RESUMO
Adenosine-to-inosine RNA editing has been proposed to be involved in a bacterial anti-phage defense system called RADAR. RADAR contains an adenosine triphosphatase (RdrA) and an adenosine deaminase (RdrB). Here, we report cryo-EM structures of RdrA, RdrB, and currently identified RdrA-RdrB complexes in the presence or absence of RNA and ATP. RdrB assembles into a dodecameric cage with catalytic pockets facing outward, while RdrA adopts both autoinhibited tetradecameric and activation-competent heptameric rings. Structural and functional data suggest a model in which RNA is loaded through the bottom section of the RdrA ring and translocated along its inner channel, a process likely coupled with ATP-binding status. Intriguingly, up to twelve RdrA rings can dock one RdrB cage with precise alignments between deaminase catalytic pockets and RNA-translocation channels, indicative of enzymatic coupling of RNA translocation and deamination. Our data uncover an interesting mechanism of enzymatic coupling and anti-phage defense through supramolecular assemblies.
Assuntos
Trifosfato de Adenosina , RNA , Adenosina Desaminase/genéticaRESUMO
Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life1,2. Mammalian cells produce one CDN, 2'3'-cGAMP, through cyclic GMP-AMP synthase after detecting cytosolic DNA signals3-7. 2'3'-cGAMP, as well as bacterial and synthetic CDN analogues, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses8-21. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is dependent on the solute carrier SLC19A122,23. Moreover, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics24,25, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs, folate and antifolate is unclear. Here we report cryo-electron microscopy structures of human SLC19A1 (hSLC19A1) in a substrate-free state and in complexes with multiple CDNs from different sources, a predominant natural folate and a new-generation antifolate drug. The structural and mutagenesis results demonstrate that hSLC19A1 uses unique yet divergent mechanisms to recognize CDN- and folate-type substrates. Two CDN molecules bind within the hSLC19A1 cavity as a compact dual-molecule unit, whereas folate and antifolate bind as a monomer and occupy a distinct pocket of the cavity. Moreover, the structures enable accurate mapping and potential mechanistic interpretation of hSLC19A1 with loss-of-activity and disease-related mutations. Our research provides a framework for understanding the mechanism of SLC19-family transporters and is a foundation for the development of potential therapeutics.
Assuntos
Microscopia Crioeletrônica , Fosfatos de Dinucleosídeos , Antagonistas do Ácido Fólico , Ácido Fólico , Nucleotídeos Cíclicos , Animais , Humanos , Fosfatos de Dinucleosídeos/metabolismo , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Mamíferos/metabolismo , Nucleotídeos Cíclicos/metabolismo , Proteína Carregadora de Folato Reduzido/química , Proteína Carregadora de Folato Reduzido/genética , Proteína Carregadora de Folato Reduzido/metabolismo , Proteína Carregadora de Folato Reduzido/ultraestruturaRESUMO
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.
Assuntos
COVID-19 , Interações entre Hospedeiro e Microrganismos , Proteólise , SARS-CoV-2 , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas Virais , Humanos , Células CACO-2 , COVID-19/enzimologia , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/virologia , Células HEK293 , Interferons/imunologia , Fases de Leitura Aberta , Ligação Proteica , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
SARS-CoV-2 belongs to the subgenus Sarbecovirus, which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections.IMPORTANCEThe ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections.
Assuntos
SARS-CoV-2 , Proteínas Virais , Viroses , Humanos , Enzimas Desubiquitinantes , Interferons/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.
Assuntos
Apoptose , Aterosclerose , Células Espumosas , Canais Iônicos , Macrófagos , Fagocitose , Animais , Canais Iônicos/metabolismo , Canais Iônicos/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/genética , Camundongos , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Tiofenos/farmacologia , Masculino , Espécies Reativas de Oxigênio/metabolismo , Placa Aterosclerótica/patologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/genética , Mitocôndrias/metabolismo , Pirazinas , TiadiazóisRESUMO
Using a batch thermophilic anaerobic system established with 60 mL serum bottles, the mechanism on how microbial enrichments obtained from magnetite-amended paddy soil via repeated batch cultivation affected methane production from acetate was investigated. Magnetite-amended enrichments (MAEs) can improve the methane production rate rather than the methane yield. Compared with magnetite-unamended enrichments, the methane production rate in MAE was improved by 50%, concomitant with the pronounced electrochemical response, high electron transfer capacity, and fast acetate degradation. The promoting effects might be ascribed to direct interspecies electron transfer facilitated by magnetite, where magnetite might function as electron conduits to link the acetate oxidizers (Anaerolineaceae and Peptococcaceae) with methanogens (Methanosarcinaceae). The findings demonstrated the potential application of MAE for boosting methanogenic performance during thermophilic anaerobic digestion.
Assuntos
Euryarchaeota , Óxido Ferroso-Férrico , Anaerobiose , Metano/metabolismo , Transporte de Elétrons , Acetatos/metabolismo , Euryarchaeota/metabolismo , Reatores BiológicosRESUMO
The host restriction factor APOBEC3G (A3G) inhibits an extensive variety of viruses, including retroviruses, DNA viruses, and RNA viruses. Our study shows that A3G inhibits enterovirus 71 (EV71) and coxsackievirus A16 (CA16) via competitively binding the 5' untranslated region (UTR) with the host protein poly(C)-binding protein 1 (PCBP1), which is required for the replication of multiple EVs. However, whether A3G inhibits other EVs in addition to EV71 and CA16 has not been investigated. Here, we demonstrate that A3G could inhibit the replication of EVD68, which requires PCBP1 for its replication, but not CA6, which does not require PCBP1 for replication. Further investigation revealed that the nucleic-acid-binding activity of A3G is required for EVD68 restriction, similar to the mechanism presented for EV71 restriction. Mechanistically, A3G competitively binds to the cloverleaf (1 to 123 nucleotides [nt]) and the stem-loop IV (234 to 446 nt) domains of the EVD68 5' UTR with PCBP1, thereby inhibiting the 5' UTR activity of EVD68; by contrast, A3G does not interact with CA6 5' UTR, resulting in no effect on CA6 replication. Moreover, the nonstructural protein 2C, encoded by EVD68, overcomes A3G suppression by inducing A3G degradation via the autophagy-lysosome pathway. Our findings revealed that A3G might have broad-spectrum antiviral activity against multiple EVs through this general mechanism, and they might provide important information for the development of an anti-EV strategy. IMPORTANCE As the two major pathogens causing hand, foot, and mouth disease (HFMD), enterovirus 71 (EV71) and coxsackievirus A16 (CA16) attract a lot of attention for the study of their pathogenesis, their involvement with cellular proteins, and so on. However, other EVs such as CA6 and EVD68 constantly occur sporadically or have spread worldwide in recent years. Therefore, more information related to these EVs is needed in order to develop a broad-spectrum anti-EV inhibitor. In this study, we first reveal that the protein poly(C)-binding protein 1 (PCBP1), involved in PV- and EV71 virus replication, is also required for the replication of EVD68, but not for the replication of CA6. Next, we found that the host-restriction factor A3G specifically inhibits the replication of EVD68, but not the replication of CA6, by competitively binding to the 5' UTR of EVD68 along with PCBP1. Our findings broaden knowledge related to EV replication and the interplay between EVs and host factors.
Assuntos
Regiões 5' não Traduzidas/fisiologia , Desaminase APOBEC-3G/metabolismo , Proteínas de Ligação a DNA/metabolismo , Enterovirus Humano D/fisiologia , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Desaminase APOBEC-3G/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Enterovirus Humano A/fisiologia , Células HEK293 , Humanos , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants have caused hundreds of thousands of deaths and shown serious social influence worldwide. Jilin Province, China, experienced the first wave of the outbreak from December 2020 to February 2021. Here, we analyzed the genomic characteristics of the SARS-CoV-2 outbreak in Jilin province using a phylogeographic tree and found that clinical isolates belonged to the B.1 lineage, which was considered to be the ancestral lineage. Several dominant SARS-CoV-2 specific linear B cell epitopes that reacted with the convalescent sera were also analysed and identified using a peptide microarray composed of S, M, and E proteins. Moreover, the serum of convalescent patients infected with SARS-CoV-2 showed neutralizing activity against four widely spreading SARS-CoV-2 variants; however, significant differences were observed in neutralizing activities against different SARS-CoV-2 variants. These data provide important information on genomic characteristics, linear epitopes, and neutralizing activity of SARS-CoV-2 outbreak in Jilin Province, China, which may aid in understanding disease patterns and regional aspects of the pandemic.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/epidemiologia , Soroterapia para COVID-19 , Epitopos de Linfócito B/genética , Surtos de Doenças , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
African swine fever (ASF) leads to high mortality in domestic pigs and wild boar, and it is caused by the African swine fever virus (ASFV). Currently, no commercially available vaccine exists for its prevention in China. In this study, we engineered a pseudorabies recombinant virus (PRV) expressing ASFV CD2v and p54 proteins (PRV-∆TK-(CD2v)-∆gE-(p54)) using CRISPR/Cas9 and homologous recombination technology. PRV-∆TK-(CD2v)-∆gE-(p54) effectively delivers CD2v and p54, and it exhibits reduced virulence. Immunization with PRV-∆TK-(CD2v)-∆gE-(p54) neither induces pruritus nor causes systemic infection and inflammation. Furthermore, a double knockout of the TK and gE genes eliminates the depletion of T, B, and monocytes/macrophages in the blood caused by wild-type viral infection, decreases the proliferation of granulocytes to eliminate T-cell immunosuppression from granulocytes, and enhances the ability of the immune system against PRV infection. An overexpression of CD2v and p54 proteins does not alter the characteristics of PRV-∆TK/∆gE. Moreover, PRV-∆TK-(CD2v)-∆gE-(p54) successfully induces antibody production via intramuscular (IM) vaccination and confers effective protection for vaccinated mice upon challenge. Thus, PRV-∆TK-(CD2v)-∆gE-(p54) demonstrates good immunogenicity and safety, providing highly effective protection against PRV and ASFV. It potentially represents a suitable candidate for the development of a bivalent vaccine against both PRV and ASFV infections.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Herpesvirus Suídeo 1 , Pseudorraiva , Suínos , Animais , Camundongos , Herpesvirus Suídeo 1/genética , Vírus da Febre Suína Africana/genética , Febre Suína Africana/prevenção & controle , Granulócitos , Sus scrofaRESUMO
Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) possesses multiple biological activities such as virus restriction, innate immunity regulation, and autoimmunity. Our previous study demonstrated that SAMHD1 potently inhibits the replication of enterovirus 71 (EV71). In this study, we observed that SAMHD1 also restricts multiple enteroviruses (EVs), including coxsackievirus A16 (CA16) and enterovirus D68 (EVD68), but not coxsackievirus A6 (CA6). Mechanistically, SAMHD1 competitively interacted with the same domain in VP1 that binds to VP2 of EV71 and EVD68, thereby interfering with the interaction between VP1 and VP2 , and therefore viral assembly. Moreover, we showed that the SAMHD1 T592A mutant maintained the EV71 inhibitory effect by attenuating the interaction between VP1 and VP2, whereas the T592D mutant failed to. We also demonstrated that SAMHD1 could not inhibit CA6 because a different binding site is required for the SAMHD1 and VP1 interaction. Our findings reveal the mechanism of SAMHD1 inhibition of multiple EVs, and this could potentially be important for developing drugs against a broad range of EVs. IMPORTANCE Enterovirus causes a wide variety of diseases, such as hand, foot, and mouth disease (HFMD), which is a severe public problem threatening children under 5 years. Therefore, identifying essential genes which restrict EV infection and exploring the underlying mechanisms are necessary to develop an effective strategy to inhibit EV infection. In this study, we report that host restrictive factor SAMHD1 has broad-spectrum antiviral activity against EV71, CA16, and EVD68 independent of its well-known deoxynucleoside triphosphate triphosphohydrolase (dNTPase) or RNase activity. Mechanistically, SAMHD1 restricts EVs by competitively interacting with the same domain in VP1 that binds to VP2 of EVs, thereby interfering with the interaction between VP1 and VP2, and therefore viral assembly. In contrast, we also demonstrated that SAMHD1 could not inhibit CA6 because a different binding site is required for the SAMHD1 and CA6 VP1 interaction. Our study reveals a novel mechanism for the SAMHD1 anti-EV replication activity.
Assuntos
Proteínas do Capsídeo/metabolismo , Infecções por Enterovirus/prevenção & controle , Enterovirus/crescimento & desenvolvimento , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Montagem de Vírus/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Imunidade Inata/imunologia , Ligação Proteica , Proteína 1 com Domínio SAM e Domínio HD/genéticaRESUMO
The propagation distance confines the development of precise time-frequency transmission using optical frequency combs due to the dispersion of the link. Here we disseminate a fiber re-circulating loop to emulate 504-km comb-based transmission. An optical filter in combination with a spool of dispersion compensation fiber is utilized to restrict the dispersion effect. The residual instability reached 4.0 × 10-14 at 1 s and 7.32 × 10-18 at 10,000 s over the 504-km link. The result indicated that this approach could meet the demand for long-haul clock transmission and comparison in the giant fiber-optic gyroscope to detect the seismic and gravitational potential.
RESUMO
SAMHD1 possesses multiple functions, but whether cellular factors regulate SAMHD1 expression or its function remains not well characterized. Here, by investigating why cultured RD and HEK293T cells show different sensitivity to enterovirus 71 (EV71) infection, we demonstrate that SAMHD1 is a restriction factor for EV71. Importantly, we identify TRIM21, an E3 ubiquitin ligase, as a key regulator of SAMHD1, which specifically interacts and degrades SAMHD1 through the proteasomal pathway. However, TRIM21 has no effect on EV71 replication itself. Moreover, we prove that interferon production stimulated by EV71 infection induces increased TRIM21 and SAMHD1 expression, whereas increasing TRIM21 overrides SAMHD1 inhibition of EV71 in cells and in a neonatal mouse model. TRIM21-mediated degradation of SAMHD1 also affects SAMHD1-dependent restriction of HIV-1 and the regulation of interferon production. We further identify the functional domains in TRIM21 required for SAMHD1 binding and the ubiquitination site K622 in SAMHD1 and show that phosphorylation of SAMHD1 at T592 also blocks EV71 restriction. Our findings illuminate how EV71 overcomes SAMHD1 inhibition via the upregulation of TRIM21.
Assuntos
Antivirais , HIV-1 , Animais , Células HEK293 , Humanos , Camundongos , Proteína 1 com Domínio SAM e Domínio HD/genética , UbiquitinaçãoRESUMO
NF-κB-interacting long noncoding RNA (NKILA) was recently identified as a negative regulator of NF-κB signaling and plays an important role in the development of various cancers. It is well known that NF-κB-mediated activation of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-driven gene expression is required for HIV-1 transcription and reactivation of latency. However, whether NKILA plays essential roles in HIV-1 replication and latency is unclear. Here, by ectopic expression and silencing experiments, we demonstrate that NKILA potently inhibits HIV-1 replication in an NF-κB-dependent manner by suppressing HIV-1 LTR promoter activity. Moreover, NKILA showed broad-spectrum inhibition on the replication of HIV-1 clones with different coreceptor tropisms as well as on LTR activity of various HIV-1 clinical subtypes. Chromatin immunoprecipitation (ChIP) assays revealed that NKILA expression abolishes the recruitment of p65 to the duplicated κB binding sites in the HIV-1 LTR. NKILA mutants disrupting NF-κB inhibition also lost the ability to inhibit HIV-1 replication. Notably, HIV-1 infection or reactivation significantly downregulated NKILA expression in T cells in order to facilitate viral replication. Downregulated NKILA was mainly due to reduced acetylation of histone K27 on the promoter of NKILA by HIV-1 infection, which blocks NKILA expression. Knockdown of NKILA promoted the reactivation of latent HIV-1 upon phorbol myristate acetate (PMA) stimulation, while ectopic NKILA suppressed the reactivation in a well-established clinical model of withdrawal of azidothymidine (AZT) in vitro These findings improve our understanding of the functional suppression of HIV-1 replication and latency by NKILA through NF-κB signaling.IMPORTANCE The NF-κB pathway plays key roles in HIV-1 replication and reactivation of HIV-1 latency. A regulator inhibiting NF-κB activation may be a promising therapeutic strategy against HIV-1. Recently, NF-κB-interacting long noncoding RNA (NKILA) was identified to suppress the development of different human cancers by inhibiting IκB kinase (IKK)-induced IκB phosphorylation and NF-κB pathway activation, whereas the relationship between NKILA and HIV-1 replication is still unknown. Here, our results show that NKILA inhibits HIV-1 replication and reactivation by suppressing HIV-1 long terminal repeat (LTR)-driven transcription initiation. Moreover, NKILA inhibited the replication of HIV-1 clones with different coreceptor tropisms. This project may reveal a target for the development of novel anti-HIV drugs.
Assuntos
HIV-1/fisiologia , NF-kappa B/metabolismo , RNA Longo não Codificante/metabolismo , Latência Viral/fisiologia , Replicação Viral/fisiologia , Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/virologia , Imunoprecipitação da Cromatina , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/fisiologia , HIV-1/genética , Humanos , Fosforilação , RNA Longo não Codificante/genética , RNA Longo não Codificante/farmacologia , Transdução de Sinais/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Guanylate binding protein 5 (GBP5) belongs to the GTPase subfamily, which is mainly induced by interferon gamma (IFN-γ) and is involved in many important cellular processes, including inflammasome activation and innate immunity against a wide variety of microbial pathogens. However, it is unknown whether GBP5 inhibits respiratory syncytial virus (RSV) infection. In this study, we identified GBP5 as an effector of the anti-RSV activity of IFN-γ and found that in children, the weaker immune response, especially the weaker IFN-γ response and the decreased GBP5 expression, leads to RSV susceptibility. Furthermore, we revealed that GBP5 reduced the cell-associated levels of the RSV small hydrophobic (SH) protein, which was identified as a viroporin. In contrast, overexpression of the SH protein rescued RSV replication in the presence of GBP5. The GBP5-induced decrease in intracellular SH protein levels is because GBP5 promotes the release of the SH protein into the cell culture. Moreover, the GBP5 C583A mutants with changes at the C terminus or the GBP5 ΔC mutant lacking the C-terminal region, which impairs GBP5 localization in the Golgi, could not inhibit RSV infection, whereas the GTPase-defective GBP5 maintained RSV inhibition, suggesting that Golgi localization but not the GTPase activity of GBP5 is required for RSV inhibition. Interestingly, we found that RSV infection or RSV G protein downregulates GBP5 expression by upregulating DZIP3, an E3 ligase, which induces GBP5 degradation through the K48 ubiquitination and proteasomal pathways. Thus, this study reveals a complicated interplay between host restrictive factor GBP5 and RSV infection and provides important information for understanding the pathogenesis of RSV.IMPORTANCE RSV is a highly contagious virus that causes multiple infections in infants within their first year of life. It can also easily cause infection in elderly or immunocompromised individuals, suggesting that individual differences in immunity play an important role in RSV infection. Therefore, exploring the pathogenic mechanisms of RSV and identifying essential genes which inhibit RSV infection are necessary to develop an effective strategy to control RSV infection. Here, we report that the IFN-inducible gene GBP5 potently inhibits RSV replication by reducing the cell-associated levels of the RSV small hydrophobic (SH) protein, which is a viroporin. In contrast, the RSV G protein was shown to upregulate the expression of the DZIP3 protein, an E3 ligase that degrades GBP5 through the proteasomal pathway. Our study provides important information for the understanding of the pathogenic mechanisms of RSV and host immunity as well as the complicated interplay between the virus and host.
Assuntos
Proteínas de Ligação ao GTP/genética , Interações Hospedeiro-Patógeno/genética , Interferon gama/genética , Infecções por Vírus Respiratório Sincicial/genética , Vírus Sincicial Respiratório Humano/genética , Proteínas Oncogênicas de Retroviridae/genética , Adulto , Criança , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Proteínas de Ligação ao GTP/imunologia , Regulação da Expressão Gênica , Complexo de Golgi/imunologia , Complexo de Golgi/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Inflamassomos/genética , Inflamassomos/imunologia , Interferon gama/imunologia , Masculino , Mutação , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Oncogênicas de Retroviridae/imunologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
This paper reports on the generation of a 100 MHz repetition rate, 1.7 mW average power and femtosecond deep-ultraviolet (DUV) 243 nm laser source. The infra-red output of a broadband Titanium-Sapphire (TiSa) laser containing 729 nm light is mixed with its second harmonic in a ß-barium borate (BBO) crystal. By manipulating the group delay dispersion (GDD), we customize the spectral shape of TiSa resonator to improve conversion efficiency. This DUV laser is employed for direct frequency comb spectroscopy of hydrogen.
RESUMO
The 4D-printing technology is applied to fabricate a shape memory peripheral stent with good biocompatibility, which sustains long-term drug release. The star polymer s-PCL is prepared by ring opening polymerization of ε-caprolactone with the -OH of ß-cyclodextrin (ßCD) as initiator, and then the s-PCL is modified with acrylate endgroup which allows the polymerization under UV light to form the crosslinking network c-PCL. Attributed to the feature of the high crosslinked structure and chemical nature of polycaprolactone (PCL) and ßCD, the composite exhibits appropriate tensile strength and sufficient elasticity and bursting pressure, and it is comparable with great saphenous vein in human body. The radial support of the 4D-printed stent is 0.56 ± 0.11 N and is equivalent to that of commercial stent. The cell adhesion and proliferation results show a good biocompatibility of the stent with human umbilical vein endothelial cells. Due to the presence of ßCD, the wettability and biocompatibility of the materials are improved, and the sustained paclitaxel release based on the host-guest complexion shows the potential of the drug-loaded stent for long-term release. This study provides a new strategy to solve the urgent need of small-diameter scaffolds to treat critical limb ischemia.
Assuntos
Células Endoteliais , beta-Ciclodextrinas , Materiais Biocompatíveis , Humanos , Poliésteres , Impressão Tridimensional , StentsRESUMO
Increasing evidence indicates ATP1B3, one of the regulatory subunits of Na+ /K+ -ATPase, is involved in numerous viral propagations, such as HIV and EV71. However, the function and mechanism of ATP1B3 on hepatitis B virus (HBV) propagation is unknown. Here, we demonstrated that ATP1B3 overexpression reduced the quantity of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in supernatants of HBV expression plasmids cotransfected HepG2 cells. Correspondingly, small interfering RNA and short hairpin RNA mediated ATP1B3 silencing promoted HBsAg and HBeAg expression in the supernatants of HBV expression plasmids transfected HepG2 cells. Mechanically, we reported that ATP1B3 expression could activate nuclear factor-κB (NF-κB) pathway by inducing the expression, phosphorylation, and nuclear import of P65 for the first time. And NF-κB inhibitor (Bay11) impaired the restraint of ATP1B3 on HBV replication. This counteraction effect of Bay11 proved that ATP1B3-induced NF-κB activation was crucial for HBV restriction. Accordingly, we observed that anti-HBV factors interferon-α (IFN-α) and interleukin-6 (IL-6) production were increased in HepG2 cells after the NF-κB activation. It suggested that ATP1B3 suppressed HBsAg and HBeAg by NF-κB/IFN-α and NF-κB/IL-6 axis. Further experiments proved that ATP1B3 overexpression induced anti-HBV factor BST-2 expression by NF-κB/IFN-α axis in HepG2 cells but not HEK293T cells, and ATP1B3 silencing downregulated BST-2 messenger RNA level in HepG2 cells. As an HBV restriction factor, BST-2 cooperated with ATP1B3 to antagonize HBsAg but not HBeAg in HepG2 cells. Our work identified ATP1B3 as a novel candidate of HBV restrictor with unrevealed mechanism and we highlighted it might serve as a potential therapeutic molecule for HBV infection.
Assuntos
Antígenos CD/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD/genética , Sobrevivência Celular , Citocinas/genética , Citocinas/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Células HEK293 , Células Hep G2 , Antígenos de Superfície da Hepatite B/genética , Antígenos E da Hepatite B/genética , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Ligação a RNA , ATPase Trocadora de Sódio-Potássio/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Replicação ViralRESUMO
BACKGROUND: African swine fever (ASF) leads to high mortality in domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). Currently, no vaccine is commercially available for prevention, and the epidemic is still spreading. Here, we constructed a recombinant pseudorabies virus (PRV) (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) that expresses the CD2v protein of ASFV and evaluated its effectiveness and safety as a vaccine candidate in mice. METHODS: A homologous recombination fragment containing ASFV CD2v was synthesized and co-transfected into HEK 293 T cells, a knockout vector targeting the PRV TK gene. The transfected cells were infected with PRV-ΔgE/ΔgI, and the recombinant strain (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) was obtained by plaque purification in Vero cells. The expression of ASFV CD2v in the recombinant virus was confirmed by sequencing, Western blotting, and immunofluorescence analysis, and the genetic stability was tested in Vero cells over 20 passages. The virulence, immunogenicity and protective ability of the recombinant virus were further tested in a mouse model. RESULTS: The PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain is stable in Vero cells, and the processing of CD2v does not depend on ASFV infection. The vaccination of PRV-ΔgE/ΔgI/ΔTK-(CD2v) causes neither pruritus, not a systemic infection and inflammation (with the high expression of interleukin-6 (IL6)). Besides, the virus vaccination can produce anti-CD2v specific antibody and activate a specific cellular immune response, and 100% protect mice from the challenge of the virulent strain (PRV-Fa). The detoxification occurs much earlier upon the recombinant virus vaccination and the amount of detoxification is much lower as well. CONCLUSIONS: The PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain has strong immunogenicity, is safe and effective, and maybe a potential vaccine candidate for the prevention of ASF and Pseudorabies.
Assuntos
Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/prevenção & controle , Herpesvirus Suídeo 1/genética , Proteínas Virais/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Febre Suína Africana/imunologia , Animais , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Citocinas/genética , Citocinas/imunologia , Células HEK293 , Humanos , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos ICR , Organismos Livres de Patógenos Específicos , Suínos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Células Vero , Proteínas Virais/administração & dosagem , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagemRESUMO
Although the host restriction factor APOBEC3G (A3G) has broad spectrum antiviral activity, whether A3G inhibits enterovirus 71 (EV71) has been unclear until now. In this study, we demonstrated for the first time that A3G could inhibit EV71 virus replication. Silencing A3G in H9 cells enhanced EV71 replication, and EV71 replication was lower in H9 cells expressing A3G than in Jurkat cells without A3G expression, indicating that the EV71 inhibition was A3G-specific. Further investigation revealed that A3G inhibited the 5'UTR activity of EV71 by competitively binding to the 5'UTR through its nucleic acid binding activity. This binding impaired the interaction between the 5'UTR and the host protein poly(C)-binding protein 1 (PCBP1), which is required for the synthesis of EV71 viral proteins and RNA. On the other hand, we found that EV71 overcame A3G suppression through its non-structural protein 2C, which induced A3G degradation through the autophagy-lysosome pathway. Our research provides new insights into the interplay mechanisms of A3G and single-stranded positive RNA viruses.
Assuntos
Desaminase APOBEC-3G/metabolismo , Enterovirus Humano A/fisiologia , Enterovirus Humano A/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Regiões 5' não Traduzidas , Desaminase APOBEC-3G/genética , Ligação Competitiva , Linhagem Celular , Células HEK293 , Humanos , Células Jurkat , Poli C/metabolismo , Proteólise , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4+ T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions.IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1.