Both antiviral activity and intracellular localization of chicken Mx protein depend on a polymorphism at amino acid position 631

The Mx protein is known to inhibit the multiplication of several RNA viruses. In chickens, a polymorphism at amino acid position 631 (631 aa) of Mx protein has been suggested to be involved in the antiviral ability against vesicular stomatitis virus (VSV) and influenza virus, indicating that a Ser-to-Asn substitution at 631 aa is the source of this antiviral ability. However, how the substitution at 631 aa contributes to the antiviral activity remains to be clarified. In this study, we investigated differences in antiviral activity against VSV and intracellular localization between Ser and Asn types at 631 aa of the chicken Mx protein. The results showed that chicken Mx protein with an Asn at 631 aa inhibited VSV multiplication and Mx distribution in a granular-like pattern in the cytoplasm. However, Mx carrying the Ser type did not inhibit viral growth and homogenous spread throughout the cytoplasm. Furthermore, we found that replacing Ser with Asn at 631 aa provided Mx with antiviral activity against VSV, with Mx showing granular-like distribution in the cytoplasm. These results demonstrated that a single amino acid polymorphism at 631 aa of the chicken Mx protein altered both the antiviral activity and intracellular localization.     

Partial antiviral activities of the Asn631 chicken Mx against newcastle disease virus and vesicular stomatitis virus

Conflicting data existed for the antiviral potential of the chicken Mx protein and the importance of the Asn631 polymorphism in determination of the antiviral activity. In this study we modified the chicken Mx cDNA from the Ser631 to Asn631 genotype and transfected them into COS-I cells, chicken embryonic fibroblast (CEF) or NIH 3T3 cells. The Mx protein was mainly located at the cytoplasm. The transfected cell cultures were challenged with newcastle disease virus (NDV) or vesicular stomatitis virus (VSV), cytopathic affect (CPE) inhibition assay showed that the times for development of visible and full CPE were significantly postponed by the Asn631 cDNA transfection at 48 h transfection, but not by the Ser631 cDNA transfection. Viral titration assay showed that the virus titers were significantly reduced before 72 h postinfection. CEF cells was incubated by the cell lysates extracted from the COS-I cells transfected with pcDNA-Mx/Asn631, could resist and delayed NDV infection. These data suggested the importance of the Asn631 polymorphism of the chicken Mx in determination of the antiviral activities against NDV and VSV at early stage of viral infection, which were relatively weak and not sufficient to inhibit the viral replication at late stage of viral infection.     

Native antiviral specificity of chicken Mx protein depends on amino acid variation at position 631

An attempt was made to determine whether amino acid variation at position 631 in the chicken Mx protein definitely influences antiviral specificity, using an artificial mutation technique by which a single amino acid was reciprocally substituted between Ser (AGT) and Asn (AAT) at position 631 of the negative and positive chicken Mx, respectively. Using permanently transfected 3T3 cell lines, the antiviral potential of chicken Mx against vesicular stomatitis virus infection was analysed. The results indicated that the phenotype of antiviral activity depends on the amino acid difference at position 631; that is, the genotype coding Asn at position 631 corresponds to the positive antiviral phenotype, and the genotype coding Ser corresponds to the negative phenotype. The present study has confirmed that the antiviral specificity of chicken Mx protein is determined by an amino acid substitution at the carboxy terminus.     

The Cytoplasmic Location of Chicken Mx Is Not the Determining Factor for Its Lack of Antiviral Activity

Background

Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.

Methodology/Principal Findings

Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.

Conclusions/Significance

The chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity.     

Asparagine 631 variants of the chicken Mx protein do not inhibit influenza virus replication in primary chicken embryo fibroblasts or in vitro surrogate assays

Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles.     

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.     

Population Research of Genetic Polymorphism at Amino Acid Position 631 in Chicken Mx Protein with Differential Antiviral Activity

A single amino acid substitution between Asn and Ser at position 631 in the chicken Mx protein has been reported to determine resistant and sensitive antiviral activity. In this study, we investigate whether various kinds of chicken breeds and jungle fowls carry the resistant or sensitive Mx allelic gene by using the mismatched PCR-restriction fragment length polymorphism (RFLP) technique. In total, 271 samples from 36 strains of 17 chicken breeds and from 3 kinds of jungle fowls were examined. The rates of the resistant Mx gene and sensitive gene were 59.2% and 40.8%, respectively. Only a Red jungle fowl captured in Laos carried the resistant Mx gene, and the other three Red jungle fowls from Indonesia and Gray and Green jungle fowls all had the sensitive Mx gene. These results were confirmed by the determination of amino acid sequences in the GTPase effector domain of jungle fowls.     

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.     

The zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses

Type I interferons (IFNs) signal through specific receptors to mediate expression of genes, which together confer a cellular antiviral state. Overexpression of the zinc finger antiviral protein (ZAP) imparts a cellular antiviral state against Retroviridae, Togaviridae, and Filoviridae virus family members. Since ZAP expression is induced by IFN, we utilized Sindbis virus (SINV) to investigate the role of other IFN-induced factors in ZAP's inhibitory potential. Overexpressed ZAP did not inhibit virion production or SINV-induced cell death in BHK cells deficient in IFN production (and thus IFN signaling), suggesting a role for an IFN-induced factor in ZAP's activity. IFN pretreatment in the presence of ZAP resulted in greater inhibition than IFN alone. Using mouse embryo fibroblast (MEF) cells deficient in Stat1, we showed that signaling through the IFN receptor is necessary for IFN′s enhancement of ZAP activity. Unlike in BHK cells, however, overexpressed ZAP exhibited antiviral activity in the absence of IFN. In wild-type MEFs with an intact Stat1 gene, IFN pretreatment synergized with ZAP to generate a potent antiviral response. Despite failing to inhibit SINV virion production and virus-induced cell death in BHK cells, ZAP inhibited translation of the incoming viral RNA. IFN pretreatment synergized with ZAP to further block protein expression from the incoming viral genome. We further show that silencing of IFN-induced ZAP reduces IFN efficacy. Our findings demonstrate that ZAP can synergize with another IFN-induced factor(s) for maximal antiviral activity and that ZAP's intrinsic antiviral activity on virion production and cell survival can have cell-type-specific outcomes.     

Influenza virus partially counteracts restriction imposed by tetherin/BST-2

Influenza virus infections lead to a burst of type I interferon (IFN) in the human respiratory tract, which most probably accounts for a rapid control of the virus. Although in mice, IFN-induced Mx1 factor mediates a major part of this response, the situation is less clear in humans. Interestingly, a recently identified IFN-induced cellular protein, tetherin (also known as CD317, BST-2, or HM1.24), exerts potent antiviral activity against a broad range of retroviruses, as well as several other enveloped viruses, by impeding the release of newly generated viral particles from the cell surface. Here we show that influenza virus belongs to the targets of this potent antiviral factor. Ectopic expression of tetherin strongly inhibited fully replicative influenza virus. In addition, depleting endogenous tetherin increased viral production of influenza virions, both in cells constitutively expressing tetherin and upon its induction by IFN. We further demonstrate, by biochemical and morphological means, that tetherin exerts its antiviral action by tethering newly budded viral particles, a mechanism similar to the one that operates against HIV-1. In addition, we determined that the magnitude of tetherin antiviral activity is comparable with or higher than the one of several previously identified anti-influenza cellular factors, such as MxA, ADAR1, ISG15, and viperin. Finally, we demonstrate that influenza virus reduces the impact of tetherin-mediated restriction on its replication by several mechanisms. First, the influenza virus NS1 protein impedes IFN-mediated tetherin induction. Second, influenza infection leads to a decrease of tetherin steady state levels, and the neuraminidase surface protein partly counteracts its activity. Overall, our study helps to delineate the intricate molecular battle taking place between influenza virus and its host cells.     

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.     

Persistence of betanodavirus in Barramundi brain (BB) cell line involves the induction of Interferon response

The BB cell line derived from the brain tissue of a barramundi (Lates calcarifer) that survived nervous necrosis virus (NNV) infection is persistently infected with NNV. To elucidate whether interferon (IFN) plays a role in the mechanism of NNV-persistent infection in BB cell line, a virus-negative control cell line was obtained by treating BB cells with NNV-specific rabbit antiserum for 5 subcultures. After the treatment, NNV titer or RNA or capsid protein was no longer detected in the cured BB (cBB) cells. Expression of Mx gene, encoding a type I IFN-inducible antiviral protein, was found in BB cells and cBB cells following NNV infection, but not in NNV-free cBB cells. Moreover, expression of Mx gene and antiviral activity against NNV were induced in cBB cells by the treatment with MAb-neutralized BB cell supernatant. Furthermore, NNV persistent infection was induced again in cBB cell culture if multiplicity of infection (MOI) was low (< or = 1). These experimental results indicated that IFN-like cytokines existed in the culture supernatant of BB cells, and IFN-induced response played an important role in protecting the majority of cells from virus lytic infection and regulating NNV persistence in the BB cell line.     

Interferon, Mx, and viral countermeasures

The interferon system provides a powerful and universal intracellular defense mechanism against viruses. Knockout mice defective in IFN signaling quickly succumb to all kinds of viral infections. Likewise, humans with genetic defects in interferon signaling die of viral disease at an early age. Among the known interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins belong to the dynamin superfamily of large GTPases and have direct antiviral activity. They inhibit a wide range of viruses by blocking an early stage of the viral replication cycle. Likewise, the protein kinase R (PKR), and the 2–5 OAS/RNaseL system represent major antiviral pathways and have been extensively studied. Viruses, in turn, have evolved multiple strategies to escape the IFN system. They try to go undetected, suppress IFN synthesis, bind and neutralize secreted IFN molecules, block IFN signaling, or inhibit the action of IFN-induced antiviral proteins. Here, we summarize recent findings about the astonishing interplay of viruses with the IFN response pathway.     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

鸡Mx蛋白基因诱变修饰及抗病活性

[目的]进一步研究鸡Mx蛋白第631位氨基酸的变异与鸡群抗病性的相关性.[方法]本实验利用PCR突变技术将鸡Mx蛋白基因的全长cDNA第2032位的碱基由G突变为A(既631位氨基酸的改变),并将突变的Mx基因插入真核表达载体pcDNA3.0,重组表达载体转染COS-Ⅰ细胞后,进行RT-PCR与间接免疫荧光(IFA)鉴定.[结果]对鸡Mx蛋白基因的cDNA进行PCR诱变修饰正确,构建了能够正确表达鸡Mx蛋白的重组真核表达载体;诱变修饰重组Mx蛋白对抗新城疫病毒(NDV)感染分析结果显示,重组Mx蛋白具有较强的抗新城疫病毒生物活性.[结论]为下一步研究鸡Mx蛋白的抗病机理与制备抗病转基因鸡奠定了坚实的基础.     

Influenza A Virus Does Not Encode a Tetherin Antagonist with Vpu-Like Activity and Induces IFN-Dependent Tetherin Expression in Infected Cells

The interferon-induced host cell factor tetherin inhibits release of human immunodeficiency virus (HIV) from the plasma membrane of infected cells and is counteracted by the HIV-1 protein Vpu. Influenza A virus (FLUAV) also buds from the plasma membrane and is not inhibited by tetherin. Here, we investigated if FLUAV encodes a functional equivalent of Vpu for tetherin antagonism. We found that expression of the FLUAV protein NS1, which antagonizes the interferon (IFN) response, did not block the tetherin-mediated restriction of HIV release, which was rescued by Vpu. Similarly, tetherin-mediated inhibition of HIV release was not rescued by FLUAV infection. In contrast, FLUAV infection induced tetherin expression on target cells in an IFN-dependent manner. These results suggest that FLUAV escapes the antiviral effects of tetherin without encoding a tetherin antagonist with Vpu-like activity.