Molecular Determinants for Nuclear Import of Influenza A PB2 by Importin α Isoforms 3 and 7

Abstract

SUMMARY Influenza A virus polymerase subunit PB2 is a major virulence determinant implicated in pathogenicity and host adaptation. During cross-species virus transfer from avian to mammalian cells, PB2 switches specificity from importin α3 to α7. This specificity is not recapitulated in vitro, where PB2 binds all importin α isoforms with comparably high affinity. In this paper, we investigated the structure, conformational dynamics and autoinhibition of importin α isoforms 1, 3 and 7 in complex with PB2. Our data suggest that association of PB2 with α3 and α7 is favored by reduced autoinhibition of these isoforms and by the unique structure of PB2’s NLS-domain. We propose that by recruiting importin α3 or α7 in the absence of importin β, PB2 reduces the complexity of adaptor-mediated import to a pseudo-bimolecular reaction, thereby acquiring a kinetic advantage over classical NLS-cargos that form an import complex only when importin α and β are simultaneously available.

Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits

“…Biochemical Techniques-The gene encoding mouse importin ␣1 lacking the IBB domain was cloned in pET30a vector (Novagen) as previously described (43,47). ⌬IBB-importin ␣1 was expressed in Escherichia coli strain BL21 (DE3) by induction at 30°C for 3 h with 1 mM isopropyl 1-thio-␤-D-galactopyranoside and purified on nickel-agarose resin as described (43,47).…”

“…⌬IBB-importin ␣1 was expressed in Escherichia coli strain BL21 (DE3) by induction at 30°C for 3 h with 1 mM isopropyl 1-thio-␤-D-galactopyranoside and purified on nickel-agarose resin as described (43,47). ⌬IBB-Importin ␣1 was further purified over a Superdex 200 column (GE Healthcare) equilibrated in G.F. buffer (20 mM Tris pH 8.0, 150 mM NaCl, 5 mM ␤-mercaptoethanol, and 0.2 mM PMSF).…”

“…Importin ␣ is the universal adaptor that binds NLSbearing import cargos (40). It exists in seven isoforms in humans, all structurally very similar (43), that generate a binding surface for NLSs, which can be monopartite like the SV40 T-large antigen NLS (44), or bipartite like nucleoplasmin NLS (45,46). To obtain a quantitative description of the structure, potency, and conservation of NLSs found in herpesvirus terminase subunits, in this paper we carried out a structural and bioinformatic analysis of HSV-1 pUL15 and HCMV pUL56 NLS sequences.…”

Structure of the Tuberous Sclerosis Complex 2 (TSC2) N Terminus Provides Insight into Complex Assembly and Tuberous Sclerosis Pathogenesis

“…A search with the Dali server (33) revealed the homology to other HEAT repeat proteins (Fig. 2e), including importin ␣3 (z score 18.0, rmsd 10 Å), a karyopherin involved in transport through the nuclear pore complex (34) and the core domain of TSC1 (z score 11.1, rmsd 7.5 Å) (23), and the armadillo repeat protein HspBP1 (z score 14.2, rmsd 3.0 Å) (35).…”

Influenza virus infection, interferon response, viral counter-response and apoptosis

“…Interferons (IFNs), thereafter, mediate the expression of interferon stimulated genes (ISGs). When secreted extracellularly, IFNs can also stimulate transcription of ISGs in neighboring non-infected cells to protect these cells from potential viral invasion [23][24][25]. ISGs encode various proteins with antiviral properties: ribonucleases (OASL, OAS1, ISG20) to degrade viral RNA, E3-ligases and ubiquitin-like molecules (HERC5, Trim25, and ISG15) to destroy viral proteins, metabolic enzymes (COX2, IDO and 25HC) to catalyze the production of immuno-and neuro-modulators (prostaglandin H2, kynurenine and oxysterol 25-hydroxycholesterol), as well as cytokine-processing factors (GBP1, GBP4, GBP5, MX1 and MX2) and cytokines (IL1B, IL8, IL6, CXCL10, CCL5) to activate and recruit immune cells to the site of infection [26][27][28][29][30][31].…”