Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events.monoclonal antibody ͉ potato virus X ͉ tobacco mosaic virus A lthough the ability of plants to express full-size human antibodies was discovered 17 years ago (1-3), the idea of industrial-scale antibody production in plants has been abandoned by most companies, mostly because of limitations of existing expression protocols. Stably transformed (transgenic) plants are able to express correctly folded and functional antibodies of both the IgG and IgA classes, but yields are generally very low (usually in the range of 1-40 g͞g of fresh biomass); in addition, the time necessary to generate the first grams of research antibody material is very long, requiring Ͼ2 years (4-8).Transient expression systems, on the other hand, allow production of research quantities of antibody material much faster. However, the early versions of transfection systems, such as Agrobacterium-mediated transient expression or viral vectormediated expression, cannot provide for high-level coexpression of two or several polypeptides necessary for the assembly of heterooligomeric proteins, in particular IgG antibodies (9-12).Recently, we have developed a scalable transient expression technology (magnifection) that is based on replication of viral vectors delivered to multiple parts of a plant body (systemic delivery) by Agrobacterium (13). Such a technology is in essence an en masse infiltration of whole, mature plants with a diluted agrobacteria suspension carrying T-DNAs encoding viral replicons. The magnifection process allows expression of various proteins, but, until now, it has been used to express only single-polypeptide proteins or homooligomers (14). Attempts to express two or more different polypeptides from one viral replicon failed because of drastically reduced expression levels obtained with bicistronic constructs (unpublished results).Therefore, we decided to explore expression protocols that involve two or more viral replicons. We report here a general solution for coexpression of high amounts of two heterologous polypeptides by using two d...

eIF4E and eIFiso4E T AbstractEukaryotic initiation factor eIF4E plays a pivotal role in translation initiation. As a component of the ternary eIF4F complex, eIF4E interacts with the mRNA cap structure to facilitate recruitment of the 40S ribosomal subunit onto mRNA. Plants contain two distinct cap-binding proteins, eIF4E and eIFiso4E, that assemble into different eIF4F complexes. To study the functional roles of eIF4E and eIFiso4E in tobacco, we isolated two corresponding cDNAs, NteIF4E1 and NteIFiso4E1, and used these to deplete cap-binding protein levels in planta by antisense downregulation. Antibodies raised against recombinant NteIF4E1 detected three distinct cap-binding proteins in tobacco leaf extracts; NteIF4E and two isoforms of NteIFiso4E. The three cap-binding proteins were immuno-detected in all tissues analysed and were coordinately regulated, with peak expression in anthers and pollen. Transgenic tobacco plants showing significant depletion of either NteIF4E or the twoNteIFiso4E isoforms displayed normal vegetative development and were fully fertile.Interestingly, NteIFiso4E depletion resulted in a compensatory increase in NteIF4E levels, whereas the down-regulation of NteIF4E did not trigger a reciprocal increase in NteIFiso4E levels. The antisense depletion of both NteIF4E and NteIFiso4E resulted in plants with a semi-dwarf phenotype and an overall reduction in polyribosome loading, demonstrating that both eIF4E and eIFiso4E support translation initiation in planta, which suggests their potential role in the regulation of plant growth.

The subgenomic RNA 2 of tobacco necrosis virus A (TNV sgRNA2) encodes the viral coat protein, is unpolyadenylated and presumably uncapped. Here, we show that TNV sgRNA2 is translated cap independently. This cap-independent translation requires the leader and a 140 nt element of the trailer both in wheat germ extract and in tobacco protoplasts. Similar to barley yellow dwarf virus (BYDV), the TNV 5' and 3' elements stimulate translation synergistically. Computer-aided phylogenetic analysis of the secondary structure of the TNV trailer revealed that the 3' translation element is part of a major conserved stem-loop that contains similarities to structures in the BYDV 3' translation element. These data suggest that the translation mechanisms of TNV sgRNA2 and BYDV RNA are related. To further characterize this relationship, we tested whether cooperativity exists between TNV sgRNA2 and BYDV 5' and 3' elements. We found that the TNV sgRNA2 5' element stimulates translation synergistically with the BYDV 3' element in vitro. This finding is the first evidence for conservation of structures that enable a 5'-3' interaction stimulating cap-independent translation.