E. S. Mardanova

Genome sequence and analysis of methylotrophic yeast Hansenula polymorpha DL1

BackgroundHansenula 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.ResultsWe 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.ConclusionsOur 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 recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

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.

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

ABSTRACT 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.

The 5'-Untranslated Region of the Maize Alcohol Dehydrogenase Gene Provides Efficient Translation of mRNA in Plants under Stress Conditions

The reduced level of expression of most cell proteins under stress conditions is determined by the low efficiency of cap-dependent translation of corresponding mRNAs. The maize gene encoding alcohol dehydrogenase, adh1, is a gene whose mRNA is efficiently translated in hypoxia. The reporter gene assay showed that the leader sequence of the adh1 mRNA provided for efficient translation of the reporter gfp gene in Nicotiana benthamiana cells in hypoxia or heat shock. The presence of this sequence in the 5′-UTR of mRNA did not change the level of expression under aerobic conditions, but the levels of gfp expression in hypoxia or heat shock were reduced five-to tenfold in the absence of this leader and remained unaffected when the adh leader sequence was present in the 5′-UTR. The adh1 leader sequence did not change the mRNA stability nor exhibited a promoter activity. Thus, the adh leader sequence acted as a translational enhancer, providing for efficient mRNA translation in plant cells under stress conditions. Introduction of this sequence into standard expression cassettes was proposed for the development of new systems to efficiently express the target proteins in plants under stress conditions.

Complete sequencing of potato virus X new strain genome and construction of viral vector for production of target proteins in plants

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.

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.

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.

The optimization of viral vector translation improves the production of recombinant proteins in plants

One of the most convenient methods for the fast and efficient production of target proteins in plants involves self-replicating recombinant viral vectors. We have constructed a plant viral vector based on the genome of the potato X virus. This vector contains the sequence of the 5′-untranslated region of RNA 4 of the alfalfa mosaic virus immediately upstream of the target gene. The incorporation of this sequence into the viral vector increases the production of the target protein by the recipient plant three- to fourfold owing to the increased efficiency of translation of viral subgenomic RNA comprising the target gene. The new vector can be used for the production of recombinant proteins in plants.

Expression of genes involved in DNA repair and telomere maintenance in the yeast Hansenula polymorpha DL1 under heat stress

185 Methylotrophic yeasts are widely used to study methanol metabolism, biogenesis and functions of peroxisomes, and as a “biofactory” to produce recom binant proteins. A characteristic feature of the Hansenula polymorpha DL1 strain ATCC 26012 is thermotolerance: these yeasts can grow at tempera tures up to 50°C [1]. This ability determines the higher thermal stability of proteins and nucleoprotein com plexes of these yeasts, which makes them an interest ing model object for studying the molecular processes such as DNA repair and telomere formation [2]. Pre viously, we determined the complete nucleotide sequence of the mitochondrial and nuclear genome of H. polymorpha DL1 and analyzed transcription when this strain was growth on glucose medium or methanol [3, 4].

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.