Influenza A virus nuclear export protein (NEP) plays an important role in the viral life cycle. Recombinant NEP proteins containing (His)6-tag at either N-or C-terminus were obtained by heterologous expression in Escherichia coli cells and their high propensity for aggregation was demonstrated. Dynamic light scattering technique was used to study the kinetics and properties of NEP aggregation in solutions under different conditions (pH, ionic strength, presence of low-molecular-weight additives and organic solvents). Using atomic force microscopy, the predominance of spherical aggregates in all examined NEP preparations was shown, with some amyloid-like structures being observed in the case of NEP-C protein. A number of structure prediction programs were used to identify aggregation-prone regions in the NEP structure. All-atom molecular dynamics simulations indicate a high rate of NEP molecule aggregation and reveal the regions preferentially involved in the intermolecular contacts that are located at the edges of the rod-like protein molecule. Our results suggest that NEP aggregation is determined by different types of interactions and represents an intrinsic property of the protein that appears to be necessary for its functioning in vivo.

This corrects the article DOI: 10.1134/S0006297918080096.

Entry of many viral and bacterial pathogens into host cells depends on cholesterol and/or cholesterol-enriched domains (lipid rafts) in the cell membrane. Earlier, we showed that influenza virus A matrix protein M1 contains amphipathic α-helices with exposed cholesterol-recognizing amino acid consensus (CRAC) motifs. In order to test possible functional activity of these motifs, we studied the effects of three synthetic peptides corresponding to the CRAC-containing α-helices of the viral M1 protein on the phagocytic activity of cultured mouse IC-21 macrophages. The following peptides were used: LEVLMEWLKTR (M1 α-helix 3, a.a. 39–49; further referred to as peptide 1), NNMDKAVKLYRKLK (M1 α-helix 6, a.a. 91–105; peptide 2), and GLKNDLLENLQAYQKR (M1 α-helix 13, a.a. 228–243; peptide 3). We found that all three peptides modulated interactions of IC-21 macrophages with non-opsonized 2-μm target particles. The greatest effect was demonstrated by peptide 2: in the presence of 35 μM peptide 2, the phagocytic index of IC-21 macrophages exceeded the control value by 60%; 10–11 mM methyl-β-cyclodextrin abolished this effect. Peptides 1 and 3 exerted weak inhibitory effect in a narrow concentration range of 5–10 μM. The dose-response curves could be approximated by a sum of two (stimulatory and inhibitory) components with different Hill coefficients, suggesting existence of at least two peptide-binding sites with different affinities on the cell surface. CD spectroscopy confirmed that the peptides exhibit structural flexibility in solutions. Altogether, our data indicate that amphipathic CRAC-containing peptides derived from the viral M1 protein modulate lipid raft-dependent processes in IC-21 macrophages.

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N-or C-terminal (His)6-tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

A complex structural analysis of nuclear export protein NS2 (NEP) of influenza virus A has been performed using bioinformatics predictive methods and small-angle X-ray scattering data. The behavior of NEP molecules in a solution (their aggregation, oligomerization, and dissociation, depending on the buffer composition) has been investigated. It was shown that stable associates are formed even in a conventional aqueous salt solution at physiological рН value. For the first time we have managed to get NEP dimers in solution, to analyze their structure, and to compare the models obtained using the method of the molecular tectonics with the spatial protein structure predicted by us using the bioinformatics methods. The results of the study provide a new insight into the structural features of nuclear export protein NS2 (NEP) of the influenza virus A, which is very important for viral infection development.