The Expression of Matryoshka Gene Encoding a Homologue of Kunitz Peptidase Inhibitor Is Regulated Both at the Level of Transcription and Translation.

The gene for Kunitz peptidase inhibitor-like protein (KPILP) contains nested alternative open reading frame (aORF) that controls expression of the maternal mRNA. The content of NbKPILP mRNA in intact leaves of Nicotiana benthamiana plant is low but increases significantly upon extended dark exposure or when foreign nucleic acid is overexpressed in the cells. The NbKPILP gene promoter along with the expressed nested aORF are likely to play an important role in maintaining the levels of NbKPILP mRNA. To elucidate the role of NbKPILP promoter, we isolated a fragment of N. benthamiana chromosomal DNA upstream of the NbKPILP transcription start, sequenced it, and created constructs in which reporter E. coli uidA gene coding for ß-D-glucuronidase (GUS) was placed under control of the NbKPILP promoter. By assessing the efficacy of uidA mRNA synthesis directed by the NbKPILP promoter and 35S promoter of the cauliflower mosaic virus in a transient expression system, we showed that the levels of GUS accumulation were comparable for both promoters. Prolonged incubation of the agroinjected plants in the darkness stimulated accumulation of the uidA mRNA directed by the NbKPILP promoter. Our experiments indicate that along with regulation at the transcriptional level, expression of NbKPILP mRNA can be affected by expression of the nested aORF controlled by the polypurine block (PPB) located upstream of its start codon, since introduction of mutations in the PPB resulted in significant accumulation of the NbKPILP mRNA. Nucleotide replacement in the aORF start codon led to the drastic increase in the amounts of NbKPILP mRNA and its protein product.

Trastuzumab and Pertuzumab Plant Biosimilars: Modification of Asn297-linked Glycan of the mAbs Produced in a Plant with Fucosyltransferase and Xylosyltransferase Gene Knockouts.

Plant biosimilars of anticancer therapeutic antibodies are of interest not only because of the prospects of their practical use, but also as an instrument and object for study of plant protein glycosylation. In this work, we first designed a pertuzumab plant biosimilar (PPB) and investigated the composition of its Asn297-linked glycan in comparison with trastuzumab plant biosimilar (TPB). Both biosimilars were produced in wild-type (WT) Nicotiana benthamiana plant (PPB-WT and TPB-WT) and transgenic ΔXTFT N. benthamiana plant with XT and FT genes knockout (PPB-ΔXTFT and TPB-ΔXTFT). Western blot analysis with anti-α1,3-fucose and anti-xylose antibodies, as well as a test with peptide-N-glycosidase F, confirmed the absence of α1,3-fucose and xylose in the Asn297-linked glycan of PPB-ΔXTFT and TPB-ΔXTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that PPB-WT and TPB-WT Asn297-linked glycans are mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297-linked GnGn-type glycan contains α1,3-fucose and ß1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguishes these plant biosimilars from human IgG. Analysis of TPB-ΔXTFT total carbohydrate content indicates the possibility of changing the composition of the carbohydrate profile not only of the Fc, but also of the Fab portion of an antibody produced in transgenic ΔXTFT N. benthamiana plants. Nevertheless, study of the antigen-binding capacity of the biosimilars showed that absence of xylose and fucose residues in the Asn297-linked glycans does not affect the ability of the glycomodified antibodies to interact with HER2/neu positive cancer cells.

Plant Factories for the Production of Monoclonal Antibodies.

Like animal cells, plant cells bear mechanisms for protein synthesis and posttranslational modification (glycosylation and phosphorylation) that allow them to be seriously considered as factories for therapeutic proteins, including antibodies, with the development of biotechnology. The plant platform for monoclonal antibody production is an attractive approach due to its flexibility, speed, scalability, low cost of production, and lack of contamination risk from animal-derived pathogens. Contemporary production approaches for therapeutic proteins rely on transgenic plants that are obtained via the stable transformation of plant cells as well as the transient (temporary) expression of foreign proteins. In this review, we discuss present-day approaches for monoclonal antibody production in plants (MAPP), features of carbohydrate composition, and methods for the humanization of the MAPP carbohydrate profile. MAPPs that have successfully passed preclinical studies and may be promising for use in clinical practice are presented here. Perspectives on using MAPPs are determined by analyzing their economic benefits and production rates, which are especially important in personalized cancer therapy as well as in cases of bioterrorism and pandemics.

Functional Role of Carbohydrate Residues in Human Immunoglobulin G and Therapeutic Monoclonal Antibodies.

Therapeutic monoclonal antibodies (TMA) provide an important means for treating diseases that were previously considered untreatable. Currently more than 40 full-size TMAs created primarily based on immunoglobulin G1 are widely used for treating various illnesses. Glycosylation of TMA is among other numerous factors that affect their biological activity, effector functions, immunogenicity, and half-life in the patient's serum. The importance of carbohydrate residues for activity of human serum immunoglobulin and TMA produced in animal cells is considered in this review, with emphasis given to N-glycosylation of the Fc fragment of the antibody.

Pectin methylesterase-generated methanol may be involved in tobacco leaf growth.

Plant leaves undergo a sink-source modification of intercellular macromolecular transport during the transition from carbon import to carbon export. After assessing the role of metabolite signaling in gene regulation in Nicotiana tabacum sink and source leaves, we observed increased pectin methylesterase (PME)-mediated methanol generation in immature leaves. Using suppression subtractive hybridization (SSH), we identified a number of genes whose activity changes from sink to source leaves. The most abundant SSH-identified genes appeared to be sensitive to methanol. We hypothesize that tobacco leaf maturation and the sink-source transition are accompanied by a change in mRNA levels of genes that function in methanol-dependent cell signaling.

Production of biologically active human myelocytokines in plants.

An effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hG-CSF and hGM-CSF) in Nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. The genes of target proteins were cloned into the viral vector driven by actin promoter of Arabidopsis thaliana. The expression vectors were delivered into plant cells by agroinjection. Maximal synthesis rate was detected 5 days after injection and was up to 500 and 300 mg per kg of fresh leaves for hG-CSF and hGM-CSF, respectively. The yield of purified hG-CSF and hGM-CSF was 100 and 50 mg/kg of fresh leaves, respectively. Recombinant plant-made hG-CSF and hGM-CSF stimulated proliferation of murine bone marrow and human erythroleucosis TF-1 cells, respectively, at the same rate as the commercial drugs.

Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach.

An effective procedure for specific determination of the cap structure at the 5'-terminus of mRNA and for isolation of the corresponding full-length cDNA has been developed. The procedure involves covalent attachment of an oligonucleotide template extender to the 5'-cap structure of mRNA followed by RT-PCR using M-MLV SuperScript II reverse transcriptase. In the course of reverse transcription, the enzyme 'jumps over' the cap structure and includes the sequence complementary to the oligonucleotide template extender into the 3'-end of the first cDNA strand. The cap-jumping method was successfully tested using some mammalian cellular mRNAs, genomic RNAs of tobacco mosaic virus (TMV) U1 and the recently isolated crucifer-infecting tobamovirus. Moreover, cDNA products corresponding to the genomic tobamovirus RNA were obtained from total RNA extracted from tobacco plants infected by crucifer-infecting tobamovirus or tobacco mosaic virus. Using the cap-jumping method, we have shown for the first time that genomic crucifer-infecting tobamovirus (crTMV) RNA contains a 5'-cap structure. This improved method can be recommended for the construction of full-length and 5'-end enriched cDNA libraries, identification of capped RNAs and determination of their 5'-terminal sequences.

Visualization by atomic force microscopy of tobacco mosaic virus movement protein-RNA complexes formed in vitro.

The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP-RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60-100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive 'beads-on-a-string' into a 'thick string' form that was partly resistant to RNAse. The 'thick string'-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The 'thick string' complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.

Internal initiation of translation directed by the 5'-untranslated region of the tobamovirus subgenomic RNA I(2).

Previously we reported that, unlike RNA of typical tobamoviruses, the translation of the coat protein (CP) gene of a crucifer-infecting tobamovirus (crTMV) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (IRES(CP,148)(CR)). The equivalent 148-nt sequence from TMV U1 RNA (U1(CP,148)(SP)) was incapable of promoting internal initiation. In the present work, we have found that the 228-nt region upstream of the movement protein (MP) gene of crTMV RNA (IRES(MP,228)(CR)) contained an IRES element that directed in vitro translation of the 3'-proximal reporter genes from chimeric dicistronic transcripts. Surprisingly, the equivalent 228-nt sequence upstream from the MP gene of TMV U1 directed translation of the downstream gene of a dicistronic transcripts as well. Consequently this sequence was termed IRES(MP,228)(U1). It was shown that IRES(MP,228)(CR), IRES(MP,228)(U1), and IRES(CP,148)(CR) could mediate expression of the 3'-proximal GUS gene from dicistronic 35S promoter-based constructs in vivo in experiments on transfection of tobacco protoplasts and particle bombardment of Nicotiana benthamiana leaves. The results indicated that an IRES element was located within the 75-nt region upstream of MP gene (IRES(MP,75)), which corresponded closely to the length of the 5'UTR of TMV subgenomic RNA (sgRNA) I(2). The RNA transcripts structurally equivalent to I(2) sgRNAs of TMV U1 and crTMV, but containing a hairpin structure (H) immediately upstream of IRES(MP,75) (HIRES(MP), (75)(CR)-MP-CP-3'UTR; HIRES(MP,75)(U1)-MP-CP-3'UTR), were able to express the MP gene in vitro. The capacity of HIRES(MP,75)(CR) sequence for mediating internal translation of the 3'-proximal GUS gene in vivo, in tobacco protoplasts, was demonstrated. We suggested that expression of the MP gene from I(2) sgRNAs might proceed via internal ribosome entry pathway mediated by IRES(MP) element contained in the 75-nt 5'UTR. Our results admit that a ribosome scanning mechanism of the MP gene expression from I(2) sgRNA operates concurrently.

A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein.

Plant virus-encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall-associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell-to-cell movement of tobacco mosaic virus.

Phosphorylation of tobacco mosaic virus movement protein abolishes its translation repressing ability.

Previously we showed that the ribonucleoprotein complexes (RNPs) of the TMV 30-kDa movement protein (MP) with TMV RNA are nontranslatable in vitro and noninfectious to protoplasts, but are infectious to intact plants. It has been suggested that MP-TMV RNA complexes could be converted into the translatable and replicatable form in planta in the course of passage through plasmodesmata (Karpova et al., 1997, Virology 230, 11-21). The role of TMV MP phosphorylation was investigated in terms of its capacity to modulate the translation-repressing ability of the MP. Phosphorylation of the TMV MP, either before or after RNP complex formation, caused a conversion of nontranslatable MP-RNA complexes into a form that was translatable in vitro and infectious to protoplasts and plants.

The immobilized movement proteins of two tobamoviruses form stable ribonucleoprotein complexes with full-length viral genomic RNA.

The movement proteins of two tobamoviruses (tobacco mosaic virus, TMV, common strain U1 and cruciferous TMV strain) containing amino-terminal hexahistidine affinity tags were overexpressed in Escherichia coli and purified by metal chelate affinity chromatography. Purified recombinant proteins were immobilized to a Ni(2+)-chelate adsorbent and their ability to interact with full-length genomic TMV RNA was tested. Here we report that binding of viral RNA to hexahistidine fusion movement proteins results in the formation of stable ribonucleoprotein complexes.

Direct RNA polymerase chain reaction for TMV detection in crude cell extracts.

A part of the 30,000 bp transport protein gene of tobacco mosaic virus (TMV) RNA was amplified via direct RNA PCR and via traditional reverse transcription followed by cDNA PCR. Both amplified cDNA products were restricted with NcoI or HaeIII endonucleases and identical restriction fragments were produced. Two efficient methods of viral RNA concentration from an infected tobacco leaf extract were used: both 3-3.5 M sodium acetate alone and 3 M LiCl with 4 M urea quantitatively precipitated TMV RNA from the extracts. TMV RNA thus obtained could be readily amplified by direct RNA PCR. These results demonstrate that direct RNA PCR can be applied for the detection or/and analysis of high molecular weight RNA and for diagnosis of viral infections.

The informosome-like virus-specific ribonucleoprotein (vRNP) may be involved in the transport of tobacco mosaic virus infection.

A new type of informosome-like virus-specific ribonucleoprotein (vRNP) differing from mature tobacco mosaic virus (TMV) particles in buoyant density and structure was found in TMV-infected cells (Yu. L. Dorokhov, N. M. Alexandrova, N. A. Miroshnichenko, and J. G. Atabekov, 1983, Virology 127, 237-252). Two groups of TMV ts mutants were used to discover whether there is a correlation between the vRNP formation and systemic spreading of virus infection (transport) over the infected plant. The first group of mutants (Ni118, flavum) contains a ts mutation in the coat protein gene but are capable of systemic spreading at nonpermissive temperature (tr transport); the second group of mutants (Ni2519, Ls1) cannot spread systemically at restrictive temperature (ts transport). It is shown that vRNP can be produced at restrictive temperature by tr-transport mutants but not by ts-transport mutants. The latter can produce vRNP only at a permissive temperature. The role of vRNP in long-distance transport of the virus infection is supported by two other observations: (a) upper leaves that were maintained at 5 degrees accumulate potentially infective material and material with the properties of vRNP but not virus particles and (b) plants that were simultaneously infected with Lsl and Ni118 at a non-permissive temperature exhibited long-distance transport and vRNP. These results also implicate coat protein in long-distance transport. It is suggested that vRNPs are novel types of virus-specific particles that are involved in both cell-to-cell and long-distance transport of TMV infections.

Stimulation by aurintricarboxylic acid of tobacco mosaic virus-specific RNA synthesis and production of informosome-like infection-specific ribonucleoprotein.

It was shown that aurintricarboxylic acid (ATA), a well-known inhibitor of protein synthesis, markedly stimulates the synthesis of tobacco mosaic virus (TMV)-specific RNA species of the intermediate (I) class (apparent molecular weights 1.1-1.3 x 10(6) and 0.6-0.8 x 10(6)). No stimulation by ATA of full-length genomic TMV RNA or the subgenomic TMV RNA coding for TMV coat protein was detected. Informosome-like infection-specific ribonucleoprotein (vRNP) particles different from mature TMV particles were found in the TMV-infected cells (Yu. L. Dorokhov, N. M. Alexandrova, N. A. Miroshnichenko, and J. G. Atabekov, 1983, Virology 127, 237-252). It is shown here that [3H]uridine incorporation into vRNP RNAs was markedly stimulated in the presence of ATA. vRNP can be released from the TMV-specific polyribosomes by EDTA treatment, which suggests that it is involved in the translation process. The results of the pulse-chase experiments (including those in which TMV RNA synthesis is blocked by 2-thiouracil) suggest that vRNP does not serve as a precursor for mature virion.

Isolation and analysis of virus-specific ribonucleoprotein of tobacco mosaic virus-infected tobacco.

A ribonucleoprotein fraction (vRNP) of a characteristic buoyant density greater than the buoyant density of tobacco mosaic virus (TMV) particles has been isolated from infected tissue by Cs2SO4 density gradient centrifugation. The vRNP particles appear to be TMV specific because they are synthesized in the presence of actinomycin D and have RNAs identified as genomic and I-class subgenomic (apparent Mr 1.1-1.3 x 106 and 0.60.8 x 10(6)) RNAs by their electrophoretic mobility and hybridization to plasmid-bearing RNA sequences. Polypeptides of apparent molecular weights 17,500 (TMV coat), 31,000, 37,000, and 39,000 were major constituents of vRNP. Of the minor polypeptides, those of apparent molecular weights 70,000, 68,000, 55,000, and 25,000 had electrophoretic mobilities similar to mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. vRNP from common TMV-infected plants, but not from plants infected with a mutant that did not form native coat protein, reacted with immunoglobins against TMV and TMV coat protein. Common TMV and its vRNP differed in the extent of reactivity toward the two immunoglobins, in electron microscopic appearance, and in the higher sensitivity of vRNP to ribonuclease.

Development of systemic TMV infection in upper noninoculated tobacco leaves after differential temperature treatment.

A differential temperature treatment (DTT) of infected plants ensured a partial synchronization of the process of tobacco mosaic virus (TMV) infection. The "infectious entity" enters (and spreads to) basal areas of the upper noninoculated leaves held at low temperature, and then to a distal part of these leaves and basal sites of the lower leaves. The spread of the "infectious entity" over the vascular system of the upper leaves could be followed.

Polyribosomes in protoplasts isolated from tobacco leaves.

Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.