Dataset Information

Identification of 2'-5'-Oligoadenylate Synthetase-Like Gene in Goose: Gene Structure, Expression Patterns, and Antiviral Activity Against Newcastle Disease Virus.

ABSTRACT: 2'-5'-oligoadenylate synthetase-like (OASL) is a kind of antiviral protein induced by interferons (IFNs), which plays an important role in the IFNs-mediated antiviral signaling pathway. In this study, we cloned and identified OASL in the Chinese goose for the first time. Goose 2'-5'-oligoadenylate synthetase-like (goOASL), including an ORF of 1527bp, encoding a protein of 508 amino acids. GoOASL protein contains 3 conserved motifs: nucleotidyltransferase (NTase) domain, 2'-5'-oligoadenylate synthetase (OAS) domain, and 2 ubiquitin-like (UBL) repeats. The tissue distribution profile of goOASL in 2-week-old gosling and adult goose were identified by Real-Time quantitative PCR, which revealed that the highest level of goOASL mRNA transcription was detected in the blood of adult goose and gosling. The mRNA transcription level of goOASL was upregulated in all tested tissues of duck Tembusu virus (DTMUV)-infected 3-day-old goslings, compared with control groups. Furthermore, using the stimulus Poly(I: C), ODN2006, R848, and lipopolysaccharide (LPS) as well as the viral pathogens DTMUV, H9N2 avian influenza virus (AIV), and gosling plague virus (GPV) to treat goose peripheral blood mononuclear cells (PBMCs) for 6?h, goOASL transcripts level was significantly upregulated in all treated groups. To further investigate the antiviral activity of goOASL, pcDNA3.1(+)-goOASL-His plasmid was constructed, and goOASL was expressed by the goose embryo fibroblast cells (GEFs) transfected with pcDNA3.1(+)-goOASL-His. Our research data suggested that Newcastle disease virus (NDV) replication (viral copies and viral titer) in GEFs was significantly reduced by the overexpression of goOASL protein. These data were meaningful for the antiviral immunity research of goose and shed light on the future prevention of NDV in fowl.

SUBMITTER: Yang C

PROVIDER: S-EPMC5011626 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Identification of 2'-5'-Oligoadenylate Synthetase-Like Gene in Goose: Gene Structure, Expression Patterns, and Antiviral Activity Against Newcastle Disease Virus.

Yang Chao C Liu Fei F Chen Shun S Wang Mingshu M Jia Renyong R Zhu Dekang D Liu Mafeng M Sun Kunfeng K Yang Qiao Q Wu Ying Y Chen Xiaoyue X Cheng Anchun A

Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 20160901 9

2'-5'-oligoadenylate synthetase-like (OASL) is a kind of antiviral protein induced by interferons (IFNs), which plays an important role in the IFNs-mediated antiviral signaling pathway. In this study, we cloned and identified OASL in the Chinese goose for the first time. Goose 2'-5'-oligoadenylate synthetase-like (goOASL), including an ORF of 1527bp, encoding a protein of 508 amino acids. GoOASL protein contains 3 conserved motifs: nucleotidyltransferase (NTase) domain, 2'-5'-oligoadenylate synt ...[more]

PMID: 27576097

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Project description:The interferon (IFN)-inducible 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway plays a critical role in antiviral immunity. Group A rotaviruses, including the simian SA11 strain, inhibit this pathway through two activities: an E3-ligase related activity of NSP1 that degrades proteins necessary for IFN signaling, and a phosphodiesterase (PDE) activity of VP3 that hydrolyzes the RNase L-activator 2',5'-oligoadenylate. Unexpectedly, we found that a recombinant (r) SA11 double mutant virus deficient in both activities (rSA11-VP3H797R-NSP1ΔC17) retained the ability to prevent RNase L activation. Mass spectrometry led to the discovery that NSP1 interacts with RNase L in rSA11-infected HT29 cells. This interaction was confirmed through copulldown assay of cells transiently expressing NSP1 and RNase L. Immunoblot analysis showed that infection with wild-type rSA11 virus, rSA11-VP3H797R-NSP1ΔC17 double mutant virus, or single mutant forms of the latter virus all resulted in the depletion of endogenous RNase L. The loss of RNase L was reversed by addition of the neddylation inhibitor MLN4924, but not the proteasome inhibitor MG132. Analysis of additional mutant forms of rSA11 showed that RNase L degradation no longer occurred when either the N-terminal RING domain of NSP1 was mutated or the C-terminal 98 amino acids of NSP1 were deleted. The C-terminal RNase L degradation domain is positioned upstream and is functionally independent of the NSP1 domain necessary for inhibiting IFN expression. Our studies reveal a new role for NSP1 and its E3-ligase related activity as an antagonist of RNase L and uncover a novel virus-mediated strategy of inhibiting the OAS-RNase L pathway. IMPORTANCE For productive infection, rotavirus and other RNA viruses must suppress interferon (IFN) signaling and the expression of IFN-stimulated antiviral gene products. Particularly important is inhibiting the interferon (IFN)-inducible 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway, as activated RNase L can direct the nonspecific degradation of viral and cellular RNAs, thereby blocking viral replication and triggering cell death pathways. In this study, we have discovered that the simian SA11 strain of rotavirus employs a novel strategy of inhibiting the OAS-RNase L pathway. This strategy is mediated by SA11 NSP1, a nonstructural protein that hijacks E3 cullin-RING ligases, causing the ubiquitination and degradation of host proteins essential for IFN induction. Our analysis shows that SA11 NSP1 also recognizes and causes the ubiquitination of RNase L, an activity resulting in depletion of endogenous RNase L. These data raise the possibility of using therapeutics targeting cellular E3 ligases to control rotavirus infections.

Project description:Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which poses a serious threat to the global pig industry. Interferons (IFNs) and IFN-stimulated genes (ISGs) play a key role in host antiviral defense. We have previously screened the porcine 2'-5'-oligoadenylate synthetase-like protein (pOASL) as a potential anti-CSFV ISG using a reporter CSFV. This study aimed to clarify the underlying antiviral mechanism of pOASL against CSFV. We confirmed that CSFV replication was significantly suppressed in lentivirus-delivered, pOASL-overexpressing PK-15 cells, whereas silencing the expression of endogenous pOASL by small interfering RNAs markedly enhanced CSFV growth. In addition, the transcriptional level of pOASL was upregulated both in vitro and in vivo upon CSFV infection. Interestingly, the anti-CSFV effects of pOASL are independent of the canonical RNase L pathway but depend on the activation of the type I IFN response. Glutathione S-transferase pulldown and coimmunoprecipitation assays revealed that pOASL interacts with MDA5, a double-stranded RNA sensor, and further enhances MDA5-mediated type I IFN signaling. Moreover, we showed that pOASL exerts anti-CSFV effects in an MDA5-dependent manner. In conclusion, pOASL suppresses CSFV replication via the MDA5-mediated type I IFN-signaling pathway.IMPORTANCE The host innate immune response plays an important role in mounting the initial resistance to viral infection. Here, we identify the porcine 2'-5'-oligoadenylate synthetase-like protein (pOASL) as an interferon (IFN)-stimulated gene (ISG) against classical swine fever virus (CSFV). We demonstrate that the anti-CSFV effects of pOASL depend on the activation of type I IFN response. In addition, we show that pOASL, as an MDA5-interacting protein, is a coactivator of MDA5-mediated IFN induction to exert anti-CSFV actions. This work will be beneficial to the development of novel anti-CSFV strategies by targeting pOASL.

Project description:The enzyme 2'-5' oligoadenylate synthase (OAS) is one of the key interferon-induced antiviral factors that act through inhibition of viral replication. In chickens, there is a single well-characterized OAS gene, oligoadenylate synthase-like (OASL) that has been shown to be upregulated after infection with various viruses. However, a deeper understanding of how chicken OASL acts against viral infection is still necessary. In this study, we tested the hypothesis that OASL short interfering RNA (siRNA)-mediated knockdown would decrease the host gene expression response to the Newcastle disease virus (NDV) by impacting antiviral pathways. To assess our hypothesis, a chicken fibroblast cell line (DF-1) was infected with the NDV (LaSota strain) and OASL expression was knocked down using a specific siRNA. The level of NDV viral RNA in the cells and the expression of interferon response- and apoptosis-related genes were evaluated by quantitative PCR at 4, 8, and 24 h postinfection (hpi). Knockdown of OASL increased the level of NDV viral RNA at 4, 8, and 24 hpi (P < 0.05) and eliminated the difference between NDV-infected and noninfected cells for expression of interferon response- and apoptosis-related genes (P > 0.05). The lack of differential expression suggests that knockdown of OASL resulted in a decreased response to NDV infection. Within NDV-infected cells, OASL knockdown reduced expression of signal transducer and activator of transcription 1, interferon alfa receptor subunit 1, eukaryotic translation initiation factor 2 alpha kinase 2, ribonuclease L, caspase 8 (CASP8) and caspase 9 (CASP9) at 4 hpi, CASP9 at 8 hpi, and caspase 3, CASP8, and CASP9 at 24 hpi (P < 0.05). We suggest that the increased NDV viral load in DF-1 cells after OASL knockdown was the result of a complex interaction between OASL and interferon response- and apoptosis-related genes that decreased host response to the NDV. Our results provide comprehensive information on the role played by OASL during NDV infection in vitro. Targeting this mechanism could aid in future prophylactic and therapeutic treatments for Newcastle disease in poultry.

Project description:BackgroundOligoadenylate synthetases (OASs) are widely distributed in Metazoa including sponges, fish, reptiles, birds and mammals and show large variation, with one to twelve members in any given species. Upon double-stranded RNA (dsRNA) binding, avian and mammalian OASs generate the second messenger 2'-5'-linked oligoadenylate (2-5A), which activates ribonuclease L (RNaseL) and blocks viral replication. However, how Metazoa shape their OAS repertoires to keep evolutionary balance to virus infection is largely unknown. We performed comprehensive phylogenetic and functional analyses of OAS genes from evolutionarily lower to higher Metazoa to demonstrate how the OAS repertoires have developed anti-viral activity and diversified their functions.ResultsAncient Metazoa harbor OAS genes, but lack both upstream and downstream genes of the OAS-related pathways, indicating that ancient OASs are not interferon-induced genes involved in the innate immune system. Compared to OASs of ancient Metazoa (i.e. sponge), the corresponding ones of higher Metazoa present an increasing number of basic residues on the OAS/dsRNA interaction interface. Such an increase of basic residues might improve their binding affinity to dsRNA. Moreover, mutations of functional residues in the active pocket might lead to the fact that higher Metazoan OASs lose the ability to produce 3'-5'-linked oligoadenylate (3-5A) and turn into specific 2-5A synthetases. In addition, we found that multiple rounds of gene duplication and domain coupling events occurred in the OAS family and mutations at functionally critical sites were observed in most new OAS members.ConclusionsWe propose a model for the expansion of OAS members and provide comprehensive evidence of subsequent neo-functionalization and sub-functionalization. Our observations lay the foundation for interrogating the evolutionary transition of ancient OAS genes to host defense genes and provide important information for exploring the unknown function of the OAS gene family.

Dataset Information

Identification of 2'-5'-Oligoadenylate Synthetase-Like Gene in Goose: Gene Structure, Expression Patterns, and Antiviral Activity Against Newcastle Disease Virus.

Publications

Identification of 2'-5'-Oligoadenylate Synthetase-Like Gene in Goose: Gene Structure, Expression Patterns, and Antiviral Activity Against Newcastle Disease Virus.

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