rus
Issue #2/2024
Е.V.Dubrovin,О.N.Koroleva, N.V.Kuzmina, V.L.Drutsa
STUDY OF INFLUENZA A VIRUS NUCLEAR EXPORT PROTEIN AGGREGATES USING ATOMIC FORCE MICROSCOPY
STUDY OF INFLUENZA A VIRUS NUCLEAR EXPORT PROTEIN AGGREGATES USING ATOMIC FORCE MICROSCOPY
The nuclear export protein (NEP) plays an important role in intracellular processes during infection of host cells. In this paper, aggregates of various morphologies and sizes were identified and characterized for the first time using atomic force microscopy. The results obtained may be used for development of new antiviral drugs and developing of NEP-based drug delivery systems.
Теги: atomic force microscopy influenza a virus nuclear export protein protein aggregation агрегация белка атомно-силовая микроскопия белок ядерного экспорта вирус гриппа а
INTRODUCTION
The nuclear export protein NEP plays an important role in the life cycle of influenza A virus, including export of viral ribonucleoproteins from the nucleus of the infected cell to the cell membrane, regulation of viral genomic RNA accumulation, and participation in release of budding virions [1, 2]. Previously, dynamic light scattering revealed high aggregation ability of recombinant NEP protein under various conditions [3]. A heterogeneous set of NEP aggregates was observed, with typical values of hydrodynamic radius varying from a few nanometres to ~2.5 μm. However, a detailed study of NEP aggregates morphology has not been performed. The aim of this work was to visualise and characterise wild-type NEP aggregates derived from inclusion bodies using atomic force microscopy (AFM). AFM has proven to be an effective tool for analysing various protein molecules and protein aggregates [4, 5].
RESEARCH METHODS
The NEP protein corresponding in structure to natural protein (without additional modules) was expressed in E. coli ER1821 bacterial cells containing the Eptrt-NEP plasmid, where protein gene is under control of the promoter and terminator of RNA polymerase of phage T7. After cell culture and cell disruption, protein was isolated as inclusion bodies as described in [6]. Subsequent additional purification was performed chromatographically according to procedure of [7]. Protein (concentration 0.3–3 µg/µl) was stored in buffer containing 150 mM NaCl, 20 mM HEPES, 1 mM EDTA, pH 7.5, at 4 °C.
5–10 µl of protein solution diluted 10–100 times with deionised water or buffer containing 10 mM NaCl (pH 7.0) was applied to freshly cleaved mica for 10 minutes, then washed with deionised water and dried in an air stream. AFM studies were performed on a Nanoscope-IIIa multimode atomic force microscope (Digital Instruments, USA) in intermittent contact mode in air using HA_NC silicon cantilevers (resonant frequency ~254 and 152 kHz, Tipsnano, Russia). Images were processed using FemtoScan software (Centre for Advanced Technologies, Russia) [8].
RESULTS AND DISCUSSION
Typical AFM images of the NEP protein preparation deposited on the freshly cleaved mica surface are presented in Fig.1. Globular structures of different sizes are observed, varying (in height) from ~1 to ~10 nm (Fig.2a). While globules of the order of 1 nm in height can be attributed to protein monomers [3, 9], larger objects can be attributed to various aggregates (multimers). The histogram of protein particles volume distribution shows a range of volume variation from 100 to 2000 nm3, which, taking into account the broadening of protein particles by the cantilever, may correspond to oligomeric particles consisting of several units to several tens of protein monomers.
In addition, the elongated structures, which are thin filaments with a diameter of ~1 nm and a length of 250–350 nm with globular elongated or spherical thickenings up to 14 nm high, was revealed (Fig.1b). The variety of shape and sizes sizes of NEP oligomers may indicate that the observed structures are various intermediate structures on the way to formation of larger aggregates recorded earlier in light scattering experiments [3]. Formation of rod-like and worm-like structures of NEP aggregates (Fig.1c) may indicate the ability of NEP protein to amyloid aggregation [10].
CONCLUSIONS
The morphology and size of NEP protein aggregates of influenza A virus were characterised for the first time. Presence of globular structures of different sizes, as well as fibrillar, elongated, rod-like and worm-like structures was revealed. The observed polymorphism of structures can be explained by ability of NEP to amyloid aggregation. The study of the amyloid nature of NEP protein and mechanism of formation of various (amyloid) aggregates is of great interest for understanding the physicochemical properties of NEP and their relationship with function of this protein in the process of infection of host cells. Understanding the mechanisms of protein aggregation may open the way to development of next-generation antiviral drugs. In addition, the possibilities of structured protein self-assembly can potentially be used to create nanocapsules based on natural materials for targeted drug delivery.
ACKNOWLEDGMENTS
This work was supported by the grant of the Russian Science Foundation No. 22-23-00395, https://rscf.ru/en/project/23-76-10046
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The nuclear export protein NEP plays an important role in the life cycle of influenza A virus, including export of viral ribonucleoproteins from the nucleus of the infected cell to the cell membrane, regulation of viral genomic RNA accumulation, and participation in release of budding virions [1, 2]. Previously, dynamic light scattering revealed high aggregation ability of recombinant NEP protein under various conditions [3]. A heterogeneous set of NEP aggregates was observed, with typical values of hydrodynamic radius varying from a few nanometres to ~2.5 μm. However, a detailed study of NEP aggregates morphology has not been performed. The aim of this work was to visualise and characterise wild-type NEP aggregates derived from inclusion bodies using atomic force microscopy (AFM). AFM has proven to be an effective tool for analysing various protein molecules and protein aggregates [4, 5].
RESEARCH METHODS
The NEP protein corresponding in structure to natural protein (without additional modules) was expressed in E. coli ER1821 bacterial cells containing the Eptrt-NEP plasmid, where protein gene is under control of the promoter and terminator of RNA polymerase of phage T7. After cell culture and cell disruption, protein was isolated as inclusion bodies as described in [6]. Subsequent additional purification was performed chromatographically according to procedure of [7]. Protein (concentration 0.3–3 µg/µl) was stored in buffer containing 150 mM NaCl, 20 mM HEPES, 1 mM EDTA, pH 7.5, at 4 °C.
5–10 µl of protein solution diluted 10–100 times with deionised water or buffer containing 10 mM NaCl (pH 7.0) was applied to freshly cleaved mica for 10 minutes, then washed with deionised water and dried in an air stream. AFM studies were performed on a Nanoscope-IIIa multimode atomic force microscope (Digital Instruments, USA) in intermittent contact mode in air using HA_NC silicon cantilevers (resonant frequency ~254 and 152 kHz, Tipsnano, Russia). Images were processed using FemtoScan software (Centre for Advanced Technologies, Russia) [8].
RESULTS AND DISCUSSION
Typical AFM images of the NEP protein preparation deposited on the freshly cleaved mica surface are presented in Fig.1. Globular structures of different sizes are observed, varying (in height) from ~1 to ~10 nm (Fig.2a). While globules of the order of 1 nm in height can be attributed to protein monomers [3, 9], larger objects can be attributed to various aggregates (multimers). The histogram of protein particles volume distribution shows a range of volume variation from 100 to 2000 nm3, which, taking into account the broadening of protein particles by the cantilever, may correspond to oligomeric particles consisting of several units to several tens of protein monomers.
In addition, the elongated structures, which are thin filaments with a diameter of ~1 nm and a length of 250–350 nm with globular elongated or spherical thickenings up to 14 nm high, was revealed (Fig.1b). The variety of shape and sizes sizes of NEP oligomers may indicate that the observed structures are various intermediate structures on the way to formation of larger aggregates recorded earlier in light scattering experiments [3]. Formation of rod-like and worm-like structures of NEP aggregates (Fig.1c) may indicate the ability of NEP protein to amyloid aggregation [10].
CONCLUSIONS
The morphology and size of NEP protein aggregates of influenza A virus were characterised for the first time. Presence of globular structures of different sizes, as well as fibrillar, elongated, rod-like and worm-like structures was revealed. The observed polymorphism of structures can be explained by ability of NEP to amyloid aggregation. The study of the amyloid nature of NEP protein and mechanism of formation of various (amyloid) aggregates is of great interest for understanding the physicochemical properties of NEP and their relationship with function of this protein in the process of infection of host cells. Understanding the mechanisms of protein aggregation may open the way to development of next-generation antiviral drugs. In addition, the possibilities of structured protein self-assembly can potentially be used to create nanocapsules based on natural materials for targeted drug delivery.
ACKNOWLEDGMENTS
This work was supported by the grant of the Russian Science Foundation No. 22-23-00395, https://rscf.ru/en/project/23-76-10046
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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