Comparative Analysis of Seven Viral Nuclear Export Signals (NESs) Reveals the Crucial Role of Nuclear Export Mediated by the Third NES Consensus Sequence of Nucleoprotein (NP) in Influenza A Virus Replication

Abstract

The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral replication.

NXT1, a Novel Influenza A NP Binding Protein, Promotes the Nuclear Export of NP via a CRM1-Dependent Pathway

“…This mechanism may explain our data showing that NXT1 promotes nuclear export of the influenza NP protein. We showed direct binding of NP/CRM1 [14] and NXT1/NP (Figure 1, Figure 6, and Figure 7), while other studies have reported binding of NXT1/CRM1 [39]. Our results also show that binding of CRM1 and NP to the NXT1 was not competitive, and that NXT1 promoted nuclear export of NP only in the absence of LMB.…”

“…Infectious influenza A/WSN/33 (H1N1) virus was produced by reverse genetics, as described previously [14]. FuGENE HD (Promega, Fitchburg, WI, USA) and Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) were used for plasmid transfection and Lipofectamine RNAiMAX Reagent (Invitrogen) was used for siRNA transfection throughout the study.…”

Phosphorylation Controls the Nuclear-Cytoplasmic Shuttling of Influenza A Virus Nucleoprotein

“…In our previous work, three NESs were identified as responsible for the nuclear export of NP (9). Among these NESs, NES3 is important for virus growth, because mutation of NES3 results in a failure to rescue mutant virus (35). The position of Y296 in the NP crystal structure (36) is close to that of NES3, suggesting that phosphorylation of Y296 may affect the function of the CRM1-dependent NES3.…”

Inhibition of CRM1-mediated nuclear export of influenza A nucleoprotein and nuclear export protein as a novel target for antiviral drug development

“…The nuclear export function of the NP-NES3 domain is CRM1dependent , and NP directly binds CRM1 (Chutiwitoonchai et al, 2014;Elton et al, 2001;Kakisaka et al, 2016). To verify that DP2392-E10 inhibits nuclear export by blocking the NP/CRM1 (and NEP/CRM1) interaction, we performed in vitro pull-downs of NP and CRM1 proteins in the presence of the compound.…”

“…In addition, CRM1 overexpression promotes the nuclear export of NP, but not NEP or M1 (Elton et al, 2001), and the interaction of NP with viral genomic RNA, and M1 is sufficient for nuclear export of vRNP (Huang et al, 2001). Moreover, NP directly binds to CRM1 (Chutiwitoonchai et al, 2014;Elton et al, 2001;Kakisaka et al, 2015), and the interaction between NP/CRM1 and a recent identified host factor, nuclear transport factor 2-like export protein 1 (NXT1), promotes nuclear export of NP and vRNA (Chutiwitoonchai and Aida, 2016). NP must also interact with cellular nucleolin, a major component of the nucleolar compartment, to facilitate the interaction between vRNP and the cellular nuclear export machinery (Terrier et al, 2016).…”