Interaction of the Influenza Virus Nucleoprotein with the Cellular CRM1-Mediated Nuclear Export Pathway

Abstract

Influenza virus transcription occurs in the nuclei of infected cells, where the viral genomic RNAs are complexed with a nucleoprotein (NP) to form ribonucleoprotein (RNP) structures. Prior to assembly into progeny virions, these RNPs exit the nucleus and accumulate in the cytoplasm. The mechanisms responsible for RNP export are only partially understood but have been proposed to involve the viral M1 and NS2 polypeptides. We found that the drug leptomycin B (LMB), which specifically inactivates the cellular CRM1 polypeptide, caused nuclear retention of NP in virus-infected cells, indicating a role for the CRM1 nuclear export pathway in RNP egress. However, no alteration was seen in the cellular distribution of M1 or NS2, even in the case of a mutant virus which synthesizes greatly reduced amounts of NS2. Furthermore, NP was distributed throughout the nuclei of infected cells at early times postinfection but, when retained in the nucleus at late times by LMB treatment, was redistributed to the periphery of the nucleoplasm. No such change was seen in the nuclear distribution of M1 or NS2 after drug treatment. Similar to the behavior of NP, M1 and NS2 in infected cells, LMB treatment of cells expressing each polypeptide in isolation caused nuclear retention of NP but not M1 or NS2. Conversely, overexpression of CRM1 caused increased cytoplasmic accumulation of NP but had little effect on M1 or NS2 distribution. Consistent with this, NP bound CRM1 in vitro. Overall, these data raise the possibility that RNP export is mediated by a direct interaction between NP and the cellular CRM1 export pathway.The influenza virus genome consists of eight segments of single-stranded RNA that encode a total of 10 identified polypeptides. The genomic RNA segments are of negative sense and are always found in association with viral polypeptides: the three subunits of an RNA-dependent RNA polymerase (PB1, PB2, and PA) and, in stoichiometric quantities, a single-strand RNA-binding nucleoprotein (NP) (28). In virions, these ribonucleoprotein (RNP) structures are packaged within a shell of the viral M1 polypeptide underlying the lipid bilayer, along with the hemagglutinin (HA) and neuraminidase integral membrane glycoproteins. Minor virion components include M2, a small transmembrane ion channel, and the NS2 polypeptide (28). Influenza virus particles enter the cell by receptor-mediated endocytosis. Following acidification of the endosome, the M1 polypeptide dissociates from the RNP segments and virion RNPs (vRNPs) are released into the cytoplasm (30, 31). Unusually for a virus with no DNA coding stage, influenza virus transcription occurs in the nucleus (20,22). Accordingly, after release of the RNPs into the cytoplasm, they migrate into the nucleus, in an active process that is thought to be mediated by the cellular importin ␣/␤ pathway (39). Once in the nucleus, vRNPs act as the template for synthesis of mRNAs, which are exported into the cytoplasm for translation. The vRNPs also act as the template for synthesis of full-length cRNA co...

Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors

“…If vRNPs were responsible for alterations in the recycling endosome without interference of any other viral factor, then inhibiting nuclear export of vRNPs during infection would not affect its morphology. In the presence of LMB, vRNPs were greatly retained in the nucleus (Elton et al, 2001) (Fig. 2B), compared with their cytosolic location from 8 h post infection onwards in the control (Fig.…”

“…2D and E). In previous reports, LMB addition to cells has resulted in a reduction in virus titers of 90 to 95%, indicating that sufficient vRNPs can still reach the plasma membrane in order to form virions (Elton et al, 2001;Ma et al, 2001). This incomplete blockade in vRNP export might justify some degree of alteration in the recycling endosome.…”

“…1C). To confirm that these changes were induced by vRNPs and not by other viral proteins, we quantified the increase in the size of Rab11 vesicles over the course of infection after inhibiting the nuclear export of vRNPs using leptomycin B (LMB), as previously described (Elton et al, 2001;Ma et al, 2001). If vRNPs were responsible for alterations in the recycling endosome without interference of any other viral factor, then inhibiting nuclear export of vRNPs during infection would not affect its morphology.…”

Role of Sulfatide in Influenza A Virus Replication

“…vRNP complexes are selectively exported from the nucleus to the cytosol through the cellular chromosome region maintenance 1 (CRM1)-mediated nuclear export pathway. 40) vRNP complexes in the cytosol are absolutely required for progeny virus formation as internal components of progeny viruses. Sulfatide expression by transfection of CGT and CST genes in COS-7 cells enhanced nuclear export of vRNP complexes in the cells infected with influenza A virus (A/Memphis/1/1971 H3N2), resulting in an increase of progeny virus production.…”

Early host response and immune signaling to 2009 pandemic influenza A (H1N1) viruses in primary cell culture models.

“…THE NEP/NS2 complex recognizes and binds to the export signal [188,189] on the vRNPs and is followed by binding of the viral matrix M1 protein and CRM1, thus linking the viral RNP with CRM1 [190][191][192]. Additionally, NS2 interacts with nucleoporins and serves as an adaptor between vRNPs and the nuclear pore complex [187,191]. The export pathway functions in a reverse manner to importin under the control of RAN cargo complex [130].…”