular/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Transporte Proteico/efeitos da radiação , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Apoptose/efeitos da radiação , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Células HeLa , Humanos , Proteínas Imediatamente Precoces/biossíntese , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Biossíntese de Proteínas , Transporte de RNA , Proteínas Recombinantes/biossíntese , Raios Ultravioleta
9.
Proc Natl Acad Sci U S A ; 108(19): 7844-9, 2011 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-21518916

RESUMO

Translational control of many mRNAs in developing metazoan embryos is achieved by alterations in their poly(A) tail length. A family of cytoplasmic poly(A)-binding proteins (PABPs) bind the poly(A) tail and can regulate mRNA translation and stability. However, despite the extensive biochemical characterization of one family member (PABP1), surprisingly little is known about their in vivo roles or functional relatedness. Because no information is available in vertebrates, we address their biological roles, establishing that each of the cytoplasmic PABPs conserved in Xenopus laevis [PABP1, embryonic PABP (ePABP), and PABP4] is essential for normal development. Morpholino-mediated knockdown of PABP1 or ePABP causes both anterior and posterior phenotypes and embryonic lethality. In contrast, depletion of PABP4 results mainly in anterior defects and lethality at later stages. Unexpectedly, cross-rescue experiments reveal that neither ePABP nor PABP4 can fully rescue PABP1 depletion, establishing that PABPs have distinct functions. Comparative analysis of the uncharacterized PABP4 with PABP1 and ePABP shows that it shares a mechanistically conserved core role in promoting global translation. Consistent with this analysis, each morphant displays protein synthesis defects, suggesting that their roles in mRNA-specific translational regulation and/or mRNA decay, rather than global translation, underlie the functional differences between PABPs. Domain-swap experiments reveal that the basis of the functional specificity is complex, involving multiple domains of PABPs, and is conferred, at least in part, by protein-protein interactions.


Assuntos
Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Sequência de Bases , Feminino , Masculino , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/genética , Proteína I de Ligação a Poli(A)/antagonistas & inibidores , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/antagonistas & inibidores , Proteínas de Ligação a Poli(A)/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vertebrados/embriologia , Vertebrados/genética , Vertebrados/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Xenopus laevis/genética
10.
Biochem Soc Trans ; 38(6): 1517-22, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21118118

RESUMO

The regulation of translation has emerged as a major determinant of gene expression and is critical for both normal cellular function and the development of disease. Numerous studies have highlighted the diverse, and sometimes related, mechanisms which underlie the regulation of global translation rates and the translational control of specific mRNAs. In the present paper, we discuss the emerging roles of the basal translation factor PABP [poly(A)-binding protein] in mRNA-specific translational control in metazoa which suggest that PABP function is more complex than first recognized.


Assuntos
Poli A/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Regulação da Expressão Gênica , Poli A/genética , Proteínas de Ligação a Poli(A)/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética
11.
PLoS Genet ; 4(9): e1000199, 2008 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-18802469

RESUMO

As genetic information is transmitted through successive generations, it passes between pluripotent cells in the early embryo and germ cells in the developing foetus and adult animal. Tex19.1 encodes a protein of unknown function, whose expression is restricted to germ cells and pluripotent cells. During male spermatogenesis, Tex19.1 expression is highest in mitotic spermatogonia and diminishes as these cells differentiate and progress through meiosis. In pluripotent stem cells, Tex19.1 expression is also downregulated upon differentiation. However, it is not clear whether Tex19.1 has an essential function in germ cells or pluripotent stem cells, or what that function might be. To analyse the potential role of Tex19.1 in pluripotency or germ cell function we have generated Tex19.1(-/-) knockout mice and analysed the Tex19.1(-/-) mutant phenotype. Adult Tex19.1(-/-) knockout males exhibit impaired spermatogenesis. Immunostaining and histological analysis revealed defects in meiotic chromosome synapsis, the persistence of DNA double-strand breaks during meiosis, and a loss of post-meiotic germ cells in the testis. Furthermore, expression of a class of endogenous retroviruses is upregulated during meiosis in the Tex19.1(-/-) testes. Increased transposition of endogenous retroviruses in the germline of Tex19.1(-/-) mutant mice, and the concomitant increase in DNA damage, may be sufficient to disrupt the normal processes of recombination and chromosome synapsis during meiosis and cause defects in spermatogenesis. Our results suggest that Tex19.1 is part of a specialised mechanism that operates in the germline to repress transposable genetic elements and maintain genomic stability through successive generations.


Assuntos
Retrovirus Endógenos/fisiologia , Deleção de Genes , Proteínas Nucleares/metabolismo , Espermatogênese , Ativação Viral , Animais , Pareamento Cromossômico , Quebras de DNA de Cadeia Dupla , Expressão Gênica , Masculino , Meiose , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas de Ligação a RNA , Espermatócitos/citologia , Espermatócitos/fisiologia , Espermatócitos/virologia
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