Nairovirus RNA sequences expressed by a Semliki Forest virus replicon induce RNA interference in tick cells

Abstract

We report the successful infection of the cell line ISE6 derived from Ixodes scapularis tick embryos by the tick-borne Hazara virus (HAZV), a nairovirus in the family Bunyaviridae. Using a recombinant Semliki Forest alphavirus replicon that replicates in these cells, we were able to inhibit replication of HAZV, and we showed that this blockage is mediated by the replication of the Semliki Forest alphavirus replicon; the vector containing the HAZV nucleoprotein gene in sense or antisense orientation efficiently inhibited HAZV replication. Moreover, expression of a distantly related nucleoprotein gene from Crimean-Congo hemorrhagic fever nairovirus failed to induce HAZV silencing, indicating that the inhibition is sequence specific. The resistance of these cells to replicate HAZV correlated with the detection of specific RNase activity and 21- to 24-nucleotide-long small interfering RNAs. Altogether, these results strongly suggest that pathogen-derived resistance can be established in the tick cells via a mechanism of RNA interference.

FIG. 1.

HAZV replicates in ISE6 cells. Semiconfluent monolayers of ISE6 cells were infected with HAZV at an MOI of 1. Mock-infected cells served as controls. (A) Virus in the culture medium was collected at different times p.i., and titers were determined. (B) Proteins from extracts of HAZV-infected or uninfected cells were analyzed after electrophoresis in 10% polyacrylamide gel and Western blot using a mouse hyperimmune ascitic fluid specific to HAZV nucleoprotein. (C) ISE6 cells uninfected (mock) or infected with HAZV were stained with polyclonal antibody specific to the nucleoprotein on day 2, 4, 6, or 8 p.i.

FIG. 2.

Viral RNA and protein synthesis were silenced in ISE6 cells infected with SFV-HAZ-Sg and superinfected with HAZV. Shown are Western blot (A) and Northern blot (B) analyses of N and S RNA expression in uninfected ISE6 cells (lane 1), cells infected with HAZV (lane 2), or cells superinfected with HAZV after being infected with SFV1 (lane 3), SFV-HAZ-Sag (lane 4), or SFV-HAZ-Sg (lane 5). Note that HAZV S RNA migrates in close proximity with the 18S rRNA which is labeled nonspecifically by the HAZV probe utilized for Northern blots.

FIG. 3.

ISE6 cells harboring SFV-HAZ-Sg possess HAZV sequence-specific RNase activity generating siRNAs. A. Sequence-specific RNase activity. Transcripts synthesized in vitro corresponding to the HAZV N sequence (left panel) or to the N sequence of Rift Valley fever virus (RVFV, right panel) were incubated in a final volume of 10 μl with lysate prepared from cells that had been infected with SFV-HAZ-Sg. After incubation during the indicated time (in minutes), RNAs were extracted and analyzed in a 4% polyacrylamide gel containing 6 M urea. B. Detection of siRNAs by RNase protection assay. Probes representing a region of HAZV S RNA (left panel) or SFV RNA (right two panels) were synthesized in vitro as ³²P-labeled RNA transcripts. Total RNAs from uninfected ISE6 cells (lanes 2, 5, and 9) or cells infected with SFV-HAZ-Sg (lanes 3, 4, 6, and 7) or SFV1 (lanes 10 and 11) and harvested at 48 h p.i. (lanes 3, 7, and 11) or 72 h p.i. (lanes 4, 6, and 10) were hybridized with the indicated probe. As controls, reactions were performed with the probes alone (lanes 1, 8, and 12). The position of the ³²P-labeled 20- and 30-nucleotide-long RNA markers is indicated (lane M).