Targeting ie‐1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkworms

Abstract

RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1 (ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L. Constitutive expression of double-stranded RNA was achieved by piggyBac-mediated transformation of Sf9 cell line with a transgene encoding double-stranded ie-1 RNA (dsie-1). Strong viral repression was seen at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of B. mori nucleopolyhedrovirus infection, with nearly 40% protection compared with nontransgenic animals. Protection was efficient at larval stages after oral infection with occlusion bodies or hemocoel injection of budded viruses. Virus injected pupae also displayed resistance. These results show that heritable RNAi can be used to protect silkworm strains from baculovirus infection.

Transactivator IE1 Is Required for Baculovirus Early Replication Events That Trigger Apoptosis in Permissive and Nonpermissive Cells

“…RNAi is an invaluable tool for defining functions of virus and host genes in lepidopteran and dipteran cells (18,25,34,48,54,58). The success of RNAi-mediated inhibition of virus gene expression is due in part to the efficiency with which in vitro-synthesized double-stranded RNA (dsRNA) enters cultured insect cells and subsequently engages the host's RNA silencing machinery (reviewed in reference 33).…”

Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

“…TAFib6, a transgenic Nistari line that expresses nonviral dsRNA, and nontransgenic Nistari and CSR2 strains were maintained as controls. The transgenic lines targeting the BmNPV ie1 gene were used and are referred to as IE 126A, IE 126B, and IE 58E, as already described (Kanginakudru et al 2007).…”

“…Total pupal protein was isolated from IE, MG(+/2), MG(+), and MG(2) transgenic and control (Nistari, CSR2, TAFib6) lines infected with 6000 OBs/larva and western blotting was carried out as described previously (Kanginakudru et al 2007). We incubated the membrane with primary antibody GP64 and secondary antibody conjugated to horseradish peroxidase.…”

“…TAFib6, a transgenic Nistari line that expresses nonviral dsRNA, and nontransgenic Nistari and CSR2 strains were maintained as controls. The transgenic lines targeting the BmNPV ie1 gene were used and are referred to as IE 126A, IE 126B, and IE 58E, as already described (Kanginakudru et al 2007).The baculovirus used for infection is the wild-type BmNPV that is usually prevalent in farmers' silkworm rearing house. The OBs were harvested from the infected hemolymph by brief centrifugation at 1000 rpm for 1 min and the pellet was washed in water and suspended in phosphate buffer saline (PBS).…”

“…The availability of a well-established method of transgenesis in Bombyx mori (Royer et al 2005;Tomita 2011) has already led to the construction of transgenic silkworm lines harboring a dsRNA-encoding transgene targeting the essential viral gene lef-1 (Isobe et al 2004) or ie-1 (Kanginakudru et al 2007). Both attempts resulted in moderate but short-lasting effects, which were also reproduced in cultured BmN cells.…”