Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2)

Abstract

contributed equally to this work During in¯uenza virus infection, viral ribonucleo proteins (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 de®nes an N-terminal domain which mediates RanGTP-dependent binding to Crm1 and a Cterminal domain which binds to the viral matrix protein M1. The 2.6 A Ê 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.

NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome

“…3a and b) is suggestive of a possible role for NP. The conserved chemical nature of the amino acid residues between influenza type A and B NS2/NEP proteins suggests a common tertiary structure (Akarsu et al, 2003), which supports the hypothesis that both type A and B NS2/ NEP proteins are mediating the regulation of transcription and replication in a similar way.…”

“…Mutation of a tryptophan at residue 78 in the C terminus of the NS2/NEP protein, previously demonstrated to be critical for binding to M1 (Akarsu et al, 2003), did not affect the ability of NS2/NEP to regulate viral RNA levels (data not shown). In addition, fractionation experiments showed that the nuclear and cytoplasmic localization of viral RNA species was not altered by NS2/NEP and leptomycin B, an inhibitor of Crm1, did not alter the effect of NS2/NEP (data not shown).…”

“…Cell lysates were analysed by SDS-PAGE followed by Western blotting. The antibodies used were anti-NS2 (Akarsu et al, 2003) and anti-rabbit horseradish peroxidase-conjugated IgG IP: 13.66.222.141…”

“…The current model of vRNP nucleocytoplasmic export involves an export complex in which NS2/NEP has an Nterminal association with Crm1 and a C-terminal association with M1, which in turn is bound to vRNPs (Akarsu et al, 2003). We performed RNP reconstitution assays in which NS2/NEP alone or both NS2/NEP and the M1 protein were co-expressed (Fig.…”

“…The nuclear export of vRNPs is carried out by the cellular export protein Crm1 (Elton et al, 2001) and there is evidence that NS2/NEP mediates the association between the two. The current model involves an export complex in which NS2/NEP has an N-terminal association with Crm1 and a C-terminal association with the viral M1 protein, which in turn is bound to vRNPs (Akarsu et al, 2003). The NS2/NEP protein also binds several nucleoporins and is thought to be responsible for recruiting the export machinery and directing the export of the complex (O'Neill et al, 1998).…”