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1.
Mx proteins are a family of large GTPases that are induced exclusively by interferon-α/β and have a broad antiviral activity against several viruses, including influenza A virus (IAV). Although the antiviral activities of mouse Mx1 and human MxA have been studied extensively, the molecular mechanism of action remains largely unsolved. Because no direct interaction between Mx proteins and IAV proteins or RNA had been demonstrated so far, we addressed the question of whether Mx protein would interact with cellular proteins required for efficient replication of IAV. Immunoprecipitation of MxA revealed its association with two closely related RNA helicases, UAP56 and URH49. UAP56 and its paralog URH49 play an important role in IAV replication and are involved in nuclear export of IAV mRNAs and prevention of dsRNA accumulation in infected cells. In vitro binding assays with purified recombinant proteins revealed that MxA formed a direct complex with the RNA helicases. In addition, recombinant mouse Mx1 was also able to bind to UAP56 or URH49. Furthermore, the complex formation between cytoplasmic MxA and UAP56 or URH49 occurred in the perinuclear region, whereas nuclear Mx1 interacted with UAP56 or URH49 in distinct dots in the nucleus. Taken together, our data reveal that Mx proteins exerting antiviral activity can directly bind to the two cellular DExD/H box RNA helicases UAP56 and URH49. Moreover, the observed subcellular localization of the Mx-RNA helicase complexes coincides with the subcellular localization, where human MxA and mouse Mx1 proteins act antivirally. On the basis of these data, we propose that Mx proteins exert their antiviral activity against IAV by interfering with the function of the RNA helicases UAP56 and URH49.  相似文献   

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Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Nairovirus within the family Bunyaviridae and is the causative agent of severe hemorrhagic fever. Despite increasing knowledge about hemorrhagic fever viruses, the factors determining their pathogenicity are still poorly understood. The interferon-induced MxA protein has been shown to have an inhibitory effect on several members of the Bunyaviridae family, but the effect of MxA against CCHFV has not previously been studied. Here, we report that human MxA has antiviral activity against CCHFV. The yield of progeny virus in cells constitutively expressing MxA was reduced up to 1,000-fold compared with control cells, and accumulation of viral genomes was blocked. Confocal microscopy revealed that MxA colocalizes with the nucleocapsid protein (NP) of CCHFV in the perinuclear regions of infected cells. Furthermore, we found that MxA interacted with NP by using a coimmunoprecipitation assay. We also found that an amino acid substitution (E645R) within the C-terminal domain of MxA resulted in a loss of MxA antiviral activity and, concomitantly, in the capacity to interact with CCHFV NP. These results suggest that MxA, by interacting with a component of the nucleocapsid, prevents replication of CCHFV viral RNA and thereby inhibits the production of new infectious virus particles.  相似文献   

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Apoptosis induction is an antiviral host response, however, influenza A virus (IAV) infection promotes host cell death. The nucleoprotein (NP) of IAV is known to contribute to viral pathogenesis, but its role in virus-induced host cell death was hitherto unknown. We observed that NP contributes to IAV infection induced cell death and heterologous expression of NP alone can induce apoptosis in human airway epithelial cells. The apoptotic effect of IAV NP was significant when compared with other known proapoptotic proteins of IAV. The cell death induced by IAV NP was executed through the intrinsic apoptosis pathway. We screened host cellular factors for those that may be targeted by NP for inducing apoptosis and identified human antiapoptotic protein Clusterin (CLU) as a novel interacting partner. The interaction between IAV NP and CLU was highly conserved and mediated through β-chain of the CLU protein. Also CLU was found to interact specifically with IAV NP and not with any other known apoptosis modulatory protein of IAV. CLU prevents induction of the intrinsic apoptosis pathway by binding to Bax and inhibiting its movement into the mitochondria. We found that the expression of IAV NP reduced the association between CLU and Bax in mammalian cells. Further, we observed that CLU overexpression attenuated NP-induced cell death and had a negative effect on IAV replication. Collectively, these findings indicate a new function for IAV NP in inducing host cell death and suggest a role for the host antiapoptotic protein CLU in this process.  相似文献   

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The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored.  相似文献   

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The interferon-induced human MxA protein belongs to the class of dynamin-like, large guanosine-5'-triphosphatases that are involved in intracellular vesicle trafficking and organelle homeostasis. MxA shares many properties with the other members of this protein superfamily, including the propensity to self-assemble and to associate with lipid membranes. However, MxA is unique in that it has antiviral activity and inhibits the replication of several RNA viruses. Here, we determined the role of membranes for the antiviral function of MxA using LaCrosse-bunyavirus (LACV). We show that MxA does not affect trafficking and sorting of viral glycoproteins but binds and mislocates the viral nucleocapsid (N) protein into membrane-associated, large perinuclear complexes. We further demonstrate that MxA localizes to a subcompartment of the smooth endoplasmic reticulum where the viral N protein accumulates. In infected MxA-expressing cells, oligomeric MxA/N complexes are formed in close association with COP-I-positive vesicular-tubular membranes. Our results suggest that this membrane compartment is the preferred place where MxA and N interact, leading to efficient sequestration and missorting of an essential viral component.  相似文献   

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Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.  相似文献   

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HIV-1 integrase (IN) catalyses integration of a DNA copy of the viral genome into the host genome. Specific interactions between retroviral IN and long terminal repeats (LTR) are required for this insertion. To characterize quantitatively the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, we used the small-angle X-ray scattering (SAXS) technique. Under certain conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity in the 3′-processing reaction. Under these conditions, tetramers were scarce. Non-specific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and non-specific ODNs had varying radii of gyration (Rg), suggesting that the specific sequence-dependent formation of IN tetramers can probably occur by dimerization of two dimers of different structure. From our data we can conclude that the DNA-induced oligomerization of HIV-1 IN is probably of importance to provide substrate specificity and to increase the enzyme activity.  相似文献   

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HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.  相似文献   

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Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication.  相似文献   

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Infections with emerging and re-emerging arboviruses are of increasing concern for global health. Tick-transmitted RNA viruses of the genus Thogotovirus in the Orthomyxoviridae family have considerable zoonotic potential, as indicated by the recent emergence of Bourbon virus in the USA. To successfully infect humans, arboviruses have to escape the restrictive power of the interferon defense system. This is exemplified by the high sensitivity of thogotoviruses to the antiviral action of the interferon-induced myxovirus resistance protein A (MxA) that inhibits the polymerase activity of incoming viral ribonucleoprotein complexes. Acquiring resistance to human MxA would be expected to enhance the zoonotic potential of these pathogens. Therefore, we screened a panel of 10 different thogotovirus isolates obtained from various parts of the world for their sensitivity to MxA. A single isolate from Nigeria, Jos virus, showed resistance to the antiviral action of MxA in cell culture and in MxA-transgenic mice, whereas the prototypic Sicilian isolate SiAr126 was fully MxA-sensitive. Further analysis identified two amino acid substitutions (G327R and R328V) in the viral nucleoprotein as determinants for MxA resistance. Importantly, when introduced into SiAr126, the R328V mutation resulted in complete MxA escape of the recombinant virus, without causing any viral fitness loss. The escape mutation abolished viral nucleoprotein recognition by MxA and allowed unhindered viral growth in MxA-expressing cells and in MxA-transgenic mice. These findings demonstrate that thogotoviruses can overcome the species barrier by escaping MxA restriction and reveal that these tick-transmitted viruses may have a greater zoonotic potential than previously suspected.  相似文献   

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