Influenza virus targets the mRNA export machinery and the nuclear pore complex

Abstract

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.

Fig. 1.

Influenza virus inhibits poly(A) RNA nuclear export. (a) MDCK cells were mock infected or infected with A/WS/33 influenza virus at an MOI of 1 for 6 and 24 h. Immunofluorescence using antibodies against influenza proteins (green) and oligo(dT) in situ hybridization (red) was performed. (b) Expression of influenza proteins in MDCKs. Cell extracts from MDCK cells infected with A/WS/33 at an MOI 1 for the indicated time points were subjected to immunoblot analysis with anti-influenza protein antibodies.

Fig. 2.

The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. (a) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. (b and c) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. (d) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a. (e) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a. (f and g) Expression levels of Nups and mRNA export factors in 293T (f) and MDCK (g) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. (h) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods.

Fig. 3.

The mRNA export inhibition induced by NS1 is reverted by increased levels of mRNA export factors. (a and b) Luciferase reporter gene expression assays were performed with 293T cells by cotransfection of reporter plasmids and plasmids encoding NXF1, p15, Rae1, Nup98, and Nup96, as indicated. (c) HeLa cells were transfected with a plasmid encoding myc-NS1 alone or cotransfected with plasmids encoding myc-NS1, GFP-NXF1, and GFP-p15. Cells were subjected to immunofluorescence with anti-myc antibody (red) followed by oligo(dT) in situ hybridization (blue). Green shows GFP-NXF1 and GFP-p15.

Fig. 4.

Low levels of Rae1 and Nup98 induce higher susceptibility to influenza virus-mediated cell death and an increase in viral replication. (a and b) Rae1^+/+ Nup98^+/+, Rae1^+/− Nup98^+/+, Rae1^+/+ Nup98^+/−, and Rae1^+/−Nup98^+/− MEFs were infected with A/WS/33 influenza virus, and cell viability was determined by comparing and quantifying bright-field microscopy (gray), DAPI (blue), and exclusion of 2 mM ethidium homodimer-1 (red). (c) The number of influenza viral particles was measured in the supernatants of the cells in a by using the hemaglutinin assay.

Fig. 5.

Selective impairment of mRNA nuclear export in Rae1 and Nup98 mutant cells. Nuclear (N) and cytoplasmic (C) RNA was isolated from Rae1^+/+ Nup98^+/+, Rae1^+/+ Nup98^+/−, Rae1^+/− Nup98^+/+, and Rae1^+/− Nup98^+/− MEFs. Relative mRNA levels were quantified by real-time RT-PCR using gene-specific primers and normalized to a set of five housekeeping genes.