High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors.

Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes' presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300-400 µg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 µg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80-100 µg per gram of fresh leaf tissue; the yield after purification was up to 20 µg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.

A plant-based transient expression system for the rapid production of highly immunogenic Hepatitis E virus-like particles.

OBJECTIVE: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. The aim of the study is the development of plant expression system for the production of virus-like particles formed by HEV capsid and the characterization of their immunogenicity. RESULTS: Open reading frame (ORF) 2 encodes the viral capsid protein and possesses candidate for vaccine production. In this study, we used truncated genotype 3 HEV ORF 2 consisting of aa residues 110 to 610. The recombinant protein was expressed in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff up to 10% of the soluble protein fraction. The yield of HEV 110-610 after purification was 150-200 µg per 1 g of green leaf biomass. The recombinant protein formed nanosized virus-like particles. The immunization of mice with plant-produced HEV 110-610 protein induced high levels of HEV-specific serum antibodies. CONCLUSIONS: HEV ORF 2 (110-610 aa) can be used as candidate for the development of a plant-produced vaccine against Hepatitis E.

Flagellin-fused protein targeting M2e and HA2 induces potent humoral and T-cell responses and protects mice against various influenza viruses a subtypes.

BACKGROUND: Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new, potentially pandemic, influenza strains. Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A current trend in the design of universal flu vaccines is the construction of recombinant proteins based on combinations of various conserved epitopes of viral proteins (M1, M2, HA2, NP). In this study, we compared the immunogenicity and protective action of two recombinant proteins which feature different designs and which target different antigens. RESULTS: Balb/c mice were immunized subcutaneously with Flg-HA2-2-4M2ehs or FlgSh-HA2-2-4M2ehs; these constructs differ in the location of hemagglutinin's HA2-2(76-130) insertion into flagellin (FliC). The humoral and T-cell immune responses to these constructs were evaluated. The simultaneous expression of different M2e and HA2-2(76-130) in recombinant protein form induces a strong M2e-specific IgG response and CD4+/ CD8+ T-cell response. The insertion of HA2-2(76-130) into the hypervariable domain of flagellin greatly increases antigen-specific T-cell response, as evidenced by the formation of multi-cytokine-secreting CD4+, CD8+ T-cells, Tem, and Tcm. Both proteins provide full protection from lethal challenge with A/H3N2 and A/H7N9. CONCLUSION: Our results show that highly conserved M2e and HA2-2(76-130) can be used as important targets for the development of universal flu vaccines. The location of the HA2-2(76-130) peptide's insertion into the hypervariable domain of flagellin had a significant effect on the T-cell response to influenza antigens, as seen by forming of multi-cytokine-secreting CD4+ and CD8+ T-cells.

The genome-wide transcription response to telomerase deficiency in the thermotolerant yeast Hansenula polymorpha DL-1.

BACKGROUND: In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity. RESULTS: Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones. CONCLUSIONS: Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.

Rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of M2 protein linked to flagellin in plants using viral vectors.

BACKGROUND: The extracellular domain of matrix protein 2 (M2e) of influenza A virus is a promising target for the development of a universal vaccine against influenza because M2e sequences are highly conserved among human influenza A strains. However, native M2e is poorly immunogenic, but its immunogenicity can be increased by delivery in combination with adjuvants or carrier particles. It was previously shown that fusion of M2e to bacterial flagellin, the ligand for Toll-like receptor (TLR) 5 and powerful mucosal adjuvant, significantly increases the immunogenicity and protective capacity of M2e. RESULTS: In this study, we report for the first time the transient expression in plants of a recombinant protein Flg-4M comprising flagellin of Salmonella typhimurium fused to four tandem copies of the M2e peptide. The chimeric construct was expressed in Nicotiana benthamiana plants using either the self-replicating potato virus X (PVX) based vector, pA7248AMV-GFP, or the cowpea mosaic virus (CPMV)-derived expression vector, pEAQ-HT. The highest expression level up to 30% of total soluble protein (about 1 mg/g of fresh leaf tissue) was achieved with the PVX-based expression system. Intranasal immunization of mice with purified Flg-4M protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza virus. CONCLUSIONS: This study confirms the usefulness of flagellin as a carrier of M2e and its relevance for the production of M2e-based candidate influenza vaccines in plants.

Plant-Produced Chimeric Hepatitis E Virus-like Particles as Carriers for Antigen Presentation.

A wide range of virus-like particles (VLPs) is extensively employed as carriers to display various antigens for vaccine development to fight against different infections. The plant-produced truncated variant of the hepatitis E virus (HEV) coat protein is capable of forming VLPs. In this study, we demonstrated that recombinant fusion proteins comprising truncated HEV coat protein with green fluorescent protein (GFP) or four tandem copies of the extracellular domain of matrix protein 2 (M2e) of influenza A virus inserted at the Tyr485 position could be efficiently expressed in Nicotiana benthamiana plants using self-replicating vector based on the potato virus X genome. The plant-produced fusion proteins in vivo formed VLPs displaying GFP and 4M2e. Therefore, HEV coat protein can be used as a VLP carrier platform for the presentation of relatively large antigens comprising dozens to hundreds of amino acids. Furthermore, plant-produced HEV particles could be useful research tools for the development of recombinant vaccines against influenza.

Genome sequence and analysis of methylotrophic yeast Hansenula polymorpha DL1.

BACKGROUND: Hansenula polymorpha DL1 is a methylotrophic yeast, widely used in fundamental studies of methanol metabolism, peroxisome biogenesis and function, and also as a microbial cell factory for production of recombinant proteins and metabolic engineering towards the goal of high temperature ethanol production. RESULTS: We have sequenced the 9 Mbp H. polymorpha DL1 genome and performed whole-genome analysis for the H. polymorpha transcriptome obtained from both methanol- and glucose-grown cells. RNA-seq analysis revealed the complex and dynamic character of the H. polymorpha transcriptome under the two studied conditions, identified abundant and highly unregulated expression of 40% of the genome in methanol grown cells, and revealed alternative splicing events. We have identified subtelomerically biased protein families in H. polymorpha, clusters of LTR elements at G + C-poor chromosomal loci in the middle of each of the seven H. polymorpha chromosomes, and established the evolutionary position of H. polymorpha DL1 within a separate yeast clade together with the methylotrophic yeast Pichia pastoris and the non-methylotrophic yeast Dekkera bruxellensis. Intergenome comparisons uncovered extensive gene order reshuffling between the three yeast genomes. Phylogenetic analyses enabled us to reveal patterns of evolution of methylotrophy in yeasts and filamentous fungi. CONCLUSIONS: Our results open new opportunities for in-depth understanding of many aspects of H. polymorpha life cycle, physiology and metabolism as well as genome evolution in methylotrophic yeasts and may lead to novel improvements toward the application of H. polymorpha DL-1 as a microbial cell factory.

Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope.

Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15-30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant "universal" vaccine against influenza A produced in plants.

Rapid Transient Expression of Receptor-Binding Domain of SARS-CoV-2 and the Conserved M2e Peptide of Influenza A Virus Linked to Flagellin in Nicotiana benthamiana Plants Using Self-Replicating Viral Vector.

The development of recombinant vaccines against SARS-CoV-2 and influenza A is an important task. The combination of the conserved influenza A antigen, the extracellular domain of the transmembrane protein M2 (M2e), and the receptor-binding domain of the SARS-CoV-2 spike glycoprotein (RBD) provides the opportunity to develop a bivalent vaccine against these infections. The fusion of antigens with bacterial flagellin, the ligand for Toll-like receptor 5 and potent mucosal adjuvant, may increase the immunogenicity of the candidate vaccines and enable intranasal immunization. In this study, we report the transient expression of RBD alone, RBD coupled with four copies of M2e, and fusions of RBD and RBD-4M2e with flagellin in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff. The yields of purified recombinant proteins per gram of fresh leaf tissue were about 20 µg for RBD, 50-60 µg for RBD-4M2e and the fusion of RBD with flagellin, and about 90 µg for RBD-4M2e fused to flagellin. Targeting to the endoplasmic reticulum enabled the production of glycosylated recombinant proteins comprising RBD. Our results show that plant-produced RBD and RBD-4M2e could be further used for the development of subunit vaccines against COVID-19 and a bivalent vaccine against COVID-19 and influenza A, while flagellin fusions could be used for the development of intranasal vaccines.

Transient expression of recombinant proteins in plants using potato virus X based vectors.

Plants become a promising biofactory for the large-scale production of recombinant proteins due to low cost, scalability, and safety. Agroinfiltration of plant leaves with a plant viral vector carrying a gene of interest is a rapid and efficient method for protein production in plants. Currently this method is in use for producing a wide range of proteins for multiple applications, including vaccine antigens, antibodies, and protein nanoparticles such as virus-like particles. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. Here, we describe potato virus X based vector pEff-GFP enabling fast and high-level expression of recombinant proteins in Nicotiana benthamiana plants. The pEff vector provides green fluorescent protein expression levels of up to 30% of total soluble protein (about 1mg per g of fresh leaf tissue) and was successfully applied for the production of the immunogens of potential clinical interest.

High-Yield Production of Receptor Binding Domain of SARS-CoV-2 Linked to Bacterial Flagellin in Plants Using Self-Replicating Viral Vector pEff.

The development of recombinant vaccines against SARS-CoV-2 is required to eliminate the COVID-19 pandemic. We reported the expression of a recombinant protein Flg-RBD comprising receptor binding domain of SARS-CoV-2 spike glycoprotein (RBD) fused to flagellin of Salmonella typhimurium (Flg), known as mucosal adjuvant, in Nicotiana benthamiana plants. The fusion protein, targeted to the cytosol, was transiently expressed using the self-replicating vector pEff based on potato virus X genome. The recombinant protein Flg-RBD was expressed at the level of about 110-140 µg per gram of fresh leaf tissue and was found to be insoluble. The fusion protein was purified using metal affinity chromatography under denaturing conditions. To increase the yield of Flg-RBD, the flow-through fraction obtained after loading of the protein sample on the Ni-NTA resin was re-loaded on the sorbent. The yield of Flg-RBD after purification reached about 100 µg per gram of fresh leaf tissue and the purified protein remained soluble after dialysis. The control flagellin was expressed in a soluble form and its yield after purification was about 300 µg per gram of fresh leaf biomass. Plant-produced Flg-RBD protein could be further used for the development of intranasal recombinant mucosal vaccines against COVID-19.

Plant-Produced Recombinant Influenza A Virus Candidate Vaccine Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin.

The development of recombinant influenza vaccines with broad spectrum protection is an important task. The combination of conservative viral antigens, such as M2e, the extracellular domain of the transmembrane protein M2, and conserved regions of the second subunit of hemagglutinin (HA), provides an opportunity for the development of universal influenza vaccines. Immunogenicity of the antigens could be enhanced by fusion to bacterial flagellin, the ligand for Toll-like receptor 5, acting as a powerful mucosal adjuvant. In this study, we report the transient expression in plants of a recombinant protein comprising flagellin of Salmonella typhimurium fused to the conserved region of the second subunit of HA (76-130 a.a.) of the first phylogenetic group of influenza A viruses and four tandem copies of the M2e peptide. The hybrid protein was expressed in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff up to 300 µg/g of fresh leaf tissue. The intranasal immunization of mice with purified fusion protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza A virus strain A/Aichi/2/68(H3N2). Our results show that M2e and hemagglutinin-derived peptide can be used as important targets for the development of a plant-produced vaccine against influenza.

Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope.

The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fecal-oral route or zoonotic. Open reading frame (ORF) 2 encodes the viral capsid protein, which is essential for virion assembly, host interaction, and inducing neutralizing antibodies. In this study, we investigated whether full-length and N- and C-terminally modified versions of the capsid protein transiently expressed in N. benthamiana plants could assemble into highly-immunogenic, virus-like particles (VLPs). We also assessed whether such VLPs can act as a carrier of foreign immunogenic epitopes, such as the highly-conserved M2e peptide from the Influenza virus. Plant codon-optimized HEV ORF2 capsid genes were constructed in which the nucleotides coding the N-terminal, the C-terminal, or both parts of the protein were deleted. The M2e peptide was inserted into the P2 loop after the residue Gly556 of HEV ORF2 protein by gene fusion, and three different chimeric constructs were designed. Plants expressed all versions of the HEV capsid protein up to 10% of total soluble protein (TSP), including the chimeras, but only the capsid protein consisting of aa residues 110 to 610 (HEV 110-610) and chimeric M2 HEV 110-610 spontaneously assembled in higher order structures. The chimeric VLPs assembled into particles with 22-36 nm in diameter and specifically reacted with the anti-M2e antibody.

The 5' untranslated region of the maize alcohol dehydrogenase gene contains an internal ribosome entry site.

Adh1, the maize gene encoding alcohol dehydrogenase ADH1, mRNA is efficiently translated in O2-deprived roots of maize, whereas many normal cellular mRNAs are poorly translated. It has been shown that adh, the 5' untranslated region of adh1 mRNA, provides effective translation of mRNA under hypoxia and heat shock conditions in Nicotiana benthamiana plants. We found that adh contains the internal ribosome entry site (IRES) active both in vivo, in N. benthamiana cells, and in vitro, in rabbit reticulocyte lysate translation system. It is widely supposed that cap-independent internal initiation may maintain efficient translation of particular cellular mRNAs under a variety of stresses and other special conditions when cap-dependent protein synthesis is impaired. We evaluated the level of IRES activity of adh and found that its contribution to the overall translation of adh-containing mRNA in plant cells is less than 1% both under normal conditions and under heat shock. The low efficiency of this IRES is inconsistent with its possible role as a main factor ensuring efficient translation of adh1 mRNA under stress conditions.

Plant-produced Recombinant Influenza A Vaccines Based on the M2e Peptide.

BACKGROUND: Influenza is a widely distributed infection that almost annually causes seasonal epidemics. The current egg-based platforms for influenza vaccine production are facing a number of challenges and are failing to satisfy the global demand in the case of pandemics due to the long production time. Recombinant vaccines are an alternative that can be quickly produced in high quantities in standard expression systems. METHODS: Plants may become a promising biofactory for the large-scale production of recombinant proteins due to low cost, scalability, and safety. Plant-based expression systems have been used to produce recombinant vaccines against influenza based on two targets; the major surface antigen hemagglutinin and the transmembrane protein M2. RESULTS: Different forms of recombinant hemagglutinin were successfully expressed in plants, and some plantproduced vaccines based on hemagglutinin were successfully tested in clinical trials. However, these vaccines remain strain specific, while the highly conserved extracellular domain of the M2 protein (M2e) could be used for the development of a universal influenza vaccine. In this review, the state of the art in developing plant-produced influenza vaccines based on M2e is presented and placed in perspective. A number of strategies to produce M2e in an immunogenic form in plants have been reported, including its presentation on the surface of plant viruses or virus-like particles formed by capsid proteins, linkage to bacterial flagellin, and targeting to protein bodies. CONCLUSION: Some M2e-based vaccine candidates were produced at high levels (up to 1 mg/g of fresh plant tissue) and were shown to be capable of stimulating broad-range protective immunity.

Combination of M2e peptide with stalk HA epitopes of influenza A virus enhances protective properties of recombinant vaccine.

BACKGROUND: Influenza infection could be more effectively controlled if a multi-purpose vaccine with the ability to induce responses against most, or all, influenza A subtypes could be generated. Conserved viral proteins are a promising basis for the creation of a broadly protective vaccine. In the present study, the immunogenicity and protective properties of three recombinant proteins (vaccine candidates), comprising conserved viral proteins fused with bacterial flagellin, were compared. METHODS: Balb/c mice were immunized intranasally with recombinant proteins comprising either one viral protein (the ectodomain of the M2 protein, 'M2e') or two viral proteins (M2e and the hemagglutinin second subunit 'HA2' epitope) genetically fused with flagellin. Further, two different consensus variants of HA2 were used. Therefore, three experimental positives were used in addition to the negative control (Flg-his). The mucosal, humoral, and T-cell immune responses to these constructs were evaluated. RESULT: We have demonstrated that insertion of the HA2 consensus polypeptide (aa 76-130), derived from either the first (HA2-1) or second (HA2-2) virus phylogenetic group, into the recombinant Flg4M2e protein significantly enhanced its immunogenicity and protective properties. Intranasal administration of the vaccine candidates (Flg-HA2-2-4M2e or Flg-HA2-1-4M2e) induced considerable mucosal and systemic responses directed at both the M2e-protein and, in general, the influenza A virus. However, the immune response elicited by the Flg-HA2-1-4M2e protein was weaker than the one generated by Flg-HA2-2-4M2e. These recombinant proteins containing both viral peptides provide complete protection from lethal challenge with various influenza viruses: A/H3N2; A/H2N2; and A/H5N1. CONCLUSION: This study demonstrates that the intranasal administration of Flg-HA2-2-4M2e recombinant protein induces a strong immune response which provides broad protection against various influenza viruses. This construct is therefore a strong candidate for development as a universal vaccine.

Efficient Transient Expression of Recombinant Proteins in Plants by the Novel pEff Vector Based on the Genome of Potato Virus X.

Agroinfiltration of plant leaves with binary vectors carrying a gene of interest within a plant viral vector is a rapid and efficient method for protein production in plants. Previously, we constructed a self-replicating vector, pA7248AMV, based on the genetic elements of potato virus X (PVX), and have shown that this vector can be used for the expression of recombinant proteins in Nicotiana benthamiana. However, this vector is almost 18 kb long and therefore not convenient for genetic manipulation. Furthermore, for efficient expression of the target protein it should be co-agroinfiltrated with an additional binary vector expressing a suppressor of post-transcriptional gene silencing. Here, we improved this expression system by creating the novel pEff vector. Its backbone is about 5 kb shorter than the original vector and it contains an expression cassette for the silencing suppressor, P24, from grapevine leafroll-associated virus-2 alongside PVX genetic elements, thus eliminating the need of co-agroinfiltration. The pEff vector provides green fluorescent protein expression levels of up to 30% of total soluble protein. The novel vector was used for expression of the influenza vaccine candidate, M2eHBc, consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen. Using the pEff system, M2eHBc was expressed to 5-10% of total soluble protein, several times higher than with original pA7248AMV vector. Plant-produced M2eHBc formed virus-like particles in vivo, as required for its use as a vaccine. The new self-replicating pEff vector could be used for fast and efficient production of various recombinant proteins in plants.

High immunogenicity of plant-produced candidate influenza vaccine based on the M2e peptide fused to flagellin.

The ectodomain of the conserved influenza matrix protein M2 (M2e) is a promising target for the development of a universal influenza vaccines. Immunogenicity of M2e could be enhanced by its fusion to bacterial flagellin, the ligand for Toll-like receptor 5. Previously we reported the transient expression in plants of a recombinant protein Flg-4M comprising flagellin fused to 4 tandem copies of the M2e. The use of self-replicating recombinant vector based on the potato virus X allowed expression of Flg-4M in Nicotiana benthaminana leaves at a very high level, up to about 1 mg/g of fresh leaf tissue. Intranasal immunization of mice with Flg-4M induced M2e-specific serum antibodies and provided protection against lethal challenge with different strains of influenza A virus. Here we show that immunization with Flg-4M not only generates a strong immune response, but also redirects the response from the carrier flagellin toward the M2e epitopes. Significant IgG response to M2e was also developed in bronchoalveolar lavages of immunized mice. Protective activity of Flg-4M upon lethal influenza challenge correlated with a decrease of virus titers in lungs relative to the control. Overall these data show the potential for the development of a plant-produced M2e-flagellin universal influenza vaccine.

Contribution of internal initiation to translation of cellular mRNAs containing IRESs.

A broad range of cellular stresses lead to the inhibition of translation. Despite this, some cellular mRNAs are selectively translated under these conditions. It is widely supposed that cap-independent internal initiation may maintain efficient translation of particular cellular mRNAs under a variety of stresses and other special conditions when cap-dependent protein synthesis is impaired. However, in spite of a large number of reports focused on the investigation of the regulation of IRES (internal ribosome entry site) activity in different tissues and under various stresses, only rarely is the real efficiency of IRES-driven translation in comparison with cap-dependent translation evaluated. When precisely measured, the efficiencies of candidate IRESs in most cases appeared to be very low and not sufficient to compensate for the reduction of cap-dependent initiation under stresses. The usually low efficiency of internal initiation of translation is inconsistent with postulated biological roles of IRESs.