Nuclear Cotransport Mechanism of Cytoplasmic Human MxB Protein

Mx proteins are large GTPases, which play a pivotal role in the interferon type I-mediated response against viral infections. The human MxA inhibits the replication of several RNA viruses and is organized in oligomeric structures. Using two different experimental approaches, the mammalian two-hybrid system and an interaction dependent nuclear translocation approach, three domains in the carboxyl-terminal moiety were identified that are involved in the oligomerization of MxA. The first consists of a carboxyl-terminal amphipathic helix (LZ1), which binds to a more proximal part of the same molecule. This intramolecular backfolding is a prerequisite for the formation of an intermolecular complex. This intermolecular interaction is mediated by two domains, a poorly defined region generated by the intramolecular interaction and a domain located between amino acids 363 and 415. Co-expression of wild-type MxA with various mutant fragments thereof revealed that the presence of the carboxyl-terminal region comprising the amphipathic helices LZ1 and LZ2 is necessary and sufficient to exert a dominant negative effect. This finding suggests that the functional interference of the carboxyl-terminal region is due to competition for binding of an as yet unidentified cellular or viral target molecules.

mentioning

confidence: 88%

Intramolecular Backfolding of the Carboxyl-terminal End of MxA Protein Is a Prerequisite for Its Oligomerization

Paolo

Hefti

Meli

et al. 1999

“…5, A and B). As a negative control, we co-transfected plasmids coding for human GFP(1-157)-MxA and human GFP(158 -238)-MxB, which are not able to form hetero-oligomers (42). In addition, coexpression of the N-and C-terminal regions of the GFP protein alone yielded no fluorescence signal (Fig.…”

Section: Subcellular Localization Of the Complex Formation Between MXmentioning

confidence: 99%

Interferon-induced Antiviral Protein MxA Interacts with the Cellular RNA Helicases UAP56 and URH49

Wisskirchen

Ludersdorfer

Müller

et al. 2011

Mx proteins are a family of large GTPases that are induced exclusively by interferon-␣/␤ 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.Mx proteins belong to a family of dynamin-like large GTPases. They play a pivotal role in the type I interferon-mediated response against a broad range of viral infections (1). The human MxA protein accumulates in the cytoplasm and has been shown to inhibit several RNA and DNA viruses, including influenza A virus (IAV), 2 vesicular stomatitis virus, Thogoto virus (THOV), and La Crosse virus (1-3). The murine Mx1 protein is a nuclear protein and inhibits the replication of several members of the orthomyxovirus family, including IAV (1).Mx proteins share a high intrinsic GTPase activity and the ability to form large oligomeric structures (4). Recently, the crystal structure of the stalk region of MxA was solved, and, in combination with similar structural data from dynamin, a model for a four-helical bundle formation of MxA was proposed (5). It is not clear whether Mx proteins exert their antiviral activity in the form of large oligomeric structures or monomers. Mutations preventing the intermolecular Mx-Mx interactions abrogate the antiviral activity (5), whereas a monomeric form of MxA with a mutation abolishing the intramolecular backfolding of the C-terminal end remains active (6 -8).Although the antiviral function of MxA has been studied extensively, little is known about the molecula...

“…26, GenBank TM accession number NM_002264) was PCR-modified with oligonucleotides AAAAAAGGATCCACCATGACCACCCCAGGA-AAAGAGAACTTT (5Ј oligonucleotide) TTTTTTGGATCCTCAAAGCT-GGAAACCTTCCATAGGAGC (3Ј oligonucleotide) to create BamHI cloning sites (in bold) on both sides of the gene coding region. After BamHI digestion the insert was cloned into FLAG-tagged pCDNA 3.1(ϩ) vector (25). All DNA manipulations were performed according to standard protocols, and the newly created gene constructs were partially sequenced.…”

Section: Cells-monolayers and Suspension Cultures Of Spodoptera Frugimentioning

confidence: 99%

Arginine/Lysine-rich Nuclear Localization Signals Mediate Interactions between Dimeric STATs and Importin α5

Fagerlund¹,

Melén²,

Kinnunen³

et al. 2002

Self Cite

168

153

Interferon stimulation results in tyrosine phosphorylation, dimerization, and nuclear import of STATs (signal transducers and activators of transcription). Proteins to be targeted into the nucleus usually contain nuclear localization signals (NLSs), which interact with importin ␣. Importin ␣ binds to importin ␤, which docks the protein complex to nuclear pores, and the complex translocates into the nucleus. Here we show that baculovirus-produced and -activated STAT1 homodimers and STAT1-STAT2 heterodimers directly interacted with importin ␣5 (NPI-1). This interaction was very stable and was dependent on lysines 410 and 413 of STAT1. Only STAT dimers that had two intact NLS elements, one in each monomer, were able to bind to importin ␣5. heterodimers. In the nucleus STATs interact with IRF-9/p48 protein to form ISGF3 complexes, which bind to well conserved interferon-stimulated response elements in the promoter regions of IFN-␣/␤-responsive genes and activate transcription (5-8). Binding of type II IFN (IFN-␥) to its receptor leads to the activation of JAK1 and JAK2 and tyrosine phosphorylation of STAT1 (also at Tyr-701). Activated STAT1 forms homodimers, which translocate into the nucleus and bind to GAS elements and activate transcription of IFN-␥-inducible genes (1, 2). Although the structure-function relationships of STATs have been carefully analyzed, the mechanisms of nuclear import of this important group of transcription factors have remained less well characterized. Recently we and others have shown that STAT1 and STAT2 have an arginine/lysine-rich nuclear localization signal (NLS) that mediates their nuclear translocation in dimeric complexes (9, 10). STAT-importinActive nuclear transport of large macromolecules occurs via the nuclear pore complex (11). Proteins to be imported into the nucleus usually contain a mono-or bipartite basic-type NLS, which binds to a specific NLS receptor, importin ␣ (12-14). The N-terminal importin ␤ binding (IBB) domain of importin ␣ interacts with importin ␤ (15), which mediates the docking of NLS-containing cargo-importin ␣/␤ complex to the cytoplasmic side of the nuclear pore, and the complex translocates into the nucleus (16,17). Inside the nucleus RanGTPase is involved in the disassembly of the cargo-importin complex (14,18,19). IFN-␥-induced nuclear import of STAT1 has been suggested to be dependent on one importin ␣ subtype, importin ␣5 (20), and the RanGTPase (21). However, the elements that regulate STAT-importin ␣5 interactions have remained elusive.In the present work we show, by using a baculovirus-reconstituted STAT activation system, that homodimeric STAT1 or heterodimeric STAT1-STAT2 complexes directly interact with importin ␣5. The interaction of STAT dimers with importin ␣ is very stable and is dependent on NLS situated in the DNA binding domain of STATs. The STAT-importin ␣5 complex consists of two importin ␣ and two STAT molecules. STAT-binding GAS oligonucleotides efficiently prevent the binding of dimeric STATs with importin ␣. We also demonstrate by co...