Bioessays. 2014 Dec;36(12):1204-12.

The alien replicon: Artificial genetic constructs to direct the synthesis of transmissible self-replicating RNAs

 

Alex V. Kochetov

Institute of Cytology & Genetics, SB RAS, Novosibirsk, Russia

Novosibirsk State University, Novosibirsk, Russia

Contact: ak@bionet.nsc.ru

 

Abstract

Artificial genetic constructs that direct the synthesis of self-replicating RNA molecules are used widely to induce gene silencing, for bioproduction, and for vaccination. Interestingly, one variant of the self-replicon has not been discussed in the literature: namely, transgenic organisms that synthesise alien replicons. For example, plant cells may be easily genetically modified to produce bacteriophages or insect viruses. Alien replicon-producing organisms (ARPOs) may serve as a unique tool for biocontrol or to selectively influence the characteristics of a target organism. The ARPO approach would have to meet strict biosafety criteria, and its practical applications are problematic. However, a discussion on ARPO applicability would be valuable to outline the full set of options available in the bioengineering toolbox. In this paper, RNA replicons for bioengineering are reviewed briefly, and the ARPO approach is discussed.

KEYWORDS: RNA-viruses; biocontrol; heterologous; replicon

PMID: 25382780

 

Supplement

ARPO construction is technically easy

Many RNA-positive single-stranded viral genomes contain specific packing signals (pac-sites) mediating their interactions with coat protein molecules following by virion assembly. The idea of ARPO is actually quite simple: if cells from the non-host organism express a genetic construct that provides the intracellular synthesis of both viral genomic RNA (as mRNA) and the coat protein, virus particle assembly may occur, and contact with the host organism may result in viral transfection followed by productive infection (see figure for illustration). Thus, an organism may produce an alien replicon in a virulent form (Alien replicon-producing organism, ARPO). The replicon is “alien” because it can’t replicate in the producing cells of non-host organism but may infect the host organism if their contact takes place.

Principal scheme of genetic construct for alien replicon production is quite simple: it should contain two promoters with appropriate transcription patterns and activities in the ARPO cells. cDNA of RNA-positive single-stranded virus (intact or modified) is placed under the control of promoter 1, separate ORF of its coat protein is placed under the direction of promoter 2. If mRNA of the viral replicon and the molecules of its coat protein contact in the cytoplasm of producing cells, the virion assembling may take place. Indeed, there may be some sequence-specific technical problems like occasional aberrant splicing or polyadenylation, as well as low stability of replicon mRNA or coat protein, but they may be overcome by conventional gene engineering tools if it is needed.

Robust and selective interaction between the RNA-replicon and the corresponding coat protein is an important pre-requisite for this process. If a virus meets these criteria, it may be considered a candidate for non-host organism synthesis and ARPO construction.

 

 

AK FIG1 Figure. Genetic construct produces two mRNAs: for viral replicon (1), and for corresponding coat protein (2). Coat protein (3) specifically packs the replicon mRNA into transmissible virus-like particles (4). Plants interact with a target organism susceptible for this virus and the replicon may spread over its population.

 

 

ARPO applications: (potential) usefulness and problems

ARPO efficiency results from the combination of a viral replicon with an opportunity to regulate its dissemination over the long-term. Two principal situations are possible: synthesis of transmissible and non-transmissible replicons. Transmissible (virulent) replicons may spread over the target organism population. The replication of non-transmissible variants is restricted by organisms directly contacting with ARPO.

Several hypothetical variants of plant-based ARPO construction and application were discussed in the paper. First example concerns the transgenic plants synthesizing virulent bacteriophage (MS2 was taken as an example). This type of ARPO may be used for producing phage-synthesizing plants for medicine or veterinary to control antibiotic-tolerant infections. If a virulent phage (or several phages) is produced in an edible plant specially designed for this purpose, it may be used as a food supplement for target organism treatment. Another variant concerns improved plant resistance against specific bacterial pathogens. Local, inducible bacteriophage production in plant cells surrounding pathogen invasion sites may enhance the efficiency of naturally occurring defence mechanisms.

A replicon that influences a target plant’s secondary metabolism characteristics is another example of ARPO use. One variant concerns the selective suppression of morphine synthesizing genes in illegally cultivated Papaver somnifera plants. It is likely to be possible through virus-inducing gene silencing (e.g., Wijekoon and Facchini, 2012). A self-replicating transmissible genetic construct that suppresses the genes in the morphine metabolic pathway and that spreads to the illegal opium poppy areas of cultivation may render heroin production unprofitable (see manuscript for details). Another example of the potential utility of ARPOs is in the area of allergies to certain tree pollens (birch, pines, peach, among others). ARPOs can be used to produce virus-like RNA-replicons inducing highly selective suppression of synthesis of allergenic proteins.

Theoretically, animal viruses may be synthesised in plants using the same type of genetic constructs: cDNAs from genomic RNA and coat protein ORF(s) controlled by plant promoters. If the producing plant belongs to the target organism diet, the viral replicon might penetrate through the gastrointestinal tract. Interestingly, mammals appear vulnerable to this way of alien replicon infiltration: it was demonstrated that VLP-protected mRNAs can penetrate into the mammal cells, and the protein synthesised by the cellular translation machinery may serve as an antigen to induce an immune response. These RNA vaccines have been discussed widely as a prospective technology.

In theory, this technology may be applied for biocontrol. Low-level inducible synthesis of a highly virulent RNA replicon that limits the reproduction rate of an infected target species may decrease the negative effects of its population overgrowth. For Lymantria dispar example, specifically designed “guard” trees might be placed randomly in the contaminated region.

To conclude, the use of transmissible RNA replicons may provide an efficient but risky method of influencing the natural populations of target species as well as some other replicon-based applications for medical and veterinary purposes. It may be applied for highly selective biocontrol, metabolic adjustment, and vaccination. Risks of ARPO application are also based on the intrinsic properties of the replicon (mutations to higher pathogenicity or novel-host variants may not be excluded, the replicon dissemination might proceed beyond the planned area, etc.). However, consideration of this approach is useful because of two reasons: (1) it is important to outline the full set of options available in the bioengineering toolbox; (2) in some situations, using risky techniques may be the only method of preventing intolerable consequences. For example, a natural population of a target organism may come under threat of extinction due to the rise of a new pathogen. In this case, ARPOs may be used for the long-term spread of RNA vaccines in its populations.

 

 

 

 

 

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