Expression of tick-borne encephalitis virus nonstructural protein 1 stimulates the secretion of extracellular vesicles capable of activating IL-1β production

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Abstract

Despite active research, so far the detailed mechanisms of TBEV pathogenesis have not been fully disclosed. Recently, extracellular vesicles, especially small-sized vesicles, which have been shown to play an important role in the pathogenesis of many viruses, have attracted the attention of scientists. In this study, we investigated the effect of nonstructural protein 1 (NS1) expression of tick-borne encephalitis virus on the release of extracellular vesicles by cells, and assessed the possibility of these vesicles affecting other cells. NS1 expression by TBEV was found to increase the release of extracellular vesicles by HEK293T cells; however, no changes in the size profile of released vesicles were detected. In addition, NS1 is detected in both large and small vesicle size fractions. It was found that NS1 TBEV is not present inside the vesicles, but is associated with their outer surface. Small-sized vesicles derived from the culture medium of NS1-expressing HEK293T cells are able to induce an increase in mRNA content and interleukin-1β (IL-1β) secretion in human neuroblastoma SHSY5Y cells. The results indicate the involvement of NS1 protein and vesicles in the development of neuroinflammation and are important for understanding the mechanisms of tick–borne encephalitis virus pathogenesis.

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Е. S. Starodubova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: estarodubova@yandex.ru
Russian Federation, Moscow, 119991

A. A. Latanova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: estarodubova@yandex.ru
Russian Federation, Moscow, 119991

Yu. V. Kuzmenko

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: estarodubova@yandex.ru
Russian Federation, Moscow, 119991

V. I. Popenko

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: estarodubova@yandex.ru
Russian Federation, Moscow, 119991

V. L. Karpov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: estarodubova@yandex.ru
Russian Federation, Moscow, 119991

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2. Fig. 1. Immunoblotting of HEK293T cells (lanes 1 and 8) transfected with pVax (lanes 1–6) or pVax-NS1 (lanes 8–13) and culture medium fractions obtained by differential centrifugation at 500 g (lanes 2 and 9), 3000 g (lanes 3 and 10), 10,000 g (lanes 4, 5 and 11, 12) and pelleting with a commercial reagent (lanes 6 and 13). The position of the molecular weight marker bands (lanes 7 and 14) is indicated on the right.

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3. Fig. 2. Localization of NS1 in vesicles of different sizes. a – Immunoblotting of HEK293T cells (lanes 1 and 6) transfected with pVax (lanes 1–4) or pVax NS1 (lanes 6–9) and fractions obtained by differential centrifugation of the culture medium at 3,000 g (lanes 2 and 7), 10,000 g (lanes 3 and 8) and by precipitation with a commercial reagent (lanes 4 and 9). The position of the molecular mass marker bands (lane 5) is indicated on the right. b – Immunoblotting of the fraction of small extracellular vesicles treated with proteinase K with or without Triton X-100.

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4. Fig. 3. Analysis of the fraction of small vesicles obtained with a commercial reagent from the culture medium of HEK293T cells transfected with the pVax plasmid or the pVax plasmid encoding NS1. a – Granulometry profile of vesicle preparations obtained on the basis of dynamic light scattering analysis (distribution of light scattering intensity (intensity, %) by hydrodynamic diameters (size, nm)). b – Granulometry profile of vesicle preparations obtained on the basis of nanoparticle trajectory analysis. c – Micrograph of small vesicle preparations obtained using transmission electron microscopy.

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5. Fig. 4. Gel filtration on Sepharose CL-2B of small vesicle fractions and proteins from the medium of cells transfected with the pVax vector or plasmid encoding NS1. a – Elution profile of small vesicles (particle concentration in fractions measured by NTA) and proteins (protein concentration according to Bradford). b – Dot blot analysis of fractions with antibodies to NS1 (clone 4C4). Fraction numbers are given at the top and on the sides.

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6. Fig. 5. Analysis of the vesicle fraction obtained by gel filtration on a Sepharose CL-2B column from the culture medium of HEK293T cells transfected with the pVax plasmid or the pVax plasmid encoding NS1. a – Granulometry profile of vesicle preparations obtained on the basis of dynamic light scattering analysis (distribution of light scattering intensity (intensity, %) by hydrodynamic diameters (size, nm)). b – Granulometry profile of vesicle preparations obtained by nanoparticle tracking analysis. c – Micrographs of vesicle preparations of pVax (left) and NS1 (right) samples obtained using transmission electron microscopy. d – Immunoblotting of vesicle preparations of NS1 samples (lane 1) with antibodies to NS1, TSG101, and GM130. The position of the molecular mass marker bands (lane 2) is shown on the right.

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7. Fig. 6. Effect of extracellular vesicles on SHSY5Y neuroblastoma cells. Relative content of mRNA (a) and IL-1β secreted into the medium (b). Statistically significant differences p < 0.05 (*).

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