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1.
Signal Transduct Target Ther ; 6(1): 300, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381015

RESUMO

Elderly people and patients with comorbidities are at higher risk of COVID-19 infection, resulting in severe complications and high mortality. However, the underlying mechanisms are unclear. In this study, we investigate whether miRNAs in serum exosomes can exert antiviral functions and affect the response to COVID-19 in the elderly and people with diabetes. First, we identified four miRNAs (miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p) through high-throughput sequencing and quantitative real-time PCR analysis, that are remarkably decreased in the elderly and diabetic groups. We further demonstrated that these miRNAs, either in the exosome or in the free form, can directly inhibit S protein expression and SARS-CoV-2 replication. Serum exosomes from young people can inhibit SARS-CoV-2 replication and S protein expression, while the inhibitory effect is markedly decreased in the elderly and diabetic patients. Moreover, three out of the four circulating miRNAs are significantly increased in the serum of healthy volunteers after 8-weeks' continuous physical exercise. Serum exosomes isolated from these volunteers also showed stronger inhibitory effects on S protein expression and SARS-CoV-2 replication. Our study demonstrates for the first time that circulating exosomal miRNAs can directly inhibit SARS-CoV-2 replication and may provide a possible explanation for the difference in response to COVID-19 between young people and the elderly or people with comorbidities.


Assuntos
COVID-19/genética , Diabetes Mellitus/genética , MicroRNAs/genética , Glicoproteína da Espícula de Coronavírus/genética , Adulto , Fatores Etários , Idoso , COVID-19/sangue , COVID-19/patologia , COVID-19/virologia , China , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Estudos de Coortes , Diabetes Mellitus/sangue , Diabetes Mellitus/patologia , Diabetes Mellitus/virologia , Exercício Físico , Exossomos/genética , Exossomos/metabolismo , Exossomos/virologia , Feminino , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , SARS-CoV-2/genética , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/sangue , Replicação Viral
2.
Vet Microbiol ; 260: 109163, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311269

RESUMO

Small ruminant morbillivirus (SRMV) is a highly contagious and economically important viral disease of small domestic and wild ruminants. Difficulty with its stable proliferation in ovis aries-derived cells has led to a relative lag in the study of its natural immunity and pathogenesis. Here we report the antiviral properties of ZAP against SRMV, a single-stranded negative-stranded RNA virus of the genus Morbillivirus. ZAP expression was significantly induced in sheep endometrial epithelial cells following SRMV infection. ZAP inhibited SRMV replication in cells after infection, while its overexpression in Vero-SLAM cells significantly increased their resistance to SRMV replication. The ZAP protein co-localized with SRMV RNA in the cytoplasm and ZAP-responsive elements were mapped to the 5' untranslated region of SRMV nucleocapsid, phosphoprotein, matrix, and fusion. In summary, ZAP confers resistance to SRMV infection by directly targeting viral RNA and inhibiting viral replication. Our findings further extend the ranges of viral targets of ZAP and help elucidate the mechanism of SRMV replication.


Assuntos
Infecções por Morbillivirus/veterinária , Morbillivirus/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Chlorocebus aethiops , Endométrio/virologia , Células Epiteliais/virologia , Feminino , Células HEK293 , Humanos , Infecções por Morbillivirus/virologia , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Ovinos , Células Vero , Replicação Viral
3.
Viruses ; 13(3)2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807534

RESUMO

The mitochondrial antiviral-signaling protein (MAVS, also known as VISA, IPS-1, or CARDIF) plays an essential role in the type I interferon (IFN) response and in retinoic acid-inducible gene I (RIG-I) mediated antiviral innate immunity in mammals. In this study, the caprine MAVS gene (caMAVS, 1566 bp) was identified and cloned. The caMAVS shares the highest amino acid similarity (98.1%) with the predicted sheep MAVS. Confocal microscopy analysis of partial deletion mutants of caMAVS revealed that the transmembrane and the so-called Non-Characterized domains are indispensable for intracellular localization to mitochondria. Overexpression of caMAVS in caprine endometrial epithelial cells up-regulated the mRNA levels of caprine interferon-stimulated genes. We concluded that caprine MAVS mediates the activation of the type I IFN pathway. We further demonstrated that both the CARD-like domain and the transmembrane domain of caMAVS were essential for the activation of the IFN-ß promotor. The interaction between caMAVS and caprine RIG-I and the vital role of the CARD and NC domain in this interaction was demonstrated by co-immunoprecipitation. Upon infection with the Peste des Petits Ruminants Virus (PPRV, genus Morbillivirus), the level of MAVS was greatly reduced. This reduction was prevented by the addition of the proteasome inhibitor MG132. Moreover, we found that viral protein V could interact and colocalize with MAVS. Together, we identified caMAVS as a RIG-I interactive protein involved in the activation of type I IFN pathways in caprine cells and as a target for PPRV immune evasion.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Indutores de Interferon/imunologia , Peste dos Pequenos Ruminantes/imunologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Animais , Chlorocebus aethiops , Células Epiteliais , Cabras , Células HEK293 , Humanos , Interferon Tipo I/imunologia , Células Vero
4.
Cell Res ; 31(6): 631-648, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33782530

RESUMO

RNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not solved the problem of efficient in vivo siRNA delivery. Here, we present a proof-of-principle strategy that reprogrammes host liver with genetic circuits to direct the synthesis and self-assembly of siRNAs into secretory exosomes and facilitate the in vivo delivery of siRNAs through circulating exosomes. By combination of different genetic circuit modules, in vivo assembled siRNAs are systematically distributed to multiple tissues or targeted to specific tissues (e.g., brain), inducing potent target gene silencing in these tissues. The therapeutic value of our strategy is demonstrated by programmed silencing of critical targets associated with various diseases, including EGFR/KRAS in lung cancer, EGFR/TNC in glioblastoma and PTP1B in obesity. Overall, our strategy represents a next generation RNAi therapeutics, which makes RNAi therapy feasible.

5.
Vet Microbiol ; 249: 108858, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32980631

RESUMO

Rabbit hemorrhagic disease virus (RHDV), a member of Caliciviridae family, causes a highly contagious disease in rabbits. The RHDV replication mechanism is poorly understood due to the lack of a suitable culture system in vitro. This study identified RHDV 5' and 3' extremities (Ex) RNA binding proteins from the rabbit kidney cell line RK-13 based on a pull-down assay by applying a tRNA scaffold streptavidin aptamer. Using mass spectrometry (MS), several host proteins were discovered which interact with RHDV 5' and 3' Ex RNA. The ribosomal protein S5 (RPS5) was shown to interact with RHDV 3' Ex RNA directly by RNA-pulldown and confocal microscopy. To further investigate the role of RPS5 in RHDV replication, small interfering RNAs for RPS5 and RPS5 eukaryotic expression plasmids were used to change the expression level of RPS5 in RK-13 cells and the results showed that the RHDV replication and translation levels were positively correlated with the expression level of RPS5. It was also verified that RPS5 promoted RHDV replication by constructing RPS5 stable overexpression cell lines and RPS5 knockdown cell lines. In summary, it has been identified that RPS5 interacted with the RHDV 3' Ex RNA region and played a role in virus replication. These results will help to understand the mechanism of RHDV replication.


Assuntos
Infecções por Caliciviridae/veterinária , Vírus da Doença Hemorrágica de Coelhos/metabolismo , Proteínas Ribossômicas/metabolismo , Replicação Viral/genética , Infecções por Caliciviridae/virologia , Regulação Viral da Expressão Gênica , Vírus da Doença Hemorrágica de Coelhos/genética
6.
Vet Microbiol ; 240: 108529, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31902498

RESUMO

Rabbit hemorrhagic disease (RHD) is an acute, inflammatory, septic, and devastating infectious disease caused by Rabbit hemorrhagic disease virus (RHDV), which poses a serious threat to the rabbit industry. RHDV2 (GI.2/RHDVb), a recently reported new variant could cause RHD in wild populations, but also RHDV-vaccinated rabbits. For now, both RHDV and RHDV2 are the main causes of RHD. To develop a new subunit vaccine that could protect rabbits against both classic RHDV and RHDV2 infections, we constructed a recombinant baculovirus (Bac-classic RHDV VP60-RHDV2 VP60) containing the VP60 genes of classic RHDV and RHDV2. Both VP60 genes were well expressed simultaneously in Spodoptera frugiperda cells (Sf9) after infection with the recombinant baculovirus. Transmission electron microscopy showed that the recombinant VP60 self-assembled into virus-like particles (VLPs). The antigenicity and immunogenicity of the bivalent VLPs vaccine were examined with animal experiments. Our results demonstrated that both the humoral and cellular immune responses were efficiently induced in rabbits by a subunit vaccine based on the recombinant baculovirus. In addition, all rabbits immunized with the bivalent VLPs vaccine survived after challenged with classic RHDV, and showed no clinical signs of RHD, whereas all the rabbits in the negative control group died from classic RHDV infection and showed typical clinical signs of RHD. In summary, our results indicated that the recombinant baculovirus carrying two VP60 genes is a candidate construct from which to develop a bivalent VLPs vaccine against both classic RHDV and RHDV2 infections.


Assuntos
Infecções por Caliciviridae/veterinária , Vírus da Doença Hemorrágica de Coelhos/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Baculoviridae , Infecções por Caliciviridae/prevenção & controle , Citocinas/imunologia , Feminino , Vírus da Doença Hemorrágica de Coelhos/genética , Imunidade Celular , Imunidade Humoral , Masculino , Coelhos , Células Sf9 , Organismos Livres de Patógenos Específicos , Spodoptera , Vacinação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas Virais/genética
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