RESUMO
Besides regular cap-dependent translation of mRNA, eukaryotes exploit internal initiation of translation driven by internal ribosome entry sites (IRESs). It is supposed that internal initiation provides translation of cellular mRNAs under stress conditions where the cap-dependent initiation is reduced. A number of IRESs have been characterized in mammalian mRNAs, but only a few examples are known in lower eukaryotes, particularly in yeasts. Here we identified two IRESs in the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1. These sites are located in 5'-untranslated regions of genes HPODL_02249 and HPODL_04025 encoding a hypothetical membrane protein and actin-binding protein, respectively. In Saccharomyces cerevisiae cells, both IRESs drive expression of a second gene of a bicistronic mRNA, as well as translation of hairpin-containing monocistronic mRNA. The possibility of spurious splicing or presence of a cryptic promoter in the IRES sequences was ruled out, indicating that expression of a second gene of a bicistronic mRNA was IRES-dependent. We evaluated IRES activity of both elements and found that under normal physiological conditions its contribution to the overall translation of the respective mRNAs in yeast cells is about 0.3-0.4%. Therefore, these results suggest that the IRES-dependent translation initiation mechanism exists in Hansenula polymorpha.
Assuntos
Sítios Internos de Entrada Ribossomal/genética , RNA Mensageiro/metabolismo , Saccharomycetales/genética , Regiões 5' não Traduzidas , Sequência de Bases , Bases de Dados Genéticas , Genes Reporter , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Biossíntese de Proteínas/genética , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Saccharomyces cerevisiae/metabolismoRESUMO
Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1-2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vacinas contra Influenza/metabolismo , Influenza Humana/prevenção & controle , Nicotiana/metabolismo , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Animais , Vetores Genéticos , Antígenos do Núcleo do Vírus da Hepatite B/genética , Humanos , Imunoglobulina G/metabolismo , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Camundongos , Dados de Sequência Molecular , Nanotecnologia , Tamanho da Partícula , Potexvirus/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Vacinas Sintéticas/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
One of the most efficient methods for fast and efficient production of the target proteins in plants is based on the use of self-replicating recombinant viral vectors. We constructed phytoviral vector based on the genome of potato X virus containing the sequence of 5'-untranslated region of RNA 4 of alfalfa mosaic virus immediately upstream of the target gene. We demonstrated that incorporation of this sequence into the viral vector results in 3-4 fold elevation of the level of production of the target protein in plant due to increased efficiency of translation of viral subgenomic RNA comprising the target gene. The new vector may be used for production of recombinant proteins in plants.
Assuntos
Vetores Genéticos , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Potexvirus/genética , RNA Viral/biossíntese , Proteínas Recombinantes/biossíntese , Vírus do Mosaico da Alfafa/genética , Plantas Geneticamente Modificadas/genética , Rhizobium/genética , Nicotiana/genética , TransfecçãoRESUMO
The complete nucleotide sequence of the genome of a new potato virus X (PVX) strain Tula isolated by us has been determined. Based on comparison of the PVX Tula nucleotide sequence with the sequences of 12 other PVX strains, this strain was assigned to the European cluster of PVX strains. Phylogenetic analysis revealed the same phylogeny for both full genome sequences and nucleotide sequences of polymerase and coat protein genes, suggesting that the PVX evolution did not involve recombination between different strains. The full-size cDNA copy of the PVX Tula genome was cloned and the accumulation of the viral coat protein in infected Nicotiana benthamiana was shown to be about twofold higher than for the PVX strain UK3. Based on the PVX Tula genome, a new vector which contained the target gene instead of the removed triple transport gene block and the coat protein gene has been constructed for expression of target proteins in plants. The productivity of the new vector was about 1.5-2-fold higher than the productivity of the vector of the same structure based on the standard PVX strain genome. The new viral vector can be used for superproduction of recombinant proteins in plants.
Assuntos
Vetores Genéticos , Genoma Viral , Potexvirus/genética , Proteínas Recombinantes/biossíntese , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , DNA Complementar/química , Genômica , Conformação de Ácido Nucleico , Filogenia , Potexvirus/classificação , RNA Viral/química , Nicotiana/genética , Nicotiana/virologiaRESUMO
Reduced level of expression of most cell proteins under stress conditions is determined by low efficiency of cap-dependent translation of corresponding mRNAs. The maize gene encoding alcohol dehydrogenase, adh1, is an example of a gene which mRNA is efficiently translated under hypoxia. Using reporter gene assay we showed that the leader sequence of adh1 mRNA, provides efficient translation of reporter gene gfp in Nicotiana benthamiana cells under hypoxia and heat shock. The presence of this leader sequence in 5' UTR of mRNA does not change the level of expression in aerobic conditions, but under hypoxia and heat shock the levels of reporter gfp expression were reduced about 5-10 fold in the absence of leader and remained unaffected in its presence in 5'UTR. We found that this leader sequence does not change the level of mRNA stability and does not exhibit promoter activity. Consequently, leader sequence acts as translational enhancer providing efficient translation of mRNA in plant cells under stress conditions. Introduction of this sequence into standard expression cassettes may be used for development of new systems of expression of target proteins in plants, efficient under stress conditions.