Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

ISG15 is one of the most strongly induced genes upon viral infection, interferon (IFN) stimulation, and lipopolysaccharide (LPS) stimulation, and only one copy has been found in mammals so far. Here two fish ISG15 genes, termed CaISG15-1 and CaISG15-2, have been cloned and sequenced from UV-inactivated GCHV (grass carp haemorrhagic virus)-infected and IFN-produced CAB cells (crucian carp Carassius auratus blastulae embryonic cells) by suppression subtractive hybridization. The full-length cDNA sequences of two crucian carp ISG15 encode a 155-amino-acid protein and a 161-amino-acid protein, both of which show 78.9% identity overall and possess the characteristic structures of mammalian ISG15 proteins including two tandem ubiquitin-like domains and the C-terminal canonical LRLRGG motif. In CAB cells treated with different stimuli including active virus, UV-inactivated GCHV and IFN containing supernatant (ICS), the expression of both CaISG15-1 and CaISG15-2 was upregulated but displayed different kinetics. Poly I:C and LPS were also able to induce an increase in mRNA for both genes. In CAB cells responsive to active GCHV, UV-inactivated GCHV, CAB ICS, Poly I:C and LPS, CaISG15-1 was upregulated more significantly than CaISG15-2. These results suggest that there are two ISG15 homologues in crucian carp, both of which might play distinct roles in innate immunity against viral and bacterial infection.     

鲫Nub1基因的克隆及特征分析

Nub1(NEDD8 Ultimate Buster-1)是近年来发现的一种干扰素诱导表达基因,过量表达该基因能抑制细胞生长。研究通过RACE-PCR方法从产生干扰素的鲫囊胚培养细胞(Carassius auratus blastulae embryonic cells,CAB)中克隆出鲫Nub1基因。鲫Nub1全长cDNA为2298bp,编码一个由589个氨基酸残基组成的蛋白。推导的鲫NUB1蛋白具有哺乳类同源蛋白保守的结构域,包括N端的UBL结构域和C端两个UBA结构域,与已知的哺乳类包括人和小鼠、鸟类和两栖类的同源蛋白具有41%-45%的相似性。诱导表达分析显示PolyI:C和LPS均能诱导CAB细胞Nub1mRNA的上调,表明Nub1基因在鱼类的抗病免疫反应中发挥某种重要作用。       

Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells.

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.     

The Testicular Antiviral Defense System: Localization, Expression, and Regulation of 2′5′ Oligoadenylate Synthetase, Double-Stranded RNA-activated Protein Kinase, and Mx Proteins in the Rat Seminiferous Tubule

Although the involvement of viruses in alterations of testicular function and in sexually transmitted diseases is well known, paradoxically, the testicular antiviral defense system has virtually not been studied. The well known antiviral activity of interferons (IFNs) occurs via the action of several IFN-induced proteins, among which the 2′5′ oligoadenylate synthetase (2′5′ A synthetase), the double-stranded RNA-activated protein kinase (PKR), and the Mx proteins are the best known. To explore the antiviral capacity of the testis and to study the testicular action of IFNs, we looked for the presence and regulation of these three proteins in isolated seminiferous tubule cells, cultured in the presence or in the absence of IFN α, IFN γ, or Sendai virus. In all conditions tested, the meiotic pachytene spermatocytes and the post-meiotic early spermatids lacked 2′5′ A synthetase, PKR, and Mx mRNAs and proteins. In contrast, Sertoli cells constitutively expressed these mRNAs and proteins, and their levels were greatly increased after IFN α or Sendai virus exposure. While peritubular cells were also able to markedly express 2′5′ A synthetase, PKR, and Mx mRNA and proteins after IFN α or viral exposure, only PKR was constitutively present in these cells. Interestingly, IFN γ had no effect on peritubular cells' 2′5′ A synthetase and Mx production but it enhanced Mx proteins in Sertoli cells. In conclusion, this study reveals that the seminiferous tubules are particularly well equipped to react to a virus attack. The fact that the two key tubular elements of the blood–testis barrier, namely, Sertoli and peritubular cells, were found to assume this protection allows the extension of the concept of blood–testis barrier to the testicular antiviral defense.     

Enhanced grass carp reovirus resistance of Mx-transgenic rare minnow (Gobiocypris rarus)

In the interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins have direct antiviral activity and inhibit a wide range of viruses by blocking an early stage of the viral genome replication cycle. However, antiviral activity of piscine Mx remains unclear in vivo. In the present study, an Mx-like gene was cloned, characterized and gene-transferred in rare minnow Gobiocypris rarus, and its antiviral activity was confirmed in vivo. The full length of the rare minnow Mx-like cDNA is 2241 bp in length and encodes a polypeptide of 625 amino acids with an estimated molecular mass of 70.928 kDa and a predicted isoelectric point of 7.33. Analysis of the deduced amino acid sequence indicated that the mature peptide contains an amino-terminal tripartite GTP-binding motif, a dynamin family signature sequence, a GTPase effector domain and two carboxy-terminal leucine zipper motifs, and is the most similar to the crucian carp (Carassius auratus) Mx3 sequence with an identity of 89%. Both P0 and F1 generations of Mx-transgenic rare minnow demonstrated very significantly high survival rate to GCRV infection (P < 0.01). The mRNA expression of Mx gene was consistent with survival rate in F1 generation. The virus yield was also concurrent with survival time using electron microscope technology. Rare minnow has Mx gene(s) of its own but introducing more Mx gene improves their resistance to GCRV. Mx-transgenic rare minnow might contribute to control the GCRV diseases.     

斑马鱼一个IFIT 家族基因的鉴定及启动子分析

IFIT 家族由一类受干扰素诱导表达并具有TPR 结构域的蛋白组成, 但是在鱼类关于IFIT 基因的研究还很少。研究利用哺乳类IFIT 家族基因IFI56 的序列搜索斑马鱼基因组数据库鉴定出一个未知基因, 该基因具有哺乳类IFIT 家族保守的基因组结构, 编码蛋白具有保守的TPR 结构域, 暂命名为IFIT-A。RT-PCR 分析表明, Poly I:C 能够诱导IFIT-A 基因转录水平上调。与哺乳类IFIT 家族基因相似, 斑马鱼IFIT-A 启动子存在ISG 基因特有的典型ISRE 结构域。荧光素酶活性实验揭示Poly I:C 和重组IFN 蛋白能激活斑马鱼IFIT-A 启动子活性。此外, 过量表达IFN 调控因子IRF3 和IRF7 能诱导斑马鱼IFIT-A 启动子活性。实验结果证明IFIT-A基因是斑马鱼IFIT 家族成员, IRF3 和IRF7 在其诱导表中具有重要调控作用。       

Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus

Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.     

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.     

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.     

Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.     

cDNA structures and regulation of two interferon-induced human Mx proteins.

Human cells treated with interferon synthesize two proteins that exhibit high homology to murine Mx1 protein, which has previously been identified as the mediator of interferon-induced cellular resistance of mouse cells against influenza viruses. Using murine Mx1 cDNA as a hybridization probe, we have isolated cDNA clones originating from two distinct human Mx genes, designated MxA and MxB. In human fibroblasts, expression of MxA and MxB is strongly induced by alpha interferon (IFN-alpha), IFN-beta, Newcastle disease virus, and, to a much lesser extent, IFN-gamma, MxA and MxB proteins have molecular masses of 76 and 73 kilodaltons, respectively, and their sequences are 63% identical. A comparison of human and mouse Mx proteins revealed that human MxA and mouse Mx2 are the most closely related proteins, showing 77% sequence identity. Near their amino termini, human and mouse Mx proteins contain a block of 53 identical amino acids and additional regions of very high sequence similarity. These conserved sequences are also present in a double-stranded RNA-inducible fish gene, which suggests that they may constitute a functionally important domain of Mx proteins. In contrast to mouse Mx1 protein, which accumulates in the nuclei of IFN-treated mouse cells, the two human Mx proteins both accumulate in the cytoplasm of IFN-treated cells.