Gene amplification and expression by RNA viruses and potential for further application to plant gene transfer

The great majority of plant viruses encapsidate messenger-sense ssRNA and have no natural DNA phase in their life cycle. Despite their RNA nature, essentially any desired change can be introduced into such genomes by using recombinant DNA techniques with suitably constructed, expressible viral cDNA clones. For some viruses such as brome mosaic virus, these methods have been used to define the sequences controlling RNA-directed genomic RNA replication and the expression of internal genes via subgenomic mRNAs. The results suggest a surprising degree of genetic flexibility, which appears to be reflected in the varied gene complements and genetic organizations of presumably related plant and animal RNA viruses sharing conserved replication genes. Foreign genes inserted in such RNA virus genomes can be amplified and expressed to a high level in transfected plant cells. In addition to the potential use of such viruses as episomal expression vectors, it should be possible to couple the viral pathways of RNA-dependent RNA synthesis to amplify and to further regulate the expression of genes transformed into plant chromosomes.     

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.     

Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana

In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest. Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells. Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems. Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein. Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants. Several plant virus vectors have been developed for the expression of foreign proteins. Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants. One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome. Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses.     

Use of viral replicons for the expression of genes in plants

Autonomously replicating virus-based vectors have been investigated as a means of introducing heterologous genes into plants. This approach has a number of potential advantages over stable genetic transformation, particularly in terms of speed and levels of expression that can be obtained. Several groups of plant viruses, with genomes consisting of both DNA and RNA, have been investigated as possible gene vectors. In the case of DNA viruses, it has generally been possible to identify nonessential regions of the genome that can be replaced by foreign sequences. However, there appear to be limitations on the size of insert which can be tolerated. In the case of RNA viruses, replacement of viral sequences usually has a drastic effect on the viability. However, in several cases it has proved possible to substantially increase the size of the viral genome by the direct insertion of additional sequences while still retaining the ability of the viruses to multiply and spread in plants. These RNA virus-based systems appear to have the greatest potential as gene vectors.     

Viral Vectors for Gene Delivery and Expression in the CNS

Viral vectors have emerged as an important tool for manipulating gene expression in the adult mammalian brain. The adult brain is composed largely of nondividing cells, and therefore DNA viruses have become the vehicle of choice for neurobiologists interested in somatic gene transfer. Recombinant viral vectors based upon adenovirus or herpes simplex virus have been created in which a gene essential for viral replication is removed and a gene of interest is inserted in the viral genome. While this eliminates pathogenicity due to viral replication, retention of viral genes and continued expression of these genes may limit the potential of the current generation of vectors. Defective viral vectors represent a different approach, in which only viral recognition signals are used to allow packaging of foreign DNA into a viral coat while eliminating the possibility of viral gene expression within target cells. The defective HSV vector has been used to transfer genes into the adult rat brain. This vector has also been used for analysis of the preproenkephalin promoterin vivo,and important regions of this promoter have been identified using this technique. A modification ofin situPCR has been developed as an adjunctive tool for sensitively documenting the presence of vector DNA within target cells duringin vivopromoter studies. Finally, the adenoassociated virus vector has been used as the first fully defective DNA viral vector, which also eliminates any contamination by helper viruses. This vector can transfer genes into the mammalian brain and has shown significant behavioral recovery in a rodent model of Parkinson's disease. Future work will undoubtedly result in still more diverse and improved vectors; however, these studies have documented the importance of viral vectors to both basic neurobiology and the potential treatment of neurologic disease.     

Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.