CHD3 facilitates vRNP nuclear export by interacting with NES1 of influenza A virus NS2.
Ontology highlight
ABSTRACT:
SUBMITTER: Hu Y
PROVIDER: S-EPMC4323543 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Emerging influenza D viruses (IDVs), the newest member in the genus Orthomyxovirus family, which can infect and transmit in multiple mammalian species as its relatives the influenza A viruses (IAVs). Additional studies of biological characteristics of IDVs are needed; here, we studied the characteristics of IDV nonstructural protein 2 (NS2), which shares the lowest homology to known influenza proteins. First, we generated reassortant viruses via reverse genetics to analyze the segment compatibility and gene interchangeability between IAVs and IDVs. Next, we investigated the locations and exact sequences of nuclear export signals (NESs) of the IDV NS2 protein. Surprisingly, three separate NES regions were found to contribute to the nuclear export of an eGFP fusion protein. Alanine scanning mutagenesis identified critical amino acid residues within each NES, and co-immunoprecipitation experiments demonstrated that their nuclear export activities depend on the CRM1-mediated pathway, particularly for the third NES (136-146aa) of IDV NS2. Interestingly, the third NES was important for the interaction of NS2 protein with CRM1. The findings in this study contribute to the understanding of IDV NS2 protein's role during nucleocytoplasmic transport of influenza viral ribonucleoprotein complexes (vRNPs) and will also facilitate the development of novel anti-influenza drugs targeting nuclear export signals of IDV NS2 protein.
| S-EPMC7600798 | biostudies-literature
Project description:Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.
| S-EPMC3554077 | biostudies-literature
Project description:During influenza virus infection, viral ribonucleoproteins (vRNPs) are replicated in the nucleus and must be exported to the cytoplasm before assembling into mature viral particles. Nuclear export is mediated by the cellular protein Crm1 and putatively by the viral protein NEP/NS2. Proteolytic cleavage of NEP defines an N-terminal domain which mediates RanGTP-dependent binding to Crm1 and a C-terminal domain which binds to the viral matrix protein M1. The 2.6 A crystal structure of the C-terminal domain reveals an amphipathic helical hairpin which dimerizes as a four-helix bundle. The NEP-M1 interaction involves two critical epitopes: an exposed tryptophan (Trp78) surrounded by a cluster of glutamate residues on NEP, and the basic nuclear localization signal (NLS) of M1. Implications for vRNP export are discussed.
| S-EPMC212717 | biostudies-literature
Project description:The nonstructural NS2 proteins of autonomous parvoviruses are known to act in a host cell-dependent manner and to play a role in viral DNA replication, efficient translation of viral mRNA, and/or encapsidation. Their exact function during the parvovirus life cycle remains, however, still obscure. We report here the characterization of the interaction with the NS2 proteins from the parvovirus minute virus of mice (MVM) and rat as well as mouse homologues of the human CRM1 protein, a member of the importin-beta family recently identified as an essential nuclear export factor. Using the two-hybrid system, we could detect the interaction between the carboxy-terminal region of rat CRM1 and each of the three isoforms of NS2 (P [or major], Y [or minor], and L [or rare]). NS2 proteins were further shown to interact with the full-length CRM1 by coimmunoprecipitation experiments using extracts from both mouse and rat cell lines. Our data show that CRM1 preferentially binds to the nonphosphorylated isoforms of NS2. Moreover, we observed that the treatment of MVM-infected cells with leptomycin B, a drug that specifically inhibits the CRM1-dependent nuclear export pathway, leads to a drastic accumulation of NS2 proteins in the nucleus. Both NS2 interaction with CRM1 and nuclear accumulation upon leptomycin B treatment strongly suggest that these nonstructural viral proteins are actively exported out of the nuclei of infected cells via a CRM1-mediated nuclear export pathway.
| S-EPMC104304 | biostudies-literature
Project description:Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.
| S-EPMC7117665 | biostudies-literature
Project description:Influenza virus is a major human and animal pathogen causing seasonal epidemics and occasional pandemics in the human population that are associated with significant morbidity and mortality. Influenza A virus, a member of the orthomyxovirus family, contains an RNA genome with a coding capacity for a limited number of proteins. In addition to ensuring the structural integrity of virions, these viral proteins facilitate the replication of virus in the host cell. Consequently, viral proteins often evolve to perform multiple functions, the influenza A virus nuclear export protein (NEP) (also referred to as non-structural protein 2, or NS2) being an emerging example. NEP was originally implicated in mediating the nuclear export of viral ribonucleoprotein (RNP) complexes, which are synthesized in the infected cell nucleus and are assembled into progeny virions at the cell membrane. However, since then, new and unexpected roles for NEP during the influenza virus life cycle have started to emerge. These recent studies have shown NEP to be involved in regulating the accumulation of viral genomic vRNA and antigenomic cRNA as well as viral mRNA synthesized by the viral RNA-dependent RNA polymerase. Subsequently, this regulation of viral RNA transcription and replication by NEP was shown to be an important factor in the adaptation of highly pathogenic avian H5N1 influenza viruses to the mammalian host. Unexpectedly, NEP has also been implicated in recruiting a cellular ATPase to the cell membrane to aid the efficient release of budding virions. Accordingly, NEP is proposed to play multiple biologically important roles during the influenza virus life cycle.
| S-EPMC3516560 | biostudies-literature
Project description:The NS1 protein of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 functions to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression, but its mechanisms of action remain to be fully elucidated. We show here that NS1 forms an inhibitory complex with NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5, which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex. Increased levels of NXF1, p15, or Rae1 revert the mRNA export blockage induced by NS1. Furthermore, influenza virus down-regulates Nup98, a nucleoporin that is a docking site for mRNA export factors. Reduced expression of these mRNA export factors renders cells highly permissive to influenza virus replication, demonstrating that proper levels of key constituents of the mRNA export machinery protect against influenza virus replication. Because Nup98 and Rae1 are induced by interferons, down-regulation of this pathway is likely a viral strategy to promote viral replication. These findings demonstrate previously undescribed influenza-mediated viral-host interactions and provide insights into potential molecular therapies that may interfere with influenza infection.
| S-EPMC1794296 | biostudies-literature
Project description:Influenza viruses antagonize key immune defence mechanisms via the virulence factor non-structural protein 1 (NS1). A key mechanism of virulence by NS1 is blocking nuclear export of host messenger RNAs, including those encoding immune factors1-3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor complex, nuclear RNA export factor 1-nuclear transport factor 2-related export protein 1 (NXF1-NXT1), which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1-NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1-NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1-NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. In sum, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.
| S-EPMC6754785 | biostudies-literature
Project description:The process of host factor-mediated nucleocytoplasmic transport is critical for diverse cellular events in eukaryotes and the life cycle of viruses. We have previously identified a chromosome region maintenance 1-independent nuclear export signal (NES) at the C terminus of the large form of hepatitis delta antigen (HDAg), designated NES(HDAg-L) that is required for the assembly of hepatitis delta virus (HDV) (C.-H. Lee et al., J. Biol. Chem. 276:8142-8148, 2001). To look for interacting proteins of the NES(HDAg-L), yeast two-hybrid screening was applied using the GAL4-binding domain fused to the NES(HDAg-L) as bait. Among the positive clones, one encodes a protein, designated NESI [NES(HDAg-L) interacting protein] that specifically interacted with the wild-type NES(HDAg-L) but not with the export/package-defective HDAg-L mutant, NES*(HDAg-L), in which Pro-205 has been replaced by Ala. Northern blot analysis revealed NESI as the gene product of a 1.9-kb endogenous mRNA transcript that is present predominantly in human liver tissue. NESI consists of 467 amino acid residues and bears a putative actin-binding site and a bipartite nuclear localization signal. Specific interaction between HDAg-L and NESI was further confirmed by coimmunoprecipitation and immunofluorescence staining. Overexpression of antisense NESI RNAs inhibited the expression of NESI and abolished HDAg-L-mediated nuclear export and assembly of HDV genomic RNA. These data indicate a critical role of NESI in the assembly of HDV through interaction with HDAg-L.
| S-EPMC1143724 | biostudies-literature
Project description:Influenza virus replication relies on the functions of host factors. In our previous study, we identified host factors involved in virus replication and began analyses of their roles in this process. In this study, we focused on Nucleoporin 93 (NUP93) and revealed its importance in influenza virus replication. NUP93 knockdown mediated by siRNAs reduced viral replication and decreased the efficiency of the early step of the viral life cycle. NUP93 did not appear to be important for virus binding, internalization, or the nuclear import of viral ribonucleoprotein (vRNP); however, in NUP93-depleted cells, viral RNA accumulated in the nucleus. These results suggest that NUP93 is involved in the nuclear export of viral RNA.
| S-EPMC6066526 | biostudies-literature