Белок ядерного экспорта (NEP) играет важную роль во внутриклеточных процессах при инфицировании клеток хозяина. В данной работе впервые с помощью атомно-силовой микроскопии выявлены и охарактеризованы агрегаты различной морфологии и размеров. Полученные результаты могут быть востребованы для разработки новых противовирусных препаратов и создания систем доставки лекарственных средств на основе NEP.

Journal Article Atomic Force Microscopy Investigation of Influenza A Virus Nuclear Export Protein Aggregation Get access EV Dubrovin, EV Dubrovin Lomonosov Moscow State University, Leninskie gory, 1-2, Moscow, Russian FederationFederal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, Moscow, Russian Federation Corresponding author: dubrovin@polly.phys.msu.ru Search for other works by this author on: Oxford Academic Google Scholar ON Koroleva, ON Koroleva Lomonosov Moscow State University, Leninskie gory, 1-2, Moscow, Russian Federation Search for other works by this author on: Oxford Academic Google Scholar AO Golovko, AO Golovko Lomonosov Moscow State University, Leninskie gory, 1-2, Moscow, Russian Federation Search for other works by this author on: Oxford Academic Google Scholar NV Kuzmina, NV Kuzmina Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia Search for other works by this author on: Oxford Academic Google Scholar DV Klinov, DV Klinov Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, Moscow, Russian Federation Search for other works by this author on: Oxford Academic Google Scholar VL Drutsa VL Drutsa Lomonosov Moscow State University, Leninskie gory, 1-2, Moscow, Russian Federation Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 25, Issue S2, 1 August 2019, Pages 1342–1343, https://doi.org/10.1017/S143192761900744X Published: 01 August 2019

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.

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.

Hepatitis C virus (HCV) is characterized by considerable genetic variability and, as a consequence, it has 6 genotypes and multitude of subtypes. HCV envelope glycoproteins are involved in the virion formation; the correct folding of these proteins plays the key role in virus infectivity. Glycosylation at certain sites of different genotypes HCV glycoproteins shows substantial differences in functions of the individual glycans (Goffard et al., 2005; Helle et al., 2010) [1], [2]. In this study, differential glycosylation sites of HCV genotype 1b envelope proteins in insect and mammalian cells was demonstrated. We showed that part of glycosylation sites was important for folding of the proteins involved in the formation of viral particles. Point mutations were introduced in the protein N-glycosylation sites of HCV (genotype 1b) and the mutant proteins were analyzed using baculovirus expression system in mammalian and insect cells. Our data showed that, in contrast to HCV 1a and 2a, the folding of HCV 1b envelope proteins E2 (sites N1, N2, N10) and E1 (sites N1, N5) was disrupted, however that did not prevent the formation of virus-like particles (VLP) with misfolded glycoproteins having densities typical for HCV particles containing RNA fragments. Experimental data are supported by mathematical modeling of the structure of E1 mutant variants.

Diverse morphology of aggregates of amyloidogenic proteins has been attracting much attention in the last few years, and there is still no complete understanding of the relationships between various types of aggregates. In this work, we propose the model, which universally explains the formation of morphologically different (wormlike and rodlike) aggregates on the example of a sigma(70) subunit of RNA polymerase, which has been recently shown to form amyloid fibrils. Aggregates were studied using AFM in solution and depolarized dynamic light scattering. The obtained results demonstrate comparably low Young's moduli of the wormlike structures (7.8-12.3 MPa) indicating less structured aggregation of monomeric proteins than that typical for beta-sheet formation. To shed light on the molecular interaction of the protein during the aggregation, early stages of fibrillization of the sigma(70) subunit were modeled using all-atom molecular dynamics. Simulations have shown that the sigma(70) subunit is able to form quasi-symmetric extended dimers, which may further interact with each other and grow linearly. The proposed general model explains different pathways of sigma(70) subunit aggregation and may be valid for other amyloid proteins.

Белки оболочки вируса гепатита С (ВГС) E1 и E2, являясь компонентами вириона, участвуют в формировании инфекционных частиц вируса в зараженной клетке. Детальное строение частицы ВГС остается малоизученным, причем наименее изучен процесс сборки вирионов и их выход из клетки. Предполагается, что свойства вириона зависят от гликозилирования белковой оболочки вируса в клетке, а гликаны в некоторых сайтах гликозилирования этих белков важны для их функционирования и прохождения жизненного цикла ВГС. N-гликаны гликопротеинов могут влиять на формирование вирусных частиц, связывание вируса с клеткой и патогенез гепатита С. Мы изучили влияние гликанов на сворачивание гликопротеина Е2, образование функциональных гликопротеиновых комплексов и формирование вирусных частиц в клетках насекомых и млекопитающих. С этой целью в сайты N-гликозилирования Е2 вируса гепатита С (генотип 1б штамм 274933RU) вводили точечные мутации и анализировали мутантные белки в бакуловирусной системе экспрессии. Удаление единичных сайтов гликозилирования гликопротеина Е2, за исключением сайта N6, не сказывалось на эффективности его синтеза в клетках насекомых Sf9, а электрофоретическая подвижность мутантных белков возрастала пропорционально снижению числа сайтов гликозилирования. В отличие от клеток Sf9, уровень синтеза гликопротеина Е2 ВГС в клетках Hek293T человека зависел от присутствия гликанов в сайтах гликозилирования N1 и N8. В то же время удаление гликанов в сайтах N1, N2 и N10 приводило к накоплению непродуктивных димеров Е1Е2 в виде агрегатов и подавлению продуктивной сборки вирусоподобных частиц как в клетках насекомых, так и в клетках млекопитающих. Удаление единичных сайтов гликозилирования Е2 ВГС не влияло на синтез РНК структурных белков и образование вирусоподобных частиц в клетках насекомых и млекопитающих.

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.

Envelope proteins of HCV play a major role in virus lifecycle. These proteins are main components of the virion. They are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation which is supposed to play a role in their stability, in the assembly of the functional HCV glycoprotein heterodimer, protein folding and viral entry. The role of N-linked glycosylation sites in HCV E1 protein in structural proteins assembly was analyzed by site-directed mutagenesis in a model system--insect cells producing three viral structural proteins with formation of virus-like particles. Removing of single N-linked glycosylation sites in HCV E1 protein does not affect the efficiency of its expression in insect Sf9 cells. E1 electrophoretic mobility is increasing in parallel with decreasing the number of glycosylation sites. The destroying of glycosylation sites N1 or N5 in E1 influences the assembly of noncovalent glycoprotein heterodimer E1E2--the prototype of natural complex incorporated in virion. The lack of glycans in N1 and N5 sites of E1 was shown to affect the efficiency of its expression in mammalian HEK293 T cells.

The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryp-tophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3′-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.

To study the functional role of the spacer region between. two consensus -10 and -35 elements of promoters, 'recognized by E. coli RNA polymerase, the model promoter-like DNA duplexes containing non nucleotide inserts (mimicking 17-mer spacer) either in one or both strands, were constructed. The modified duplexes can form the heparin-resistant binary complexes with RNA polymerase. The DNA duplex with nonnucleotide insert in the template strand can specifically direct the synthesis of mRNA in the in vitro run-off transcription assays.

A method for isolation of a highly purified preparation of E. coli RNA polymerase core enzyme was developed based on IMPACT technology and dissociation of the RNA polymerase complex with σ70 subunit. Washing of the immobilized RNA polymerase with 5–10 mM solution of glutamate (pH 5.0–5.5) completely removed the σ70 subunit from the holoenzyme and decreased amounts of protein admixtures. The possibility of reconstruction of the RNA polymerase holoenzyme directly on the affinity column was demonstrated. Activities of the resulting RNAP core enzyme preparations were tested by in vitro transcription. Some amino acids and their mixtures were shown to influence the in vitro transcription. The findings indicate that changes in the transcription efficiency in the presence of amino acids should be associated with a specific destruction of the interaction between σ70 subunit and the core enzyme.

Three proteins, namely: "core" protein C and glycoproteins E1 and E2, are main structural proteins forming a hepatitis C vius (HCV) virion. The virus structure and assembly, a role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Using recombinant baculoviruses expressing HCV structural protein genes in insect cells the specific structural proteins at the level of 25-35% relative to a common cell protein content, heterodimers of the glcoproteins, and HCV-like particles have been obtained. It has been demonstrated that recombinant proteins C, E1, and E2 go through the posttranslation modification, the glycoproteins form the non-covalent heterodimer, and HCV-like particles are located in endoplasmatic reticulum membrains of infected cells. An ability of the expressed proteins for forming E1E2 dimers and HCV-like particles was used for studying the role of E1 protein glcosylation upon expression and processing of the glycoproteins.