A recent paper in Frontiers in Physiology by Taning et al. demonstrated the feasibility of engineering an insect virus into a delivery system capable of eliciting a gene silencing response, through the RNAi pathway, both in vitro and in vivo conditions.
In their study, Taning et al. engineered Flock House virus (FHV), an insect virus composed of two positive-sense single stranded RNAs that encode a RNA-dependent polymerase (RNA1) and a viral capsid protein precursor (RNA2). Since FHV infection results in the production of viral siRNAs, the authors’ rationale was to insert a foreign sequence in the FHV genome to deliver specific RNAi effects in infected cells. For this purpose, the authors developed a recombinant FHV expressing selected Drosophila melanogaster target gene sequences and evaluated whether it could replicate and suppress gene expression in D. melanogaster S2 cells and adult insects.
The authors used a plasmid containing the full FHV RNA1 genome and inserted a polylinker, into position 3037 bp, in order to introduce a reporter gene (enhanced green fluorescent protein, eGFP) and a D. melanogaster target sequence for dsRNA production during viral replication. The plasmid was also designed with a ribozyme sequence derived from hepatitis delta virus (HDV), attached to its 3’ end, necessary for the formation of competent templates for self-directed RNA replication. By using a CuSO4 inducible pMT vector system, primary transcripts of FHV RNA1 were expressed in the cytoplasm of D. melanogaster S2 cells. The S2 cells were also transfected with a recombinant plasmid containing the FHV RNA2 sequence, tailed at its 3’ end by the HDV ribozyme sequence to generate competent templates for capsid protein expression. The expression of both FHV RNA1 and RNA2, in the same S2 cell, allowed for the formation of functional virions.
After confirming the expression of the recombinant FHV RNA1 and RNA2 transcripts in the S2 cells by fluorescence and PCR, Taning et al. showed that the resulting infective recombinant virus were able to induce gene silencing both in vitro (S2 cells) and in vivo (adult flies). The authors used an unpurified virus supernatant to infect virus-free S2 cells and adult flies, observing a decrease in the transcript level of the targeted genes, when a visible increase in cell or insect mortality was detected.
RNAi has become a useful tool in entomology for functional genomics, as well as a potential instrument for controlling insect pests populations. Nevertheless, cellular uptake of dsRNA is a factor that affects RNAi silencing efficiency in numerous insect species. The use of modified viruses with a broad host range, such as FHV, could be an effective delivery system in insect species refractory to the uptake of dsRNA.
Taning Clauvis N. T., Christiaens Olivier, Li XiuXia, Swevers Luc, Casteels Hans, Maes Martine, Smagghe Guy. (2018) Engineered Flock House Virus for Targeted Gene Suppression Through RNAi in Fruit Flies (Drosophila melanogaster) in Vitro and in Vivo. Frontiers in Physiology 9:805, doi: 10.3389/fphys.2018.00805