RESUMEN
Nucleoprotein (N) is well known for its function in the encapsidation of the genomic RNAs of negative-strand RNA viruses, which leads to the formation of ribonucleoproteins that serve as templates for viral transcription and replication. However, the function of the N protein in other aspects during viral infection is far from clear. In this study, the N protein of snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was proved to be ubiquitinated mainly via K63-linked ubiquitination. We identified nine host E3 ubiquitin ligases that interacted with SHVV N, among which seven E3 ubiquitin ligases facilitated ubiquitination of the N protein. Further investigation revealed that only two E3 ubiquitin ligases, Siah E3 ubiquitin protein ligase 2 (Siah2) and leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1), mediated K63-linked ubiquitination of the N protein. SHVV infection upregulated the expression of Siah2 and LRSAM1, which maintained the stability of SHVV N. Besides, overexpression of Siah2 or LRSAM1 promoted SHVV replication, while knockdown of Siah2 or LRSAM1 inhibited SHVV replication. Deletion of the ligase domain of Siah2 or LRSAM1 did not affect their interactions with SHVV N but reduced the K63-linked ubiquitination of SHVV N and SHVV replication. In summary, Siah2 and LRSAM1 mediate K63-linked ubiquitination of SHVV N to facilitate SHVV replication, which provides novel insights into the role of the N proteins of negative-strand RNA viruses. IMPORTANCE: Ubiquitination of viral protein plays an important role in viral replication. However, the ubiquitination of the nucleoprotein (N) of negative-strand RNA viruses has rarely been investigated. This study aimed at investigating the ubiquitination of the N protein of a fish rhabdovirus SHVV (snakehead vesiculovirus), identifying the related host E3 ubiquitin ligases, and determining the role of SHVV N ubiquitination and host E3 ubiquitin ligases in viral replication. We found that SHVV N was ubiquitinated mainly via K63-linked ubiquitination, which was mediated by host E3 ubiquitin ligases Siah2 (Siah E3 ubiquitin protein ligase 2) and LRSAM1 (leucine-rich repeat and sterile alpha motif containing 1). The data suggested that Siah2 and LRSAM1 were hijacked by SHVV to ubiquitinate the N protein for viral replication, which exhibited novel anti-SHVV targets for drug design.
Asunto(s)
Nucleoproteínas , Ubiquitina-Proteína Ligasas , Ubiquitinación , Vesiculovirus , Replicación Viral , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Animales , Nucleoproteínas/metabolismo , Nucleoproteínas/genética , Vesiculovirus/fisiología , Vesiculovirus/metabolismo , Vesiculovirus/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Células HEK293 , Proteínas Virales/metabolismo , Proteínas Virales/genética , Línea Celular , Infecciones por Rhabdoviridae/virología , Infecciones por Rhabdoviridae/metabolismo , Enfermedades de los Peces/virología , Enfermedades de los Peces/metabolismoRESUMEN
Phosphoprotein (P), co-factor of the polymerase (large protein, L) of single-stranded negative-sense RNA viruses, is phosphorylated during viral infection and its phosphorylation has been reported to play important roles in viral replication. However, the function of P phosphorylation in viral replication is still far from clear. Snakehead vesiculovirus (SHVV) is a kind of fish rhabdovirus that has caused serious economic losses in snakehead fish culture in China without any effective preventive or therapeutical measures currently. In this study, 4D label-free phosphoproteomics sequencing of SHVV-infected cells identified five phosphorylated sites on SHVV P, among which threonine 160 (T160) was proved to be phosphorylated. Overexpression of wild-type P, but not P-T160A or P-T160E mutant, promoted SHVV replication, suggesting that the T160 phosphorylation on the P protein is critical for SHVV replication. Moreover, we found that T160A or T160E mutation on SHVV P had no effect on the interactions of P-nucleoprotein (N), P-P, or P-L. Further study revealed that p38 mitogen-activated protein kinase (p38MAPK) and glycogen synthase kinase 3 (GSK3) interacted with SHVV P and mediated the T160 phosphorylation. Besides, overexpression of p38MAPK or GSK3 facilitated, while knockdown or activity inhibition of p38MAPK or GSK3 suppressed, SHVV replication. Overall, p38MAPK- and GSK3-mediated phosphorylation of the P protein at T160 is required for SHVV replication, which provided targets for designing anti-SHVV drugs and developing live-attenuated SHVV vaccines. Our study helps understand the role of P phosphorylation in the replication of single-stranded negative-sense RNA viruses. IMPORTANCE Phosphorylation of viral proteins plays important roles in viral replication. Currently, the role of phosphorylation of phosphoprotein (P) in the replication of single-stranded negative-sense RNA viruses is far from clear. Identification of the phosphorylated sites on viral P protein and the related host kinases is helpful for developing live-attenuated vaccines and designing antiviral drugs. This study focused on identifying the phosphorylated sites on P protein of a fish rhabdovirus SHVV, determining the related host kinases, and revealing the effects of the phosphorylated sites and kinases on SHVV replication. We found that SHVV P was phosphorylated at T160, which was mediated by the kinases p38MAPK and GSK3 to promote SHVV replication. This study is the first time to study the role of P phosphorylation in fish rhabdovirus replication.
Asunto(s)
Glucógeno Sintasa Quinasa 3 , Infecciones por Rhabdoviridae , Animales , Fosforilación , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Peces , Vesiculovirus/genética , Proteínas Virales/genética , Replicación Viral , Fosfoproteínas/genéticaRESUMEN
Asp-Glu-Ala-Asp (DEAD) box helicase 3 X-linked (DDX3X) plays important regulatory roles in the replication of many viruses. However, the role of DDX3X in rhabdovirus replication has seldomly been investigated. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was used to study the role of DDX3X in rhabdovirus replication. DDX3X was identified as an interacting partner of SHVV phosphoprotein (P). The expression level of DDX3X was increased at an early stage of SHVV infection and then decreased to a normal level at a later infection stage. Overexpression of DDX3X promoted, while knockdown of DDX3X using specific small interfering RNAs (siRNAs) suppressed, SHVV replication, indicating that DDX3X was a proviral factor for SHVV replication. The N-terminal and core domains of DDX3X (DDX3X-N and DDX3X-Core) were determined to be the regions responsible for its interaction with SHVV P. Overexpression of DDX3X-Core suppressed SHVV replication by competitively disrupting the interaction between full-length DDX3X and SHVV P, suggesting that full-length DDX3X-P interaction was required for SHVV replication. Mechanistically, DDX3X-mediated promotion of SHVV replication was due not to inhibition of interferon expression but to maintenance of the stability of SHVV P to avoid autophagy-lysosome-dependent degradation. Collectively, our data suggest that DDX3X is hijacked by SHVV P to ensure effective replication of SHVV, which suggests an important anti-SHVV target. This study will help elucidate the role of DDX3X in regulating the replication of rhabdoviruses. IMPORTANCE Growing evidence has suggested that DDX3X plays important roles in virus replication. In one respect, DDX3X inhibits the replication of viruses, including hepatitis B virus, influenza A virus, Newcastle disease virus, duck Tembusu virus, and red-spotted grouper nervous necrosis virus. In another respect, DDX3X is required for the replication of viruses, including hepatitis C virus, Japanese encephalitis virus, West Nile virus, murine norovirus, herpes simplex virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because DDX3X has rarely been investigated in rhabdovirus replication, this study aimed at investigating the role of DDX3X in rhabdovirus replication by using the fish rhabdovirus SHVV as a model. We found that DDX3X was required for SHVV replication, with the mechanism that DDX3X interacts with and maintains the stability of SHVV phosphoprotein. Our data provide novel insights into the role of DDX3X in virus replication and will facilitate the design of antiviral drugs against rhabdovirus infection.
Asunto(s)
ARN Helicasas DEAD-box , Perciformes , Fosfoproteínas , Vesiculovirus , Replicación Viral , Animales , ARN Helicasas DEAD-box/genética , Peces , Perciformes/virología , ARN Interferente Pequeño , Vesiculovirus/patogenicidad , Vesiculovirus/fisiología , Proteínas ViralesRESUMEN
Cyprinid herpesvirus 3 (CyHV-3) has caused severe economic losses to carp culture, but its pathogenicity is far from clear. Our previous study has revealed that microRNA (miR)-722 was upregulated during CyHV-3 infection, indicating that miR-722 might play an important role in CyHV-3 replication. In this study, we found that overexpression of miR-722 inhibited CyHV-3 replication and promoted IFN expression. The putative target gene of miR-722 was searched over the CyHV-3 genome, and ORF89 was identified and validated as a target gene of miR-722. Overexpression of ORF89 markedly reduced the expression of IFN and IFN-stimulated genes. Mechanistically, ORF89 interacted with and degraded IFN regulatory factor 3 (IRF3), and inhibited the entry of IRF3 into the nucleus by suppressing the dimerization of IRF3. Moreover, ORF89-mediated suppression of IFN expression could be restored by adding miR-722. To our knowledge, our findings confirm a novel virus-host combat, in which CyHV-3 evades host antiviral immunity by its ORF89 protein, whereas host miR-722, upregulated on CyHV-3 infection, targets ORF89 to impede CyHV-3 replication.
Asunto(s)
Evasión Inmune , MicroARNs , Factor 3 Regulador del Interferón/genética , Proteínas Virales/genética , MicroARNs/genéticaRESUMEN
The frequent outbreak of grass carp hemorrhagic disease caused by grass carp reovirus (GCRV), especially the mainly prevalent type II GCRV (GCRV-II), has seriously affected the grass carp culture in China. However, its pathogenic mechanism is still far from clear. In this study, the GCRV-II outer capsid protein VP35 was used as bait to capture interacting partners from Ctenopharyngon idellus kidney (CIK) cells, and heat shock protein 90 (Hsp90) was selected and confirmed interacting with VP35 through the C-terminal domain of Hsp90. Knockdown of Hsp90 or inhibition of Hsp90 activity suppressed GCRV-II proliferation, demonstrating that Hsp90 is an essential factor for GCRV-II proliferation. The confocal microscopy and flow cytometry showed that Hsp90 localized at both membrane and cytoplasm of CIK cells. The entry of GCRV-II into CIK cells was efficiently blocked by incubating the cells with Hsp90 antibody or by pretreating the virus with recombinant Hsp90 protein. Whereas overexpression of Hsp90 in CIK cells, grass carp ovary (GCO) cells, or 293T cells promoted GCRV-II entry, indicating that the membrane Hsp90 functions as a receptor of GCRV-II. Furthermore, Hsp90 interacted with clathrin and mediated GCRV-II entry into CIK cells through clathrin endocytosis pathway. In addition, we found that the cytoplasmic Hsp90 acted as a chaperone of VP35 because inhibition of Hsp90 activity enhanced VP35 polyubiquitination and degraded VP35 through the proteasome pathway. Collectively, our data suggest that Hsp90 functions both as a receptor for GCRV-II entry and a chaperone for the maturation of GCRV-II VP35, thus ensuring efficient proliferation of GCRV-II. IMPORTANCE Identification of viral receptors has always been the research hot spot in virus research field as receptor functions at the first stage of viral infection, which can be designed as efficient antiviral drug targets. GCRV-II, the causative agent of the grass carp epidemic hemorrhagic disease, has caused tremendous losses in grass carp culture in China. To date, the receptor of GCRV-II remains unknown. This study focused on identifying cellular receptor interacting with the GCRV-II outer capsid protein VP35, studying the effects of their interaction on GCRV-II proliferation, and revealing the underlying mechanisms. We demonstrated that Hsp90 acts both as a receptor of GCRV-II by interacting with VP35 and as a chaperone for the maturation of VP35, thus ensuring efficient proliferation of GCRV-II. Our data provide important insights into the role of Hsp90 in GCRV-II life cycle, which will help understand the mechanism of reovirus infection.
Asunto(s)
Proteínas de la Cápside , Enfermedades de los Peces , Proteínas de Choque Térmico , Infecciones por Reoviridae , Reoviridae , Animales , Anticuerpos Antivirales/metabolismo , Proteínas de la Cápside/metabolismo , Carpas/virología , Proliferación Celular , Clatrina/metabolismo , Enfermedades de los Peces/virología , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores Virales/metabolismo , Reoviridae/fisiología , Infecciones por Reoviridae/veterinariaRESUMEN
Mitochondrial antiviral signaling protein (MAVS) is as an adaptor in RIG-I like receptor (RLR) signaling, which plays the key role in interferon (IFN) production during host antiviral innate immune activation. MAVS is fine tuned to avoid excess IFN production, which have been extensively studied in human and mammals. However, the regulation of MAVS in teleost still remains obscure. In this manuscript, we cloned ring finger protein 5 (bcRNF5) of black carp (Mylopharyngodon piceus) and characterized this teleost E3 ubiquitin ligase as a negative regulator of MAVS. The coding region of bcRNF5 consists of 615 nucleotides which encode 205 amino acids, containing two trans-membrane domain (TM) and a ring-finger domain (RING). The transcription regulation of bcRNF5 varies in host cells in response to stimulations of LPS, poly (I:C), grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). bcRNF5 migrates around 22 KDa in immunoblot (IB) assay and distributes mainly in cytoplasm by immunofluorescent (IF) staining test. Moreover, bcRNF5 significantly inhibits bcMAVS-mediated IFN promoter transcription. In addition, both IF and co-immunoprecipitation assay showed that bcRNF5 interacts with bcMAVS. Furthermore, bcMAVS-mediated antiviral ability is distinctly impaired by bcRNF5. Taken together, these results conclude that bcRNF5, as a negative regulator of the MAVS-mediated IFN signaling, may play a key role in host protection upon virus infection in black carp.
Asunto(s)
Carpas , Enfermedades de los Peces , Reoviridae , Animales , Humanos , Carpas/metabolismo , Reoviridae/fisiología , Inmunidad Innata , Ubiquitina-Proteína Ligasas , Antivirales , Proteínas de Peces , Mamíferos/metabolismo , Proteínas de Unión al ADNRESUMEN
Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction caused by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction, and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensures the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help us to understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. IMPORTANCE It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help us to understand the polymerase function of negative-stranded RNA viruses.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Vesiculovirus/fisiología , Proteínas Virales/metabolismo , Replicación Viral , Animales , Células Cultivadas , Peces , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Estabilidad Proteica , ARN Viral/biosíntesis , Infecciones por Rhabdoviridae/veterinaria , Infecciones por Rhabdoviridae/virología , Vesiculovirus/metabolismoRESUMEN
Spring viremia of carp virus (SVCV) is a highly pathogenic Vesiculovirus in the common carp. The phosphoprotein (P protein) of SVCV is a multifunctional protein that acts as a polymerase cofactor and an antagonist of cellular interferon (IFN) response. Here, we report the 1.5-Å-resolution crystal structure of the P protein central domain (PCD) of SVCV (SVCVPCD). The PCD monomer consists of two ß sheets, an α helix, and another two ß sheets. Two PCD monomers pack together through their hydrophobic surfaces to form a dimer. The mutations of residues on the hydrophobic surfaces of PCD disrupt the dimer formation to different degrees and affect the expression of host IFN consistently. Therefore, the oligomeric state formation of the P protein of SVCV is an important mechanism to negatively regulate host IFN response.IMPORTANCE SVCV can cause spring viremia of carp with up to 90% lethality, and it is the homologous virus of the notorious vesicular stomatitis virus (VSV). There are currently no drugs that effectively cure this disease. P proteins of negative-strand RNA viruses (NSVs) play an essential role in many steps during the replication cycle and an additional role in immunosuppression as a cofactor. All P proteins of NSVs are oligomeric, but the studies on the role of this oligomerization mainly focus on the process of virus transcription or replication, and there are few studies on the role of PCD in immunosuppression. Here, we present the crystal structure of SVCVPCD A new mechanism of immune evasion is clarified by exploring the relationship between SVCVPCD and host IFN response from a structural biology point of view. These findings may provide more accurate target sites for drug design against SVCV and provide new insights into the function of NSVPCD.
Asunto(s)
Fosfoproteínas/química , Rhabdoviridae/química , Proteínas Virales/química , Animales , Cristalografía por Rayos X , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina betaRESUMEN
Periplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we deployed cryo-electron tomography (cryo-ET) to visualize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We show, for the first time, that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the "spoke and hub" structure in B. burgdorferi. This structure not only strengthens structural rigidity of the round-shaped C-ring but also appears to rotate with the C-ring. Our studies provide structural insights into the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the basis for the development of novel therapeutic strategies against several pathogenic spirochetes.
Asunto(s)
Adenosina Trifosfatasas/ultraestructura , Borrelia burgdorferi/fisiología , Flagelos/fisiología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/química , Borrelia burgdorferi/metabolismo , Citoplasma , Tomografía con Microscopio Electrónico/métodos , Flagelos/metabolismo , Flagelos/ultraestructura , Periplasma/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Sistemas de Secreción Tipo III/ultraestructuraRESUMEN
The outer membrane protein U (OmpU) is a conserved outer membrane protein in a variety of pathogenic Vibrio species and has been considered as a vital protective antigen for vaccine development. Vibrio mimicus (V. mimicus) is the pathogen causing ascites disease in aquatic animals. In this study, the prokaryotically expressed and purified His-tagged OmpU of V. mimicus (His-OmpU) was used as a subunit vaccine. The formalin inactivated V. mimicus, purified His tag (His-tag), and PBS were used as controls. The vaccinated yellow catfish were challenged with V. mimicus at 28 days post-vaccination, and the results showed that the His-OmpU and inactivated V. mimicus groups exhibited much higher survival rates than the His-tag and PBS groups. To fully understand the underlying mechanism, we detected the expression levels of several immune-related genes in the spleen of fish at 28 days post-vaccination and 24 h post-challenge. The results showed that most of the detected immune-related genes were significantly upregulated in His-OmpU and inactivated V. mimicus groups. In addition, we performed the serum bactericidal activity assay, and the results showed that the serum from His-OmpU and inactivated V. mimicus groups exhibited much stronger bactericidal activity against V. mimicus than those of His-tag and PBS groups. Finally, the serum agglutination antibody was detected, and the antibody could be detected in His-OmpU and inactivated V. mimicus groups with the antibody titers increasing along with the time post-vaccination, but not in His-tag or PBS group. Our data reveal that the recombinant OmpU elicits potent protective immune response and is an effective vaccine candidate against V. mimicus in yellow catfish.
Asunto(s)
Adhesinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Enfermedades de los Peces/inmunología , Inmunogenicidad Vacunal , Vibriosis/veterinaria , Vibrio mimicus/inmunología , Animales , Bagres , Vacunas de Subunidad/inmunología , Vibriosis/inmunologíaRESUMEN
Leader RNA, a kind of virus-derived small noncoding RNA, has been proposed to play an important role in regulating virus replication, but the underlying mechanism remains elusive. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus causing high mortality to the cultured snakehead fish in China, was used to unveil the molecular function of leader RNA. High-throughput small RNA sequencing of SHVV-infected cells showed that SHVV produced two groups of leader RNAs (named legroup1 and legroup2) during infection. Overexpression and knockout experiments reveal that legroup1, but not legroup2, affects SHVV replication. Mechanistically, legroup1-mediated regulation of SHVV replication was associated with its interaction with the viral nucleoprotein (N). Moreover, the nucleotides 6-10 of legroup1 were identified as the critical region for its interaction with the N protein, and the amino acids 1-45 of N protein were proved to confer its interaction with the legroup1. Taken together, we identified two groups of SHVV leader RNAs and revealed a role in virus replication for one of the two types of leader RNAs. This study will help understand the role of leader RNA in regulating the replication of negative-stranded RNA viruses.
Asunto(s)
Regiones no Traducidas 5'/fisiología , Vesiculovirus/fisiología , Replicación Viral/genética , Animales , Células Cultivadas , Mapeo Cromosómico , Femenino , Peces/virología , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Novirhabdovirus/fisiología , Proteínas de la Nucleocápside/genética , ARN Pequeño no Traducido/fisiología , ARN Viral/genética , ARN Viral/fisiología , Análisis de Secuencia de ARN , Vesiculovirus/genéticaRESUMEN
BACKGROUND: In recent years, interest in Bacillus velezensis has increased significantly due to its role in many industrial water bioremediation processes. In this study, we isolated and assessed the transcriptome of Bacillus velezensis LG37 (from an aquaculture pond) under different nitrogen sources. Since Bacillus species exhibit heterogeneity, it is worth investigating the molecular mechanism of LG37 through ammonia nitrogen assimilation, where nitrogen in the form of molecular ammonia is considered toxic to aquatic organisms. RESULTS: Here, a total of 812 differentially expressed genes (DEGs) from the transcriptomic sequencing of LG37 grown in minimal medium supplemented with ammonia (treatment) or glutamine (control) were obtained, from which 56 had Fold Change ≥2. BLAST-NCBI and UniProt databases revealed 27 out of the 56 DEGs were potentially involved in NH4+ assimilation. Among them, 8 DEGs together with the two-component regulatory system GlnK/GlnL were randomly selected for validation by quantitative real-time RT-PCR, and the results showed that expression of all the 8 DEGs are consistent with the RNA-seq data. Moreover, the transcriptome and relative expression analysis were consistent with the transporter gene amtB and it is not involved in ammonia transport, even in the highest ammonia concentrations. Besides, CRISPR-Cas9 knockout and overexpression glnK mutants further evidenced the exclusion of amtB regulation, suggesting the involvement of alternative transporter. Additionally, in the transcriptomic data, a novel ammonium transporter mnrA was expressed significantly in increased ammonia concentrations. Subsequently, OEmnrA and ΔmnrA LG37 strains showed unique expression pattern of specific genes compared to that of wild-LG37 strain. CONCLUSION: Based on the transcriptome data, regulation of nitrogen related genes was determined in the newly isolated LG37 strain to analyse the key regulating factors during ammonia assimilation. Using genomics tools, the novel MnrA transporter of LG37 became apparent in ammonia transport instead of AmtB, which transports ammonium nitrogen in other Bacillus strains. Collectively, this study defines heterogeneity of B. velezensis LG37 through comprehensive transcriptome analysis and subsequently, by genome editing techniques, sheds light on the enigmatic mechanisms controlling the functional genes under different nitrogen sources also reveals the need for further research.
Asunto(s)
Amoníaco/metabolismo , Bacillus/genética , Bacillus/metabolismo , Proteínas Bacterianas/fisiología , Nitrógeno/metabolismo , Bacillus/crecimiento & desarrollo , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , TranscriptomaRESUMEN
Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.
Asunto(s)
Orthomyxoviridae/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/ultraestructura , Cristalografía por Rayos X , Virus de la Influenza A/química , Proteínas de la Membrana/metabolismo , Membranas/metabolismo , Orthomyxoviridae/fisiología , Polimerizacion , Proteínas Virales/metabolismo , Virión/metabolismo , Liberación del Virus/fisiologíaRESUMEN
MicroRNAs (miRNAs) are small noncoding RNAs that have been reported to play important roles in virus replication. Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead and has caused heavy economical losses in cultured snakehead fish in China. Our previous study has revealed that miR-214 inhibited SHVV replication, but the underline mechanism was not completely understood. In this study, glycogen synthase (GS) gene was identified as a target gene of miR-214. Overexpression of miR-214 reduced cellular GS gene expression. Knockdown of GS by siRNA, similar to the overexpression of miR-214, inhibited SHVV replication. Moreover, we found that siGS-mediated inhibition of SHVV replication could be restored by reducing cellular miR-214 level via using miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting GS. This study provided information for understanding the molecular mechanism of SHVV pathogenicity and a potential antiviral strategy against SHVV infection.
Asunto(s)
Enfermedades de los Peces/fisiopatología , Proteínas de Peces/genética , Glucógeno Sintasa/genética , MicroARNs/genética , Perciformes , ARN Viral/genética , Infecciones por Rhabdoviridae/veterinaria , Animales , Enfermedades de los Peces/virología , Proteínas de Peces/metabolismo , Glucógeno Sintasa/metabolismo , MicroARNs/metabolismo , ARN Viral/metabolismo , Infecciones por Rhabdoviridae/fisiopatología , Infecciones por Rhabdoviridae/virología , Vesiculovirus/genética , Vesiculovirus/fisiologíaRESUMEN
Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1â¯cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1â¯cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1â¯cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1â¯cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.
Asunto(s)
Autofagia , Glutamina/metabolismo , Disulfuro de Glutatión/metabolismo , Perciformes/virología , Vesiculovirus/fisiología , Animales , Butionina Sulfoximina , Línea Celular , Glutatión , Perciformes/fisiología , Infecciones por Rhabdoviridae/metabolismo , Infecciones por Rhabdoviridae/veterinaria , Replicación ViralRESUMEN
Cyclophilin A (CypA) is a ubiquitously expressed cellular protein and involves in diverse pathological conditions, including infection and inflammation. CypA acts as a key factor in the replication of several viruses. However, little is known about the role of CypA in the replication of the red-spotted grouper nervous necrosis virus (RGNNV). In the present report, grouper CypA (GF-CypA) was cloned from the grouper fin cell line (GF-1) derived from orange-spotted grouper (Epinephelus coioides). Sequence analysis found that GF-CypA open reading frame (ORF) of 495 bp encodes a polypeptide of 164 amino acids residues with a molecular weight of 17.4â¯kDa. The deduced amino acid sequence shared highly conserved regions with CypA of other animal species, showing that GF-CypA is a new member of Cyclophilin A family. We observed that GF-CypA was up-regulated in the GF-1â¯cells infected with RGNNV. Additionally, overexpression of CypA could significantly inhibit the replication of RGNNV in GF-1â¯cells. By contrast, when the GF-CypA was knock-downed by siRNA in GF-1â¯cells, the replication of RGNNV was enhanced. Furthermore, the expressions of pro-inflammatory factors, such as TNF-2, TNF-α, IL-1b, and ISG-15, were increased in GF-CypA transfected GF-1â¯cells challenged with RGNNV, indicating that GF-CypA might be involved in the regulation of the host pro-inflammatory factors. Altogether, we conclude that GF-CypA plays a vital role in the inhibitory effect of RGNNV replication that might be modulating the cytokines secretion in GF-1â¯cells during RGNNV infection. These results will shed new light on the function of CypA in the replication of RGNNV and will pave a new way for the prevention of the infection of RGNNV in fish.
Asunto(s)
Lubina/genética , Lubina/inmunología , Ciclofilina A/genética , Ciclofilina A/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ciclofilina A/química , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Nodaviridae/fisiología , Filogenia , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/veterinaria , Alineación de Secuencia/veterinaria , Replicación ViralRESUMEN
Recent advances in cryo-electron tomography (cryo-ET) have allowed direct visualization of the initial interactions between bacteriophages and their hosts. Previous studies focused on phage infection in Gram-negative bacteria but it is of particular interest how phages penetrate the thick, highly cross-linked Gram-positive cell wall. Here we detail structural intermediates of phage Φ29 during infection of Bacillus subtilis. Use of a minicell-producing strain facilitated in situ tomographic reconstructions of infecting phage particles. Φ29 initially contacts the cell wall at an angle through a subset of the twelve appendages, which are attached to the collar at the head proximal portion of the tail knob. The appendages are flexible and switch between extended and downward conformations during this stage of reversible adsorption; appendages enzymatically hydrolyze wall teichoic acids to bring the phage closer to the cell. A cell wall-degrading enzyme at the distal tip of the tail knob locally digests peptidoglycan, facilitating penetration of the tail further into the cell wall, and the phage particle reorients so that the tail becomes perpendicular to the cell surface. All twelve appendages attain the same "down" conformation during this stage of adsorption. Once the tail has become totally embedded in the cell wall, the tip can fuse with the cytoplasmic membrane. The membrane bulges out, presumably to facilitate genome ejection into the cytoplasm, and the deformation remains after complete ejection. This study provides the first visualization of the structural changes occurring in a phage particle during adsorption and genome transfer into a Gram-positive bacterium.
Asunto(s)
Bacillus subtilis/ultraestructura , Bacteriófagos/ultraestructura , Microscopía por Crioelectrón/métodos , Bacillus subtilis/virología , Bacteriófagos/patogenicidad , Tomografía con Microscopio Electrónico/métodos , Análisis Multivariante , Peptidoglicano/ultraestructuraRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in the regulation of diverse biological processes in eukaryotes. Chinese perch (Siniperca chuatsi) is one of the most economically important fish species widely cultured in China. Growth is an extremely important characteristic in fish. Individual differences in body size are common in Siniperca chuatsi, which significantly influence the aquaculture production of Siniperca chuatsi. However, the underline growth-related regulatory factors, such as miRNAs, are still unknown. RESULTS: To investigate the growth-related miRNAs in Siniperca chuatsi, two RNA libraries from four growth-related tissues (brain, pituitary, liver, and muscle) of Siniperca chuatsi at 6-month stage with relatively high or low growth rates (big-size group or small-size group) were obtained and sequenced using Solexa sequencing. A total of 252 known miRNAs and 12 novel miRNAs were identified. The expression patterns of these miRNAs in big-size group and small-size group were compared, and the results showed that 31 known and 5 novel miRNAs were differently expressed (DE). Furthermore, to verify the Solexa sequencing, five DE miRNAs were randomly selected and quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that their expression patterns were consistent with those of Solexa sequencing. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of target genes of DE miRNAs was performed. It showed that the target genes were involved in multiple biological processes including metabolic process, suggesting that metabolic process played an important role in growth of fish. CONCLUSIONS: Siniperca chuatsi is a popular and economically important species in aquaculture. In this study, miRNAs in Siniperca chuatsi with different growth rates were identified, and their expression profiles were compared. The data provides the first large-scale miRNA profiles related to growth of Siniperca chuatsi, which is expected to contribute to a better understanding of the role of miRNAs in regulating the biological processes of growth and possibly useful for Siniperca chuatsi breeding.
Asunto(s)
MicroARNs/genética , Percas/crecimiento & desarrollo , Percas/genética , Análisis de Secuencia de ARN , Animales , Especificidad de ÓrganosRESUMEN
Cyclophilin A (CypA) is a key member of immunophilins that has peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a cellular receptor for immunosuppressive drug cyclosporine A (CsA), CypA is involved in various cellular activities. CypA has an important role in viral infection which either facilitates or inhibits their replication. Inhibition of CypA via inhibitors is useful for overcoming several viral infections, indicating that CypA is an attractive target for anti-viral therapy. Collectively, these facts demonstrate the critical roles of CypA in mediating or inhibiting viral infections, suggesting that CypA can be an attractive cellular target for the development of anti-viral therapy.
Asunto(s)
Antivirales/farmacología , Ciclofilina A/antagonistas & inhibidores , Ciclofilina A/fisiología , Interacciones Huésped-Patógeno/fisiología , Replicación Viral/fisiología , Humanos , Terapia Molecular Dirigida/métodos , Virosis/metabolismo , Virosis/virología , Replicación Viral/efectos de los fármacosRESUMEN
Snakeheadvesiculovirus (SHVV), a new member of the family Rhabdoviridae, has caused enormous economic losses in snakehead fish culture during the past years in China; however, little is known about the molecular mechanisms of its pathogenicity. MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in virus infection. In this study, we identified that SHVV infection downregulated miR-214 in striped snakehead (SSN-1) cells in a time- and dose-dependent manner. Notably, transfecting SSN-1 cells with miR-214 mimic significantly inhibitedSHVV replication, whereas miR-214 inhibitor promoted it, suggesting that miR-214 acted as a negative regulator of SHVV replication. Our study further demonstrated that N and P of SHVV were the target genes of miR-214. Over-expression of P, but not N, inhibited IFN-α production in SHVV-infected cells, which could be restored by over-expression of miR-214. Taken together, these results suggest that miR-214 is downregulated during SHVV infection, and the downregulated miR-214 in turn increased N and P expression and decreased IFN-α production, thus facilitating SHVV replication. This study provides a better understanding of the molecular mechanisms on the pathogenesis of SHVV and a potential antiviral strategy against SHVV infection.