Problems of VirologyProblems of VirologyВопросы вирусологии0507-40882411-2097Central Research Institute for Epidemiology44610.36233/0507-4088-195eeiuhqORIGINAL RESEARCHESОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯResearch ArticleThe role of the encephalomyocarditis virus type 1 proteins L and 2A in the inhibition of the synthesis of cellular proteins and the accumulation of viral proteins during infectionРоль белков L и 2А вируса энцефаломиокардита типа 1 в ингибировании синтеза клеточных белков и накоплении вирусных белков при инфекцииhttps://orcid.org/0000-0003-0995-7944IvinYury Yu.ИвинЮрий Юрьевич
Russian Federation

researcher, Laboratory of biochemistry, Head of the Department of development and implementation innovative and pilot technology

научный сотрудник лаборатории биохимии, начальник управления разработки и внедрения инновационных и полупромышленных технологий

ivin_uu@chumakovs.suhttps://orcid.org/0009-0008-0361-9681ButusovaAnna A.БутусоваАнна Александровна
Russian Federation

junior researcher, Laboratory of biochemistry

младший научный сотрудник лаборатории биохимии

amadreaera@gmail.comhttps://orcid.org/0009-0005-8067-6768GladnevaEkaterina E.ГладневаЕкатерина Евгеньевна
Russian Federation

junior researcher, Laboratory of biochemistry

младший научный сотрудник лаборатории биохимии

gladneva_ee@chumakovs.suhttps://orcid.org/0009-0005-5130-2695KolomijtsevaGalina Ya.КоломийцеваГалина Яковлевна
Russian Federation

Cand. Sci. (Chem.), Assoc. Prof, Head of the Department of isotope analysis

кандидат хим. наук, доцент, заведующая отделом изотопного анализа

kolom@genebee.msu.ruhttps://orcid.org/0000-0003-1613-5228KhapchaevYusuf K.ХапчаевЮсуф Хаджибекович
Russian Federation

D. Sci. (Biol.), Head of the Department for the production of polio vaccines

доктор биол. наук, начальник цеха по производству полиомиелитных вакцин

hapchaev_uh@chumakovs.suhttps://orcid.org/0000-0001-6130-4145IshmukhametovAydar A.ИшмухаметовАйдар Айратович
Russian Federation

D. Sci. (Med.), Professor, Academician of RAS, Head of the Department of organization and technology of production of immunobiological preparations

доктор медицинских наук, профессор, академик РАН, генеральный директор, заведующий кафедрой организации и технологии производства иммунобиологических препаратов

ishmukhametov@chumakovs.suFederal State Autonomous Scientific Institution M.P. Chumakov Federal Scientific Center for Research and Development of Immunobiological Drugs of the Russian Academy of Sciences (Polio Institute)ФГАНУ «Федеральный научный центр исследований и разработки иммунобиологических препаратов имени М.П. Чумакова РАН» (Институт полиомиелита)A.N. Belozersky Research Institute of Physico-Chemical Biology MSUНаучно-исследовательский институт физико-химической биологии имени А.Н. Белозерского ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова»Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical UniversityИнститут трансляционной медицины и биотехнологии ФГАОУ ВО Первый МГМУ имени И.М. Сеченова Минздрава России071120236854284440412202004122020Copyright ©; 2023, Ivin Y.Y., Butusova A.A., Gladneva E.E., Kolomijtseva G.Y., Khapchaev Y.K., Ishmukhametov A.A.Copyright ©; 2023, Ивин Ю.Ю., Бутусова А.А., Гладнева Е.Е., Коломийцева Г.Я., Хапчаев Ю.Х., Ишмухаметов А.А.2023Ivin Y.Y., Butusova A.A., Gladneva E.E., Kolomijtseva G.Y., Khapchaev Y.K., Ishmukhametov A.A.Ивин Ю.Ю., Бутусова А.А., Гладнева Е.Е., Коломийцева Г.Я., Хапчаев Ю.Х., Ишмухаметов А.А.https://creativecommons.org/licenses/by/4.0https://virusjour.crie.ru/jour/article/view/446

Introduction. Infection of cells with encephalomyocarditis virus type 1 (EMCV-1, Cardiovirus A: Picornaviridae) is accompanied by suppression of cellular protein synthesis. The main role in the inhibition of cellular translation is assigned to the L and 2A «security» proteins. The mechanism of the possible influence of the L protein on cellular translation is unknown. There are hypotheses about the mechanism of influence of 2A protein on the efficiency of cap-dependent translation, which are based on interaction with translation factors and ribosome subunits. However, the available experimental data are contradictory, obtained using different approaches, and do not form a unified model of the interaction between the L and 2A proteins and the cellular translation machinery.

Aim. To study the role of L and 2A «security» proteins in the suppression of translation of cellular proteins and the efficiency of translation and processing of viral proteins in infected cells.

Materials and methods. Mutant variants of EMCV-1 were obtained to study the properties of L and 2A viral proteins: Zfmut, which has a defective L; Δ2A encoding a partially deleted 2A; Zfmut&Δ2A containing mutations in both proteins. Translational processes in infected cells were studied by Western-blot and the pulse method of incorporating radioactively labeled amino acids (14C) into newly synthesized proteins, followed by radioautography.

Results. The functional inactivation of the 2A protein does not affect the inhibition of cellular protein synthesis. A direct correlation was found between the presence of active L protein and specific inactivation of cellular protein synthesis at an early stage of viral infection. Nonspecific suppression of the translational processes of the infected cell, accompanied by phosphorylation of eIF2α, occurs at the late stage of infection. Partial removal of the 2A protein from the EMCV-1 genome does not affect the development of this process, while inactivation of the L protein accelerates the onset of complete inhibition of protein synthesis. Partial deletion of the 2A disrupts the processing of viral capsid proteins. Suppression of L protein functions leads to a decrease in the efficiency of viral translation.

Conclusion. A study of the role of EMCV-1 L and 2A proteins during the translational processes of an infected cell, first performed using infectious viral pathogens lacking active L and 2A proteins in one experiment, showed that 2A protein is not implicated in the inhibition of cellular translation in HeLa cells; L protein seems to play an important role not only in the specific inhibition of cellular translation but also in maintaining the efficient synthesis of viral proteins; 2A protein is involved not only in primary but also in secondary processing of EMCV-1 capsid proteins.

Введение. Инфицирование клеток вирусом энцефаломиокардита типа 1 (EMCV-1, Cardiovirus A: Picornaviridae) сопровождается подавлением синтеза клеточных белков. Главную роль в ингибировании клеточной трансляции отводят «секьюрити»-белкам L и 2А. Механизм возможного влияния белка L на клеточную трансляцию неизвестен. Существуют гипотезы о механизме влияния белка 2А на эффективность кэп-зависимой трансляции, которые основаны на его взаимодействии с факторами трансляции и субъединицами рибосом. Экспериментальные данные противоречивы и не позволяют сформулировать единую модель взаимодействия белков L и 2А с трансляционным аппаратом клетки.

Цель. Изучить роль «секьюрити»-белков L и 2А в подавлении трансляции клеточных белков и эффективности трансляции и процессинга вирусных белков зараженных клеток.

Материалы и методы. Для исследования свойств вирусных белков L и 2A получены мутантные варианты EMCV-1: Zfmut, имеющий дефектный белок L; Δ2A, кодирующий частично делетированный белок 2А; Zfmut&Δ2A, содержащий мутации в обоих белках. Течение трансляционных процессов в зараженных клетках изучали c помощью вестерн-блот-гибридизации и пульс-метода включения радиоактивно меченных аминокислот (14С) в новосинтезированные белки.

Результаты. Функциональная инактивация белка 2А не влияет на ингибирование синтеза клеточных белков. Обнаружена прямая корреляция между наличием активного белка L и специфической инактивацией синтеза клеточных белков на раннем этапе вирусной инфекции. На поздних стадиях инфекции происходит неспецифическое подавление трансляционных процессов зараженной клетки, сопровождающееся фосфорилированием eIF2α. Частичное удаление белка 2А из генома EMCV-1 не влияет на течение этого процесса, в то время как инактивация белка L ускоряет наступление полного ингибирования синтеза белков. При частичной делеция белка 2А нарушается процессинг белков вирусного капсида. Подавление функций белка L приводит к снижению эффективности вирусной трансляции.

Заключение. Белок 2А не влияет на ингибирование трансляции в клетках HeLa. Белок L играет важную роль не только в специфическом ингибировании клеточной трансляции, но и в поддержании эффективного синтеза вирусных белков. Белок 2А принимает участие не только в первичном, но и во вторичном процессинге капсидных белков EMCV-1.

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