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

Lingcai Zhao,Yiqing Zheng,Juan Su,Huizhi Xia,Chang Liu,Jingjin Huang,Jihui Ping

doi:10.3390/v12101100

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.

Highlights

Influenza viruses are segmented, single-stranded, negative-sense, enveloped RNA viruses belonging to the Orthomyxoviridae genus [1]
We could not generate viable influenza D viruses (IDVs) nonstructural protein 2 (NS2) recombinant viruses by reverse genetics in this way, even though recombinant virus was able to be rescued when porcine teschovirus-1 (PTV-1) 2A sequence was inserted into PR8 NS gene (Figure 1B) [16]
We explored whether IDV NS2 protein had other features that could distinguish transiently expressed truncated segments of IDV NS2 fused to enhanced green fluorescent protein (eGFP) to examine subcellular

Summary

Introduction

Single-stranded, negative-sense, enveloped RNA viruses belonging to the Orthomyxoviridae genus [1]. Four types of influenza viruses are currently recognized: the influenza A viruses (IAVs) and influenza B viruses (IBVs) contain eight gene segments, while the influenza C viruses (ICVs) and influenza D viruses (IDVs) contain seven gene segments [2]. The influenza D virus, the newest member of the Orthomyxoviridae genus, was first isolated from swine with respiratory symptoms in 2011 in the United States [3]. Subsequent surveys revealed that cattle, not pigs, were the natural reservoirs of these type D viruses [4,5]. There are a number of animals susceptible to IDV, including sheep, goats [6], ferrets [3], and guinea pigs [7]. Serological studies have shown that a low percentage of human sera have detectable titers of specific antibodies against the influenza D viruses [8,9]

Methods

Results

Discussion

Conclusion