Features of Nuclear Export Signals of NS2 Protein of Influenza D Virus

Abstract

Emerging influenza D viruses (IDVs), the newest member in the genus Orthomyxovirus family, which can infect and transmit in multiple mammalian species as its relatives the influenza A viruses (IAVs). Additional studies of biological characteristics of IDVs are needed; here, we studied the characteristics of IDV nonstructural protein 2 (NS2), which shares the lowest homology to known influenza proteins. First, we generated reassortant viruses via reverse genetics to analyze the segment compatibility and gene interchangeability between IAVs and IDVs. Next, we investigated the locations and exact sequences of nuclear export signals (NESs) of the IDV NS2 protein. Surprisingly, three separate NES regions were found to contribute to the nuclear export of an eGFP fusion protein. Alanine scanning mutagenesis identified critical amino acid residues within each NES, and co-immunoprecipitation experiments demonstrated that their nuclear export activities depend on the CRM1-mediated pathway, particularly for the third NES (136-146aa) of IDV NS2. Interestingly, the third NES was important for the interaction of NS2 protein with CRM1. The findings in this study contribute to the understanding of IDV NS2 protein’s role during nucleocytoplasmic transport of influenza viral ribonucleoprotein complexes (vRNPs) and will also facilitate the development of novel anti-influenza drugs targeting nuclear export signals of IDV NS2 protein.

Construction of an Influenza D Virus with an Eight-Segmented Genome

“…Rescue experiments with NS2-deletion mutants suggested that the NOR of NS2 contains no functional domain responsible for viral infectivity, and deletion mutants with other regions in the C-terminal region could not be generated. A recent report indicated that IDV NS2 includes three nuclear export signals (NESs) at positions 66-75, 97-107, and 136-146, which may mediate the chromosome region maintenance 1 (CRM1)-dependent nuclear export pathway, similar to IAV NS2 [30]. Our rescue experiments suggest that NS2∆NOR, which retains these NESs, might play an equivalent role to intact NS2 in viral infectivity.…”

“…The NS1 and NS2 proteins possess an overlapping identical 63-amino-acid sequence at each N-terminal region and subsequent unique sequences in the downstream region of each protein, which are translated by different reading frames, even in the Cterminal overlapped gene region (Figure 1A). According to recent reports, the IDV NS1 protein antagonizes cellular type I interferon response [29] and the NS2 protein is a nuclear export protein (NEP) that mediates the nuclear export of viral RNPs [30], similar to NS proteins of the other type viruses. However, the biological functions of these proteins have not been fully elucidated at the molecular level.…”

DDX5/METTL3-METTL14/YTHDF2 Axis Regulates Replication of Influenza A Virus

“…After staining with secondary antibodies, cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min. Finally, images were acquired using a confocal microscope (Nikon, Japan) equipped with a Plan Apo objective (60×/1.40, oil immersion; Nikon, Japan) and CFI eyepiece (10×/22; Nikon) ( 58 ).…”

Host Range, Biology, and Species Specificity of Seven-Segmented Influenza Viruses—A Comparative Review on Influenza C and D

“…This limited protection as well as the presence of diverse genetic variants in co-circulation caused re-infection, and thereby a high prevalence of IDV in cattle [147]. In the case of IDV, the third (136-146 aa) nuclear export signal (NES) of IDV NS2 among the three separate NES regions is majorly responsible for the nucleocytoplasmic transport through the chromosome region maintenance 1/Xpo1 (CRM1) pathway [262]. With the increased clinical cases of ICV-related pneumonia in children along with the absence of antiviral immunity specific to ICV, more and more studies are needed in this area.…”