The 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 infection

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Abstract

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.

About the authors

Yury Yu. Ivin

Federal 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)

Author for correspondence.
Email: ivin_uu@chumakovs.su
ORCID iD: 0000-0003-0995-7944

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

Russian Federation, 108819, Moscow

Anna A. Butusova

Federal 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)

Email: amadreaera@gmail.com
ORCID iD: 0009-0008-0361-9681

junior researcher, Laboratory of biochemistry

Russian Federation, 108819, Moscow

Ekaterina E. Gladneva

Federal 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)

Email: gladneva_ee@chumakovs.su
ORCID iD: 0009-0005-8067-6768

junior researcher, Laboratory of biochemistry

Russian Federation, 108819, Moscow

Galina Ya. Kolomijtseva

A.N. Belozersky Research Institute of Physico-Chemical Biology MSU

Email: kolom@genebee.msu.ru
ORCID iD: 0009-0005-5130-2695

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

Russian Federation, 119234, Moscow

Yusuf K. Khapchaev

Federal 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)

Email: hapchaev_uh@chumakovs.su
ORCID iD: 0000-0003-1613-5228

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

Russian Federation, 108819, Moscow

Aydar A. Ishmukhametov

Federal 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); Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University

Email: ishmukhametov@chumakovs.su
ORCID iD: 0000-0001-6130-4145

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

Russian Federation, 108819, Moscow; 117418, Moscow

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2. Fig. 1. Scheme of mutations introduced into the sequence of L and 2A proteins of the EMCV-1 to obtain mutant viruses Zfmut, Δ2A and Zfmut&Δ2A (a); Changes in the plaque phenotype of the obtained mutant viruses Δ2A and Zfmut&Δ2A during passages on BHK-21 cell culture (b)

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3. Fig. 2. The location of the amino acid residue at position 99 of the VP2 protein (a). Comparison of the reproduction cycles of wild-type EMCV-1 (WT) and mutant virus with the amino acid substitution of threonine for alanine at position 99 of the VP2 protein (Thr/VP2_99/Ala) (b).

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4. Fig. 3. Accumulation of infectious viral particles (a) and copies of viral genomic RNA (b) during the reproduction cycle of WT, Zfmut, Δ2A and Zfmut&Δ2A viruses in HeLa cells. The number of PFU was determined by plaque titration in BHK-21 cells. The number of copies of viral RNA was determined by real-time PCR

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5. Fig. 4. Autoradiography of synthesized proteins in HeLa cells infected with WT, Zfmut, Δ2A, and Zfmut&Δ2A viruses. Protein translation inhibitors: Ars-sodium arsenite (100nM), T-2 toxin (50nM). Mock – uninfected cells without inhibitors. Signals corresponding to viral proteins are marked with arrows.

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6. Fig. 5. The level of incorporation of 14C-labeled amino acids into synthesized proteins during infection of HeLa cells with WT, Zfmut, Δ2A and Zfmut&Δ2A viruses. A value of 1 corresponds to the level of incorporation of 14C-labeled amino acids into the proteins of an uninfected culture (a). Level of phosphorylation of translation initiation factor eIFAα in HeLa cells during infection with WT, Zfmut, Δ2A and Zfmut&Δ2A viruses. A value of 1 corresponds to the level of eIFAα phosphorylation in uninfected cells (b). Values obtained by processing the results of immunoblotting. Image processing was carried out using the ImageJ program.

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7. Fig. 6. Study of the accumulation of proteins of the studied viruses during the infection of HeLa cells using specific mouse immune serum (a) and autoradiography using amino acids labeled with the 14C isotope (b). For panels a and b, the same lysates of infected cells were used. Samples: 1 – WT EMCV-1 purified by cesium chloride gradient ultracentrifugation; 2 – WT from the working pool; 3–12 – samples from HeLa cells 4h after infection with the studied viruses; Mock – uninfected cells. The number of samples 3–12 are aligned according to the content of β-actin.

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8. Fig. 7. Relative signal level of viral proteins accumulated by 4 h.p.i. of HeLa cells with WT, Zfmut, Δ2A and Zfmut&Δ2A viruses. a – signal level of non-structural proteins and precursor proteins of capsid proteins; b – signal level of capsid proteins. The data were obtained by processing the results of immunoblotting of lysates of infected cells using specific mouse serum against EMCV-1 proteins (fig. 6 a). a level of 1 corresponds to the signal level of each individual protein of the WT virus. The value of the signal of individual proteins was normalized to the signal of the WT virus. Image processing was carried out using the ImageJ program. * – p < 0.05, *** – p < 0.001.

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Copyright (c) 2023 Ivin Y.Y., Butusova A.A., Gladneva E.E., Kolomijtseva G.Y., Khapchaev Y.K., Ishmukhametov A.A.

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