Baculovirus Inhibition By RNA Interference
RNA interference (RNAi) is a recently discovered phenomenon of selective mRNA destruction leading to gene knockdown. The first hint of co-suppression was a serendipitous discovery. The unexpected observation of suppression of par-1 gene by both sense and anti-sense strands injected separately to C. elegans was initially noted as a technical aberration. Similarly, a few years later the observation of suggested the probability of RNA induced viral suppression by over expression of sense strand RNA. Apparent influence of dsRNA came to fore by the report of In this study it was shown that when a viral protease gene was expressed either in sense-strand or anti-sense strand, it repressed the viral growth, but the viral inhibition was more profound in the hybrid plants that produced dsRNA. However, it was who showed for the first time that the dsRNA was more potent in inducing gene knockdown than the sense or antisense RNA alone. They suppressed the unc genes of the C. elegans by injecting dsunc RNA. They coined the word RNA interference (RNAi) for this phenomenon. Later they showed that by soaking in dsRNA or by feeding C. elegans with bacteria harbouring dsRNA-producing construct, RNAi could be induced. Subsequently, there has been an explosion of information regarding the RNAi mechanism and the genes knocked down by this technique. RNAi is now believed to be an ancient anti-viral strategy, probably a part of which the cellular machine also utilizes for gene regulation mechanism. For example, Dicer enzyme that is necessary for the initiation of RNAi is also involved in microRNA processing from the stem-loop precursors. Further, mutation of Arabidopsis homologue of Dicer prevents accumulation of miRNAs. The precursor miRNA transcripts (pre-miRNA) are processed by a RNaseIII nuclease (e.g. Drosha), and are exported from the nucleus (by Exportin-5). Dicer cleaves miRNAs into their mature forms to initiate gene silencing. The 3' two-nucleotide overhang structure has been identified as the specificity determinant in targeting and maintaining small RNAs in the RNAi pathway. Thus the miRNA analyses by genetic and biochemical studies are providing a glimpse at the range of biological processes and phenomena regulated by RNAi. These observations lead us to believe the importance of RNAi not only as a natural weapon against pathogens, but also as a tool for exogenous induction of anti-pathogen strategy. RNAi has been well demonstrated in many arthropod species including the silkworm B. mori.
For the economically important lepidopteran insect, the silkworm Bombyx mori L, the most devastating viral disease is caused by a nucleopolyhedrovirus, B. mori NucleoPolyhedra Virus (BmNPV). More than 20% of the crop loss is due to BmNPV infection. With the aim of developing anti-viral strategy, we exploited the RNA interference (RNAi) to inhibit the expression of essential viral genes.In transient cell culture studies, the double stranded RNA (dsRNA) against target baculoviral genes are transfected to BmN cells. The cells are then infected with BmNPV and the viral inhibition is monitored by western blot.
To develop a strain of silkworm resistant to the baculovirus, transgenic silkworms having transgenes expressing dsRNA against target genes are being produced. PiggyBac mediated transgenesis, is used to integrate the transgene to the silkworm genome. The transgenics are now being tested for their viral resistance. Multiple genes of BmNPV are also being targeted to enhance the viral resistance.
Application of RNAi for Baculovirus Inhibition
Silkworm is infected by a number of pathogens including the most devastating virus, the B. mori nucleopolyhedrovirus (BmNPV). As high as 20% of the silkworm cocoon crop is lost due to the BmNPV infection annually in India. The infection is hard to prevent, partly due to sturdy nature of the polyhedra, and partly because of the strains of silkworms. The strains that are silk yield-wise productive are somewhat more susceptible to the virus. Development of a viral-resistant strain of the silkworm is thus one of the priorities for sericulturists. With the aim of developing anti-viral strategy, we exploited the RNAi to inhibit the expression of an essential viral gene, Immediate-Early-1 (ie-1). This essential viral gene is stimulated early in viral replication and regulates a number of viral genes. To test the efficacy of the RNAi technique in B. mori, we initiated a cell culture based system, in which 420 bp of double stranded RNA to target the 5'-terminal region of IE-1 mRNA (dsie-1), was transfected to two lepidopteran insect cell lines, viz. BmN and Sf9. 24 hours post transfection, the cells were infected with BmNPV or Autographa californica multiple nucleopolyhedrovirus (AcNPV), respectively, and western blot was carried out using virus specific anti-gp64 antibody. The results indicate that there was an initial inhibition of viral proliferation of up to 50% at 24 hours post infection (hpi) as compared to untransfected and virus-infected control cells. However, by 96 hpi the virus proliferation matched that of the untransfected, but virally infected control cells. To test in vivo efficacy of RNAi, dsie-1 RNA was injected into the hemolymph of silkworm larvae and 24 hours later, the silkworms were infected with BmNPV. Results show that at low level of viral infection, the dsie-1 successfully prevented viral proliferation. However, the high dose of infected viral particles led to larval deaths, comparable to the un-injected, but virally challenged control worms.
By using PiggyBac mediated transgenesis, we also obtained a transgenic Sf9 cell-line that expresses dsRNA against ie-1 gene. This polyclonal cell-line showed viral inhibition at 48 hpi. These results suggest the possibility of using RNAi in development of baculoviral resistance in the silkworm, B. mori. Using the same construct that gave transgenic Sf9 cell-lines, we have obtained the transgenic silkworms that express dsie-1. These transgenics are made into homozygous line by sib mating and will be used in viral assays. Further investigation is currently being carried out in several of these transgenic silkworms to evaluate the promotion of viral tolerance in the silkworm.