Interferon-inducible antiviral protein MxA enhances cell death triggered by endoplasmic reticulum stress

Human myxovirus resistance gene A (MxA) is a type I interferon-inducible protein and exhibits the antiviral activity against a variety of RNA viruses, including influenza virus. Previously, we reported that MxA accelerates cell death of influenza virus-infected cells through caspase-dependent and -independent mechanisms. Similar to other viruses, influenza virus infection induces endoplasmic reticulum (ER) stress, which is one of cell death inducers. Here, we have demonstrated that MxA enhances ER stress signaling in cells infected with influenza virus. ER stress-induced events, such as expression of BiP mRNA and processing of XBP1 mRNA, were upregulated in cells expressing MxA by treatment with an ER stress inducer, tunicamycin (TM), as well as influenza virus infection. TM-induced cell death was also accelerated by MxA. Furthermore, we showed that MxA interacts with BiP and overexpression of BiP reduces MxA-promoted ER stress signaling. Because cell death in virus-infected cells is one of ultimate anti-virus mechanisms, we propose that MxA-enhanced ER stress signaling is a part of the antiviral activity of MxA by accelerating cell death.     

Stimulus-dependent and domain-dependent cell death acceleration by an IFN-inducible protein, human MxA.

Human MxA is an interferon- alpha / beta (IFN-alpha/beta)-inducible protein that inhibits multiplication of influenza viruses and other RNA viruses. We reported that MxA accelerates cell death induced by apoptotic stimuli as well as influenza viral infection. However, the mechanism of MxA-mediated enhancement of cell death is not well understood. Here, we demonstrated that the cell death promotion activity of MxA was caspase dependent when cell death was induced by UV irradiation or cycloheximide (CHX). In contrast, in the case of cell death after influenza viral infection, MxA promoted both caspase-dependent and caspase-independent cell death. The C-terminal region of MxA containing the oligomerization domain was found to be responsible for promotion of the cell death induced by CHX. In the case of cell death after influenza viral infection, both C-terminal and N-terminal regions were shown to be involved in cell death promotion, although the GTP-binding and GTP-hydrolysis activity dependent on a tripartite GTP-binding motif in the N-terminal region was not required for the cell death promotion activity of MxA. These results suggest that MxA accelerates cell death induced by influenza viral infection through at least two distinct pathways.     

Alpha interferon, antiviral proteins and their value in clinical medicine

Type I interferon system is an important part of host's innate defense mechanisms against viral infections. The type I interferons mediate in part their antiviral effect via induction of various proteins. Among them the most widely known are 2'-5' oligoadenylate synthetase (2'-5' OAS) and a protein kinase (PKR). MxA, an other antiviral protein, is specifically induced by the type I interferons. The MxA protein contains the dynamin signature, which is implicated in transport processes. The MxA protein appears to block the replication of certain viruses at poorly defined steps. There are substantial differences in the antiviral activity of MxA between virus types. Indeed, the replication of vesicular stomatitis virus and influenza virus is inhibited by MxA, but not the one of type I herpes simplex virus. Measurements of interferon alpha and MxA levels may be of high value in clinical practice. Interferon alpha can be detected by using a bioassay based on the interferon alpha ability to protect cultured cells from the cytopathic effect caused by a selected challenged virus, or by using immunological techniques. The current bioassays are the most sensitive methods but they are cumbersome and lengthy, even though simplifications have been proposed. Immunological techniques are easier, however they do not explore the biological activity of the circulating interferon. The presence of type I interferon in biological samples (serum, plasma, cerebro-spinal fluid, cultured cell supernatants) can be indirectly assessed by capability of interferon alpha to induce in vitro the synthesis of MxA in a dose dependent manner in cultured cells. Following to the lysis of the cells, the induced MxA can be quantitated and hence the type I-interferon concentration can be determinated in samples. The quantitation of MxA protein in peripheral blood lysates can be useful as a specific marker of acute viral infections. A minute amount of whole blood (15 mul) is sufficient which facilitates its use in pediatrics. The specifically type-I-interferons inducible MxA protein is also a potential useful marker in the management of interferon alpha-treatment. Moreover, the detection of interferon alpha and antiviral proteins constitute an indirect approach for investigating the hypothesis of the role of viruses in chronic diseases with suspected infectious aetiology.     

不同H5N1亚型高致病性禽流感病毒诱导IFN-α/β和MxA mRNA表达

目的: 用两株鹅源H5N1禽流感病毒(AIV)接种A549细胞,比较两毒株在细胞中的增殖情况,以及病毒诱导细胞干扰素(INF)-α/β和黏病毒抗性蛋白A(MxA) mRNA表达的情况。方法: 将两组病毒悬液接种A549细胞,按Reed-Muench法计算两毒株的组织培养半数感染量(TCID₅₀),PCR扩增检测细胞中病毒的HA和M1片段。Real-time PCR方法检测细胞中IFN-α/β和MxA mRNA表达。结果: 两株病毒均能感染A549细胞, AIV128诱导细胞的IFN-α/β和MxA mRNA表达均较AIV75高。结论: H5N1亚型AIV75和AIV128毒株能在人肺泡癌上皮细胞A549中复制,细胞IFN-α/β和MxA mRNA的表达诱导依赖于病毒的复制。     

A persistent variant of influenza C virus fails to interact with actin filaments during viral assembly.

C/AA-pi virus, a variant of influenza C/Ann Arbor/1/50 virus, establishes persistent infections in MDCK cells, characterized by low levels of progeny production. During viral assembly, nucleoprotein (NP) was found homogeneously distributed over cytoplasmic and nuclear compartments and matrix (M) protein was likewise localized in a barely structured fashion. In contrast, infections with nonpersistent influenza A, B and C viruses produced cytoplasmic granular structures, which typically consisted of colocalized NP and M proteins. Studies on the in vitro interaction between NP and M proteins revealed identical binding capacities comparing influenza C wild-type virus with the persistent variant. Cytochalasin D treatment of infected cells demonstrated that NP protein of the wild-type virus, but not of the persistent variant, was distinctly associated with cellular actin filaments. Moreover, the assembly characteristics of wild-type virus were modulated in the presence of recombinant persistent-type NP protein towards a behaviour similar to persistent infection. Cell type specificity was particularly illustrated in C/AA-pi virus-infected Vero cells, which did not support viral persistence, but produced granular wild-type-like complexes. Thus, interaction between NP, M and actin proteins (i) is a basic part of the viral assembly process, (ii) is dominantly modulated by NP protein and (iii) is specifically altered in the case of persistent infection.     

Avian cells expressing the murine Mx1 protein are resistant to influenza virus infection

The cDNA encoding the murine Mx1 protein, a mediator of resistance to influenza virus, was inserted into a replication-competent avian retroviral vector in either the sense (referred to as Mx+) or the antisense (referred to as Mx-) orientation relative to the viral structural genes. Both vectors produced virus retaining the Mx insert (Mx recombinant viruses referred to as Mx+ and Mx-) following transfection into chicken embryo fibroblasts (CEF). Mx protein of the appropriate size and nuclear localization was expressed only in CEF cells infected with the Mx+ virus. Mx expression was observed in all Mx(+)-infected cells and was stable during long-term culture. Cells infected with the Mx+ virus were resistant to infection by human influenza A/WSN/33 (H1N1) and avian influenza viruses A/Turkey/Wisconsin/68 (H5N9) and A/Turkey/Massachusetts/65 (H6N2), but were susceptible to infection by the enveloped RNA viruses Sindbis and vesicular stomatitis virus (VSV). Normal CEF and cells infected with the Mx virus were susceptible to influenza A, Sindbis, and VSV. The synthesis of influenza proteins, especially the larger polymerase and hemagglutinin proteins, was reduced in Mx+ retrovirus-infected cells superinfected by influenza A.     

Blood MxA protein as a marker for respiratory virus infections in young children

BackgroundType I interferon induced MxA response can differentiate viral from bacterial infections, but MxA responses in rhinovirus or asymptomatic virus infections are not known.ObjectiveTo study MxA protein levels in healthy state and during respiratory virus infection of young children in an observational prospective cohort.Study designBlood samples and nasal swabs were collected from 153 and 77 children with and without symptoms of respiratory infections, respectively. Blood MxA protein levels were measured by an enzyme immunoassay and PCR methods were used for the detection of respiratory viruses in nasal swabs.ResultsRespiratory viruses were detected in 81% of symptomatic children. They had higher blood MxA protein levels (median [interquartile range]) than asymptomatic virus-negative children (695 [345–1370] μg/L vs. 110 [55–170] μg/L; p < 0.001). Within asymptomatic children, no significant difference was observed in MxA responses between virus-positive and virus-negative groups. A cut-off level of 175 μg/L had 92% sensitivity and 77% specificity for a symptomatic respiratory virus infection. Rhinovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, coronavirus, and human metapneumovirus infections were associated with elevated MxA responses. Asymptomatic virus-negative children vaccinated with a live virus vaccine had elevated MxA protein levels (240 [120–540] μg/L), but significantly lower than children with an acute respiratory infection, who had not received vaccinations (740 [350–1425] μg/L; p < 0.001).ConclusionBlood MxA protein levels are increased in young children with symptomatic respiratory virus infections, including rhinovirus infections. MxA is an informative general marker for the most common acute virus infections.     

The synthesis of polypeptides in influenza C virus-infected cells

The synthesis of virus-specific polypeptides was analyzed in MDCK cells infected with the JJ/50 strain of influenza C virus. In addition to three major structural proteins gp88, NP, and M, the synthesis of five polypeptides with molecular weights of 29,500 (C1), 27,500 (C2), 24,000 (C3), 19,000 (C4), and 14,000 (C5) was found in infected cells. None of these polypeptides were detected either in virions or in immunoprecipitates obtained after treatment of infected cell lysates with antiviral serum, suggesting that they are not viral structural proteins. Polypeptides C1-C5 were found to be synthesized in MDCK cells infected with different influenza C virus strains as well as in different host cell types infected with C/JJ/50. Further, it was observed that cellular protein synthesis was greatly reduced under hypertonic conditions, whereas the synthesis of C1-C5 was relatively unaffected. These results suggest that polypeptides C1-C5 are virus coded rather than host cell coded. Peptide mapping studies showed that each of polypeptides C3, C4, and C5 had a peptide composition similar to the M protein. The amount of C2 synthesized in infected cells was insufficient for mapping. This polypeptide was, however, found to rapidly disappear in pulse-chase experiments, suggesting that C2 is probably not unique but biosynthetically related to one of the other proteins. In contrast to these polypeptides, polypeptide C1 showed a map which is largely different from any major structural polypeptide. It therefore appears likely that C1 is a nonstructural protein of influenza C virus similar to the NS1 protein of influenza A and B viruses.     

Interferon-inducible Mx gene expression in cotton rats: cloning, characterization, and expression during influenza viral infection.

Mx proteins belong to the superfamily of large GTPases with antiviral activity against a wide range of RNA viruses. In vivo, the expression of Mx genes is tightly regulated by the presence of type I interferons (IFNs), and their induction has been described during several viral infections. However, because of the absence of functional Mx genes in most common laboratory strains of mice, in vivo studies of the expression of these genes during viral infection have been hampered. We have cloned the cDNAs for the cotton rat homologs of Mx1 and Mx2 genes that encode full-length proteins. Mx1 localized in the nucleus, whereas Mx2, as its human homolog MxA, localized in the cytoplasm. The expression of Mx genes in cotton rat cells was induced by type I IFNs (IFN-alpha and IFN-beta) but induced only marginally with type II IFN (IFN-gamma). In vivo, the expression of Mx genes was dramatically augmented in lungs of cotton rats infected with influenza virus. The expression of Mx genes and protein(s) was dependent on the dose of virus and the time postinfection for the analysis. Our data present for the first time a complete analysis of the kinetics of expression of these influenza resistant genes in vivo and underscore the fidelity and sensitivity of the cotton rat model for the study of influenza viral infection.     

Complete nucleotide sequence of the influenza C/California/78 virus nucleoprotein gene

The complete nucleotide sequence of RNA segment 5 of the influenza C/California/78 (C/Cal/78) virus was determined by using cloned cDNA derived from viral RNA. The gene contains 1809 nucleotides and can code for a protein of 565 amino acids with a molecular weight of 63 525. The RNA 5 protein of the influenza C/Cal/78 virus possesses two short regions which share a high degree (60–83%) of sequence homology with the nucleoproteins of influenza A and B viruses. These and other structural features of the RNA 5 protein suggest that RNA 5 of influenza C viruses codes for the nucleoprotein. The data also suggest that influenza C viruses are orthomyxoviruses, but that they are more distantly related to either type A or type B viruses than are influenza A and B viruses to each other.     

Inhibition of coxsackievirus B4 replication in stably transfected cells expressing human MxA protein

Coxsackieviruses B (CVB) (B1-B6), positive-strand RNA viruses, cause a variety of diseases. CVB4 may have a causal role in insulin-dependent diabetes mellitus. IFN-alpha inhibits CVB replication; however, the mechanism is not well known. The interferon-alpha-inducible human MxA protein exerts an antiviral activity against negative-strand RNA viruses and against Semliki Forest virus, a positive-strand RNA virus. To test the antiviral spectrum of MxA against CVB4, we took advantage of stably transfected Vero cells expressing MxA (Vero/MxA) in 98% of cells. Compared with control cells, in Vero/MxA cells, CVB4 yields were dramatically reduced and expression of the VP1 CVB protein analyzed by immunofluorescence was highly restricted. Furthermore, the accumulation of positive- and negative-strand CVB4 RNA was prevented as shown by in situ hybridization and RT-PCR. These results indicate that the antiviral activity of MxA extends to CVB4 and that its replication cycle is inhibited at an early step in Vero/MxA cells.     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

Respiratory syncytial virus strain A2 is resistant to the antiviral effects of type I interferons and human MxA.

Respiratory syncytial virus (RSV) belongs to Paramyxoviridae family of enveloped negative-strand RNA viruses and causes severe bronchiolitis and pneumonia in children younger than 2 years of age. As members of Paramyxoviridae family, RSV and parainfluenza type 3 (PIV3) have similar modes of infection and replication. A variety of negative-strand RNA virus infections, including that of PIV3, are inhibited by human MxA protein, a type I interferon (IFN)-inducible GTPase. We tested whether the MxA protein, induced either by type I human IFNs or by stable transfection of human MxA gene in human (U-87) or simian (Vero) cells, confers resistance to these cells against infection by RSV strain A2. RSV infection was resistant to antiviral effects induced by 0-10,000 U/ml type I IFNs (IFN-alpha or -beta) in both human lung epithelial, A549, and fibroblast, MRC-5 cells. RSV virus yield was reduced only by 10- to 20-fold, and viral protein synthesis was not significantly affected under conditions of IFN treatment where PIV3 yield was reduced by 1000- to 10,000-fold. Human or simian cell lines constitutively expressing MxA were protected against infection by PIV3 but not by RSV. Our results indicate that RSV A2 is resistant to the antiviral effects of MxA, even though RSV and PIV3 have similar replication strategies. In IFN-treated coinfected cultures, IFN-resistant RSV A2 did not prevent the IFN-mediated inhibition of PIV3 multiplication. Hence the resistance of RSV A2 to type I IFNs does not appear to be due to soluble factors released into the medium or a disruption in the cellular antiviral machinery brought about by RSV A2 infection.     

Mx genes show weaker primary response to virus than other interferon-regulated genes.

Some interferon (IFN)-regulated genes are induced as a primary response to virus as well as secondarily through virus-induced IFN. Here we investigated whether this dual control mechanism would also regulate the activity of the human and mouse Mx genes that encode proteins with intrinsic antiviral potentials. To distinguish between a primary response to virus and a secondary response to virus-induced IFN, we studied virus-induced Mx gene expression in cell lines that lack a functional IFN system and in cells with blocked protein synthesis. In contrast to the two IFN-regulated human genes ISG56 and ISG15, the human MxA gene showed almost no primary response to Newcastle disease virus (NDV) or influenza virus. Similarly, direct activation of the mouse Mx1 gene by NDV or influenza virus was not significant in mouse embryo cells or explanted peritoneal macrophages. A moderate primary Mx1 response to NDV was observed in the permanent cell line L1210. Lack of a strong IFN-independent Mx response to virus indicates that this mode of gene regulation does not play a significant role in Mx-mediated resistance to viral disease.     

Myxovirus resistance protein A for discriminating between viral and bacterial lower respiratory tract infections in children – The TREND study

ObjectiveDiscriminating between viral and bacterial lower respiratory tract infection (LRTI) in children is challenging, leading to an excessive use of antibiotics. Myxovirus resistance protein A (MxA) is a promising biomarker for viral infections. The primary aim of the study was to assess differences in blood MxA levels between children with viral and bacterial LRTI. Secondary aims were to assess differences in blood MxA levels between children with viral LRTI and asymptomatic controls and to assess MxA levels in relation to different respiratory viruses.MethodsChildren with LRTI were enrolled as cases at Sachs' Children and Youth Hospital, Stockholm, Sweden. Nasopharyngeal aspirates and blood samples for analysis of viral PCR, MxA, and C-reactive protein were systematically collected from all study subjects in addition to standard laboratory/radiology assessment. Aetiology was defined according to an algorithm based on laboratory and radiological findings. Asymptomatic children with minor surgical disease were enrolled as controls.ResultsMxA levels were higher in children with viral LRTI (n = 242) as compared to both bacterial (n = 5) LRTI (p <0.01, area under the curve (AUC) 0.90, 95% CI: 0.81 to 0.99), and controls (AUC 0.92, 95% CI: 0.88 to 0.95). In the subgroup of children with pneumonia diagnosis, a cutoff of MxA 430 μg/l discriminated between viral (n = 29) and bacterial (n = 4) aetiology with 93% (95% CI: 78–99%) sensitivity and 100% (95% CI: 51–100%) specificity (AUC 0.98, 95% CI: 0.94 to 1.00). The highest MxA levels were seen in cases PCR positive for influenza (median MxA 1699 μg/l, interquartile range: 732 to 2996) and respiratory syncytial virus (median MxA 1115 μg/l, interquartile range: 679 to 2489).DiscussionMxA accurately discriminated between viral and bacterial aetiology in children with LRTI, particularly in the group of children with pneumonia diagnosis, but the number of children with bacterial LRTI was low.     

Reproductive activity of the influenza A virus in the splenocytes of experimental animals with mono- and mixed infections

Virus-induced processes in organs and tissues of Syrian hamsters in relation to the influenza A virus strain used (HON1 or H3N2), age of the animals, and in the presence of mixed infection were compared. The infection of young hamsters with A/PR8/34 and A/Bangkok/1/79 viruses was shown to induce the synthesis of viral proteins NP and M in spleen cells lasting for up to 15 days (the observation period). In mixed influenza and respiratory syncytial virus infection the possibility of influenza virus genome expression did not change. After infection of mature hamsters, synthesis of virus-specific NP and M proteins in splenocytes was observed only in the animals infected with influenza A/PR8/34 virus but not in those infected with the less pathogenic influenza A/Bangkok/1/79 virus.     

Increased levels of antiviral MxA protein in peripheral blood of patients with a chronic disease of unknown etiology

Interferon alpha (IFN-alpha) is synthesized in response to viral infections. MxA protein, induced specifically by IFN-alpha and beta, expressed in peripheral blood cells, is detected more consistently than circulating IFN-alpha in serum of patients with viral infections. Thus, activation of the IFN-alpha/MxA system can be used as additional marker of the presence of a virus in patients. Therefore MxA protein and IFN-alpha levels were measured in patients with multiple sclerosis (MS), a chronic neurological disease of unknown etiology, in order to investigate the possible role of viruses in the expression of this disease. The means of MxA values obtained by using an immunochemiluminescent assay were significantly higher in blood of patients with remitting (n = 197) or relapsing (n = 39) multiple sclerosis (MS) patients and in patients with viral infections than in blood from healthy controls (n = 25) and from patients with bacterial infections (n = 12). Intra-individual variance in MxA levels in seven clinically stable remitting patients with MS was observed in the course of a follow-up, and high MxA levels were detected in three of them in blood samples collected consecutively over several months. By using an ultra sensitive assay, a higher MxA-inducer activity was obtained with sera from MS patients (n = 39) than with those from healthy controls (n = 12). Experiments with neutralizing antibodies proved that this activity in serum from patients was due to IFN-alpha, whereas IFN-alpha could not be detected by other methods. Altogether these results demonstrate that there is an activation of the IFN-alpha/MxA system in MS patients, which is consistent with the hypothesis that a viral infection may be associated with MS.