Abstract
Prior studies have suggested that insect DNA viruses are negatively affected by dicer-2-mediated RNA interference (RNAi). To examine this further, we utilized an in vitro assay to measure dicer activity in lepidopteran and dipteran cells, combined with baculoviruses expressing the RNAi suppressor B2 from Flock House virus or Aedes aegypti dicer-2 (Aedicer-2) using a constitutive heat shock promoter. Addition of cell lysates containing baculovirus-expressed B2 to lysates from dipteran (S2, Aag2) or lepidopteran (Sf9) cells inhibited endogenous dicer activity in a dose-dependent manner, while expression of Aedicer-2 restored siRNA production in Ae. albopictus C6/36 cells, which are dicer-2 defective. However, B2 expression from the constitutive heat shock promoter had no impact on baculovirus replication or virulence in cell lines or larvae that were either highly permissive (Trichoplusia ni) or less susceptible (Spodoptera frugiperda) to infection. We determined that this constitutive level of B2 expression had little to no ability to suppress dicer activity in cell lysates, but higher expression of B2, following heat shock treatment, inhibited dicer activity in all cells tested. Thus, we cannot rule out the possibility that optimized expression of B2 or other RNAi suppressors may increase baculovirus replication and expression of heterologous proteins by baculoviruses.
Highlights
The small interfering RNA pathway is an important antiviral defense mechanism in plants and arthropods, which lack antibody-based immune systems
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is capable of entering dipteran cells and expressing some of its early genes, but there is no productive infection[31,32,33]; we refer to this process as transduction rather than infection
There is evidence that the dicer-2 small interfering RNA (siRNA) pathway can target double-stranded RNAs (dsRNAs) elements produced during insect DNA virus replication
Summary
The small interfering RNA (siRNA) pathway is an important antiviral defense mechanism in plants and arthropods, which lack antibody-based immune systems. Dicer (RNase III endonuclease) enzymes, an important component of the siRNA pathway, bind and digest (or dice) long double-stranded RNAs (dsRNAs) into 21-bp siRNAs. The resulting siRNAs direct an RNA-induced silencing complex (RISC) to scavenge and enable Argonaute-mediated enzymatic degradation (slicing) of RNA in a sequence-specific manner. Several plant and insect RNA viruses encode viral suppressors of RNAi (VSRs), that (1) bind to long dsRNA or siRNAs2 to avoid cellular dsRNA detection and dicing by dicer-2, or (2) interfere with RISC complex formation and Argonaute-2 slicing activity[3]. Transient VSR expression in cells may impact microRNA processing, but, we only address the impact of the B2 VSR on dicer-2 activities Baculoviruses such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are large DNA-containing viruses that are important pathogens of insects, mainly lepidopterans. We generated a baculovirus that expressed the Aedes aegypti dicer-2 (Aedicer-2) ( from the constitutive HS promoter) and assessed the effects of expressing Aedicer-2 or B2 individually or together in permissive lepidopteran or non-permissive dipteran cells
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