[The Effects of the Hydrogen Sulfide Donor GYY4137 on the Proteasome Pool of Colorectal Cancer Cells].

Cancer cells are characterized by an increased level of metabolism and are highly dependent on the correct functioning of the processes that ensure homeostasis. Reactive sulfur species (RSS) are important molecular modulators of metabolic processes in both healthy and tumor cells. The effect of RSS and, in particular, H2S, on key cellular systems, including the ubiquitin-proteasome system (UPS), which provides the destruction of most intracellular proteins, has been shown. The main components of the UPS are proteasomes, multisubunit protein complexes, within which proteolysis occurs. At the same time, data on the effect of H2S directly on the pool of proteasomes in tumor cells are insufficient. Here, we studied the effect of incubation of SW620B8-mCherry colorectal adenocarcinoma cells expressing a fluorescently labeled proteasome subunit with 50, 100, and 200 µM of the hydrogen sulfide donor GYY4137. The effect of the substance on the proteasome pool was assessed 6, 24, 48, and 72 h after administration. It was shown that the chymotrypsin-like and caspase-like proteasome activity decreases in cells incubated with 200 µM of the GYY4137 for 24 h. This coincided with an increase in the expression of proteasome subunit genes. In lysates of cells incubated with 200 µM GYY4137 for 48 h an increase in the content of the constitutive ß5 subunit was observed and the activity of proteasomes leveled off. Following prolonged incubation with GYY4137 (72h), an increase in the expression levels of some proteasome genes was also observed, although this did not have a significant effect on the activity and subunit composition of proteasomes. Thus, the obtained data indicate the modulation of proteasome activity by the hydrogen sulfide donor and the effect of GYY4137 on transcription and translation of proteasome genes.

[Changes in the Activities and Contents of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice].

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes that differ, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer's disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (nonconstitutive proteasomes), has been shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and corresponding proteins in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). Increases by 4 and 6 folds in transcripts of the PSMB9 and PSMB8 genes encoding immune proteasome subunits were detected, as well as a significant increase in the content of immune ß-subunits (by 2.8 folds, ß1i; 2.2 folds, ß2i) in samples from 5xFAD mice at the age of 380 days, compared with samples from mice at 60 days of age. Moreover, the activation of both 20S and 26S proteasomes containing immune subunits were revealed in samples from 380 days old 5xFAD mice by electrophoresis in native conditions. This indicates activated synthesis of the immune subunits and assembly of nonconstitutive proteasomes at the terminal stage of pathology development. The obtained data, in combination with the available literature, indicate that the activation of nonconstitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, which may reflect both the development of neuroinflammation and adaptive processes in tissues induced by the accumulation of toxic protein aggegates.

[Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging].

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the ß5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the ß5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit ß5; increased expression of genes encoding immune proteasome subunits ß5i and ß1i. In tissue homogenates of aged mice, an increase in the content of immune subunits ß1i and ß2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the ß5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the ß5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

[Hepatitis C Virus Nonstructural Protein 3 Increases Secretion of Interleukin-lbeta in HEK293T Cells with a Reconstructed NLRP3 Inflammasome].

The pathology of diseases arising from infections by viruses of Flaviviridae is largely determined by the development of systemic inflammation. The cytokines interleukin-1beta and interleukin-18 play a key role in triggering inflammation. Their secretion from cells, in its turn, is induced upon activation of inflammasomes. Activation of NLRP3 (NLR pyrin domain-containing family 3) inflammasomes was detected in cells infected with Flaviviridae. Some nonstructural proteins of these viruses have been shown to be able to activate or to inhibit the NLRP3 inflammasome, in particular, through interaction with its components. In this study, a functional NLRP3 inflammasome was reconstructed in human HEK293T cells and the effect of some nonstructural proteins of individual Flaviviridae viruses on it was studied. This model did not reveal any impact of nonstructural NS1 proteins of the West Nile virus, NS3 of hepatitis C virus, or NS5 of tick-borne encephalitis virus on the inflammasome components content. At the same time, in the presence of the NS1 of the West Nile virus and NS5 of the tick-borne encephalitis virus, the level of secretion of interleukin-1beta did not change, whereas in the presence of the NS3 protein of the hepatitis C virus, it increased by 1.5 times. Thus, NS3 can be considered as one of the factors of NLRP3 inflammasome activation and inflammatory pathogenesis in chronic hepatitis C virus infection.

[Rpn4p without the DNA-Binding Domain Provides Saccharomyces cerevisiae Resistance to Oxidative Stress and Cycloheximide].

The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a key transcriptional regulator of the proteasome in Saccharomycetes yeast and is also involved in the cellular response to various stresses. In addition to proteasomal genes, Rpn4 affects the expression of several hundred other genes, including genes involved in DNA repair and oxidative stress response. At the same time, the molecular mechanisms used by Rpn4 in controlling target genes and its functioning as a regulator of the cellular response to stress remain largely unclear. The aim of this work was to determine the Rpn4 domains required to ensure cell resistance to stress. It was shown that the N-terminal and central regions of the protein contain sites required for resistance to all types of stresses. The putative nuclear localization signal does not affect the functioning of Rpn4. Unexpectedly, a protein with the deletion of both zinc finger motifs that form the DNA-binding domain provides yeast resistance to oxidative stress and cycloheximide. Moreover, we showed that Rpn4 can be recruited to the promoter regions of the regulated genes even if they do not contain its binding sites. Based on these data, it can be assumed that Rpn4 is involved in gene regulation and the cellular response to stress due to protein-protein interactions.

[Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts].

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically ß1, ß2, and ß5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits ß1, ß2, and ß5 can be substituted by the immune ßli, ß2i, and ß5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.

[Audiological profile of patients with SARS-Co-V-2 PCR-positive cases]. / Audiologicheskii profil' patsientov pri zabolevanii, vyzvannom virusom SARS-CoV-2.

COVID-19 is a new pandemic caused by a novel coronavirus, SARS-CoV-2. COVID-19 has spread throughout China and received worldwide attention. On 11 February 2020, World Health Organization (WHO) officially declared COVID-19. The clinical symptoms of COVID-19 patients may vary, more often include symptoms affected by upper and lower respiratory tract damage. In ENT practice it is used to mention rhinitis, sore throat, anosmia/hyposmia. The effect of COVID-19 is an interesting issue in audiology. There were 78 patients who were confirmed positive for COVID-19 PCR-positive cases and 30 normal non-infected subjects in our study. The patients were divided into two groups according to severity their clinical symptoms from asymptomatic COVID-19 PCR-positive cases to severe form. All patients underwent audiological evaluation included tympanometry, acoustic threshold and transient evoked otoacoustic emission (TEOAE). Although hearing sensitivity was normal among some participants, it was statistically proved that TEOAEs could pick up subtle deterioration in the outer hair cells functions and impact on the cochlear.

[Hyper-Resistance of the Bacillus licheniformis 24 Strain to Oxidative Stress Is Associated with Overexpression of Enzymatic Antioxidant System Genes].

At the International Space Station (ISS), artificial living conditions are created and maintained to satisfy human needs, these conditions are also favorable for the growth of numerous microorganisms, molds and bacteria. Among the microorganisms detected on the ISS are those from the automicroflora of crew members, and a significant number of spore-forming bacteria. In most cases, this group of microorganisms gives rise to strains that are able to colonize, grow and reproduce on interior materials and equipment of stations, and may be involved in biodestructive processes. These bacteria show increased resistance to various stress factors, for example, DNA-damaging and oxidizing agents. The molecular mechanisms of this resistance to stress are poorly understood. As part of the sanitary-microbiological monitoring of the ISS habitat, the Bacillus licheniformis 24 strain was isolated. Here, we demonstrated that this strain has increased resistance to hydrogen peroxide and Paraquat when compared to the "terrestrial" B. licheniformis B-10956 strain. B. licheniformis 24 overexpressed genes encoding enzymes that neutralize reactive oxygen species, such as KatX catalase and the superoxide dismutases SodA and SodF. Apart from this, in comparison with B. licheniformis B-10956, of B. licheniformis 24 cells had lower hydrogen sulfide production that was associated with sharply reduced expression of the cysIJ operon that encodes sulfite reductase. The results indicate that enzymatic antioxidant protective systems make a more significant contribution to the hyper-resistance of Bacillus strains to oxidizing agents than components of non-enzymatic systems, such as hydrogen sulfide.

[Biotechnological Potential of the Bacillus subtilis 20 Strain].

Bacillus subtilis bacteria play an important role in veterinary medicine, medicine, and biotechnology, and the permanently growing demand for biotechnological products fuels the improvement of the properties of biotechnological strains. B. subtilis strains with improved characteristics maybe obtained by rational design and the directed evolution technologies, or be found among newly described strains. In the course of the long-term microbiome composition studies in the Russian segment of the International Space Station, the B. subtilis 20 strain was isolated, this strain shows the capacity for rapid growth and considerable biomass accumulation, as well as increased resistance to acidification of the environment in comparison to the "terrestrial" B. subtilis 168 strain. What is more, B. subtilis 20 is hyperresistant to the DNA and protein damaging factors that are linked to the overexpression of the genes controlling DNA repair, hydrogen sulfide production, and reactive oxygen species neutralization. The described properties of B. subtilis 20 are indicative of its considerable potential as a promising producer of biologically active compounds.

[Evolution of the System of Coordinate Regulation of Proteasomal Gene Expression in the Yeast Class Saccharomycetes].

The 26S proteasome is a multisubunit ATP-dependent protease complex and is necessary for the normal function of the eukaryotic cell and its survival in stress. Twenty years ago, we, in collaboration with German researchers, were the first to experimentally describe a system for coordinated regulation of proteasomal gene expression in the yeast Saccharomyces cerevisiae. This system consists of the ScRpn4 transcription factor and its binding site, called PACE. Based on the results of a bioinformatics search in the first sequenced yeast genomes, Rpn4-like proteins and PACE-like elements were postulated for other species of the class Saccharomycetes. We experimentally characterized Rpn4-like proteins in the biotechnologically significant yeast species Komagataella pfaffii (Pichia pastoris), Yarrowia lipolytica, and Debaryomyces hansenii and the opportunistic yeast Candida glabrata. As ample information accumulates for the genome sequences of new yeast species and strains, the question arises as to how diverse the regulatory system of proteasomal genes is in terms of structure and likely mechanisms of function. In this work, a bioinformatics search for Rpn4-like proteins and PACE-like elements was conducted in 3111 strains belonging to 427 yeast species of the class Saccharomycetes. It was shown that only the DNA-binding domain is conserved among Rpn4-like proteins, in accordance with conservation of PACE elements. Certain systems were found to contain more than one Rpn4-like protein with structural differences in the DNA-binding domain or to include an autoregulation of the genes for Rpn4-like proteins. Given that Rpn4-like proteins and proteasomes play a role in the cell response to stress, the diversity of systems for the regulation of proteasomal genes was assumed to corresponds to adaptation of organisms to their living environments.

[Dynamics of the Functional Activity and Expression of Proteasome Subunits during Cellular Adaptation to Heat Shock].

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.

[Candida glabrata Rpn4-like Protein Complements the RPN4 Deletion in Saccharomyces cerevisiae].

Expression of Saccharomyces cerevisiae proteasomal genes is regulated in a coordinated manner by a system that includes the ScRpn4 transcription factor and its binding site known as PACE. Earlier we showed that, Rpn4-like proteins from the biotechnologically important yeast species Komagataella pfaffii (Pichia pastoris), Yarrowia lipolytica, and Debaryomyces hansenii are capable of complementing the RPN4 deletion in S. cerevisiae in spite of their low structural similarity to ScRpn4. The opportunistic yeast pathogen Candida glabrata has a gene coding for a Rpn4-like protein, which has not been characterized experimentally yet. The С. glabrata ortholog ScRpn4 was expressed heterologously and found to restore the stress resistance and expression of proteasomal genes in a mutant S. cerevisiae strain with a RPN4 deletion. This complementation required the unique N-terminal region of CgRpn4. The results indicate that CgRpn4 acts as a transcriptional activator of proteasomal genes. The S. cerevisiae model can be used for further structural and functional analyses of CgRpn4.

[A Plasmid-Expressed CRISPR/Cas9 System Suppresses Replication of HSV Type I in a Vero Cell Culture].

Herpesviruses are widespread in the human population. Herpes simplex virus type 1 (HSV1) alone infects more than 3.7 billion people. In most of these, the virus establishes a latent form resistant to the action of all antiviral drugs. Moreover, completely drug-resistant strains of herpesviruses are known, which has prompted the search for alternative approaches to the treatment of herpesviruses, including genome editing with prokaryotic CRISPR/Cas. The CRISPR/Cas9 system of Streptococcus pyogens effectively suppresses HSV1 infection when expressed from genome-integrated lentiviral vectors. However, there are concerns about the safety of this approach. Here we describe the system built upon the plasmid-encoded CRISPR/Cas9 targeted against UL52 and UL29 genes of the HSV1 primase-helicase complex. The construct was transfected into Vero cells with no significant cytotoxic effects detected. Complete suppression of HSV1 infection within two days was observed, raising the possibility that the proposed plasmid-expressed CRISPR/Cas9 system may be used for the screening of genes important for the HSV1 life cycle and for development of novel strategies for targeted therapy of herpesvirus infections.

[A DNA Construct That Encodes the Rabies Virus Consensus Glycoprotein with a Proteasome Degradation Signal Induces Antibody Production with IgG2A Subtype Predominance].

The possibility of enhancing the immunogenicity of the rabies virus glycoprotein antigen encoded by a DNA vaccine has been investigated. Ubiquitin-like protein FAT10 has been attached to the N-terminus of the glycoprotein to target it to the proteasome and stimulate its presentation by MHC class I. Two forms of the protein, chimeric and original, have been detected in cells transfected with the DNA construct encoding the chimeric protein. The presence of the glycoprotein on the cell surface has been detected by immunostaining of transfected cells. The production of IgG and IgG2a antibodies has been more efficiently induced in mice immunized with the plasmid that encodes the chimeric protein than in those immunized with the plas-mid that encodes unmodified glycoprotein. Moreover, the level of IgG2a antibodies exceeded the level of IgG1 antibodies, which indicates a preferential increase in the Th1 component of the immune response. The proposed DNA construct that encodes a modified glycoprotein with a proteasome degradation signal maybe a promising DNA vaccine immunogen for post-exposure prophylaxis of rabies.

Codon optimization and improved delivery/immunization regimen enhance the immune response against wild-type and drug-resistant HIV-1 reverse transcriptase, preserving its Th2-polarity.

DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid ("surrogate challenge"). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199-220 and aa 528-543. Drug-resistance mutations disrupted the epitope at aa 205-220, while the CTL epitope at aa 202-210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.

[Rabies Virus Glycoprotein with a Consensus Amino Acid Sequence and a Lysosome Targeting Signal Causes Effective Production of Antibodies in DNA-Immunized Mice].

Safe and effective anti-rabies vaccines are intensely sought worldwide. DNA vaccines have already shown their efficacy and safety and have occupied a special place in the field. Two prototype anti-rabies DNA vaccines were compared for the potential to induce virus-specific antibody production. One vector contained a codon-optimized gene with a territory-adapted consensus sequence of the rabies virus glycoprotein. The other one expressed the same glycoprotein in fusion with a c-CD63 lysosome targeting motif at the C terminus. ELISA of serum samples from immunized mice showed that the c-CD63 variant induced more efficient antibody production and shifted the IgG2a/IgG1 ratio towards the Th2-type immune response. The results gave grounds to believe that the approach successfully applied to the rabies glycoprotein may help to develop new-generation anti-rabies vaccines.

[C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein].

The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.

[Heat-shock protein HSP70 decreases activity of proteasomes in human neuroblastoma cells treated by amyloid-beta 1-42 with isomerized Asp7].

Experimental evidences indicate that heat-shock protein 70 (HSP70) can serve as a prospective therapeutic agent to treat Alzheimer's disease (AD). It has demonstrated a neuroprotective effect in vivo on mice models of AD. Moreover, HSP70 decreases oxidative stress in neurons induced by amyloid-ß (Aß42) and its more toxic form with isomerized Asp7 (isoAß42). The dysfunction of Ubiquitin-proteasome system (UPS) is observed in AD. UPS is responsible for the degradation of the majority of cellular proteins and plays an important role in protecting cells from oxidative stress. Here, we have shown that the incubation of human neuroblastoma cells SK-N-SH with isoAß42 increases the activity of intracellular proteasomes, which are the principal elements of the UPS. On the contrary, the proteasomal activity was decreased in isoAß42-treated cells in the presence of exogenous HSP70. These results highlight the existence of an interplay between Aß peptides, proteasomes, and HSP70.

[Inhibition of the expression of proteasomal genes Saccharomyces cerevisiae by artificial transcriptional repressor].

26S proteasome is an ATP-dependent protease complex that takes part in cell homeostasis maintenance by the selective degradation of regulatory and damaged proteins. The proteasomal genes expression in Saccharomyces cerevisiae yeast is coordinately regulated by the system, which consists of the Rpn4 transcription factor and its binding site, called PACE. The ability to modulate proteasomal activity by changing the expression of its genes is an essential tool that can be used in fundamental studies devoted to the mechanisms of proteasome dependent cell processes, as well as in applied research for developing strategies to correct proteasome activity in some pathological processes. In this work, we present a detailed description of our SaxBricks method that allows one to construct DNA-binding domains with custom specificity from nucleotide- specific TAL domains. Having applied the SaxBricks method, we created a modular transcriptional repressor for Rpn4-dependent genes that effectively suppresses the expression of proteasomal genes.

[Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits].

The interaction of viral proteins with host cell components plays an important role in antiviral immune response. One of the key steps of antiviral defense is the formation of immunoproteasomes. The effect of nonstructural protein 1 (NS1) of tick-borne encephalitis virus on the immunoproteasome formation was studied. It was shown that cell expression of NS1 does not reduce the efficacy of the immunoproteasome generation in response to interferon-γ stimulation and even increases the content of the immunoproteasome subunits without the interferon-γ treatment. Thus, NS1 of tick-borne encephalitis virus activates, rather than blocks the mechanisms of immune defense in the cell.