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1.
Nucleic Acids Res ; 50(4): e23, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-34850942

RESUMO

Most RNA footprinting approaches that require ribonuclease cleavage generate RNA fragments bearing a phosphate or cyclic phosphate group at their 3' end. Unfortunately, current library preparation protocols rely only on a 3' hydroxyl group for adaptor ligation or poly-A tailing. Here, we developed circAID-p-seq, a PCR-free library preparation for selective 3' phospho-RNA sequencing. As a proof of concept, we applied circAID-p-seq to ribosome profiling, which is based on sequencing of RNA fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast and highly efficient sequencing of phospho-RNA fragments from eukaryotic cells and tissues. We used circAID-p-seq to portray ribosome occupancy in transcripts, providing a versatile and PCR-free strategy to possibly unravel any endogenous 3'-phospho RNA molecules.


Assuntos
RNA , Ribossomos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fosfatos , RNA/genética , Ribossomos/genética , Análise de Sequência de RNA/métodos
2.
Nature ; 510(7504): 235-240, 2014 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-24870228

RESUMO

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.


Assuntos
Marcação de Genes/métodos , Genoma Humano/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Reparo Gênico Alvo-Dirigido/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Animais , Antígenos CD34/metabolismo , DNA Complementar/genética , Endonucleases/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Sangue Fetal/transplante , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos , Mutação/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia
3.
PLoS Pathog ; 11(7): e1005050, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26181333

RESUMO

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1° sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.


Assuntos
Antivirais/farmacologia , Capsídeo/microbiologia , HIV-2/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Integração Viral/efeitos dos fármacos , Animais , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , HIV-2/genética , HIV-2/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Vírus da Imunodeficiência Símia/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia
4.
Viruses ; 15(3)2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36992361

RESUMO

The host transmembrane protein SERINC5 is incorporated into retrovirus particles and inhibits HIV-1 infectivity. The lentiviral Nef protein counteracts SERINC5 by downregulating it from the cell surface and preventing its incorporation into virions. The ability of Nef to antagonize the host factor varies in magnitude between different HIV-1 isolates. After having identified a subtype H nef allele unable to promote HIV-1 infectivity in the presence of SERINC5, we investigated the molecular determinants responsible for the defective counteraction of the host factor. Chimeric molecules with a subtype C Nef highly active against SERINC5 were constructed to locate Nef residues crucial for the activity against SERINC5. An Asn at the base of the C-terminal loop of the defective nef allele was found in place of a highly conserved acidic residue (D/E 150). The conversion of Asn to Asp restored the ability of the defective Nef to downregulate SERINC5 and promote HIV-1 infectivity. The substitution was also found to be crucial for the ability of Nef to downregulate CD4, but not for Nef activities that do not rely on the internalization of receptors from the cell surface, suggesting a general implication in promoting clathrin-mediated endocytosis. Accordingly, bimolecular fluorescence complementation revealed that the conserved acidic residue contributes to the recruitment of AP2 by Nef. Altogether, our results confirm that Nef downregulates SERINC5 and CD4 by engaging a similar machinery and indicates that, in addition to the di-leucine motif, other residues in the C-terminal flexible loop are important for the ability of the protein to sustain clathrin-mediated endocytosis.


Assuntos
Antígenos CD4 , Linfócitos T CD4-Positivos , HIV-1 , Proteínas de Membrana , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Humanos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Proteínas de Membrana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Substituição de Aminoácidos , Células HEK293 , Células Jurkat , HIV-1/patogenicidade , Sequência de Aminoácidos , Endocitose , Clatrina , Infecções por HIV , Antígenos CD4/metabolismo , Regulação para Baixo
5.
Elife ; 102021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33594971

RESUMO

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.


Assuntos
RNA Longo não Codificante/metabolismo , Ribossomos/metabolismo , Animais , Camundongos , Células-Tronco Embrionárias Murinas , Peptídeos/metabolismo , Biossíntese de Proteínas , Proteômica , RNA Longo não Codificante/genética , Ribossomos/genética
6.
Annu Rev Virol ; 5(1): 323-340, 2018 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-30265629

RESUMO

SERINC genes encode for homologous multipass transmembrane proteins with unknown cellular function, despite being highly conserved across eukaryotes. Among the five SERINC genes found in humans, SERINC5 was shown to act as a powerful inhibitor of retroviruses. It is efficiently incorporated into virions and blocks the penetration of the viral core into target cells, by impairing the fusion process with a yet unclear mechanism. SERINC5 was also found to promote human immunodeficiency virus 1 (HIV-1) virion neutralization by antibodies, indicating a pleiotropic activity, which remains mostly unexplored. Counteracting factors have emerged independently in at least three retrovirus lineages, underscoring their fundamental importance during retrovirus evolution. Nef and S2 of primate and equine lentiviruses, and glycoGag of gammaretroviruses, act similarly by targeting SERINC5 to endosomes and excluding it from virions. Here, we discuss the features that distinguish SERINC5 from other known restriction factors, delineating a yet unique class of antiviral inhibitors.


Assuntos
HIV/imunologia , Imunidade Inata , Fatores Imunológicos/metabolismo , Vírus da Leucemia Murina/imunologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Humanos , Camundongos
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