Expression of a viral polymerase-bound host factor turns human cell lines permissive to a plant- and insect-infecting virus

Abstract

Tospoviruses are the only plant-infecting members of the Bunyaviridae family of ambisense ssRNA viruses. Tomato spotted wilt tospovirus (TSWV), the type-member, also causes mild infection on its main insect vector, Frankliniella occidentalis. Herein, we identified an F. occidentalis putative transcription factor (FoTF) that binds to the TSWV RNA-dependent RNA polymerase and to viral RNA. Using in vitro RNA synthesis assays, we show that addition of purified FoTF improves viral replication, but not transcription. Expression of FoTF deletion mutants, unable to bind the RNA-dependent RNA polymerase or viral RNA, blocks TSWV replication in F. occidentalis cells. Finally, expression of FoTF wild-type turns human cell lines permissive to TSWV replication. These data indicate that FoTF is a host factor required for TSWV replication in vitro and in vivo, provide an experimental system that could be used to compare molecular defense mechanisms in plant, insect, and human cells against the same pathogen (TSWV), and could lead to a better understanding of evolutionary processes of ambisense RNA viruses.

Fig. 1.

F. occidentalis FoTF binds to the TSWV RdRp. (A) TSWV RdRp fragments used in this study, respective domains, and their respective position in relation to the whole RdRp are shown. (B) Immunoprecipitation (IP) and Western blotting (WB) using anti-TL2249, anti-FoTF, and anti-N Abs. Abs were incubated with whole-cell lysates from TSWV-infected F. occidentalis larvae. (C) WB after GST pulldown with pGEX empty vector, pGEX-TL2249, pGEX-FoTF, pGEX-FoTFΔN, and pGEX-FoTFΔC. GST beads were applied to whole-cell lysates of TSWV-infected F. occidentalis larvae. (D) Yeast two-hybrid X-Gal filter staining assay after cotransformation with TSWV RdRp fragments (pTL1, pTL1797, pTL2249, and pTL908) or TSWV N (pN) with F. occidentalis FoTF (pFoTF); yeast growth (and β-gal expression) indicates direct interaction; negative controls were transformations with pFoTF and pTL2249 only; positive control was pLC1 (which codes for GAL4, and triggers β-gal expression without the need of any interaction). (E) FoTF map and position of deletion mutants used in this study.

Fig. 2.

RNA gel shift assay. Autoradiograph of RNA-binding reactions after addition of radiolabeled TSWV S RNA transcripts to purified FoTF only (A), TSWV N protein as positive control (B), FoTFΔN(C), BSA as a negative control (D), or FoTF plus nonlabeled TSWV S RNA (E). Protein concentrations varied from 0 to 20 μM; –, no protein added.

Fig. 3.

In vitro RNA synthesis assay. (A) Gel electrophoresis of radiolabeled TSWV RNA products after amendments to the standard reaction, as indicated in the table. After decay of the radioactivity, gels as shown in A were submitted for Northern hybridization by using probes specific to vc-S RNA (B), vc-M RNA (C), and vc-L RNA (D). (E) Probe specific to the N gene, the N mRNA, is visualized, and can be differentiated by the size (1.2 kb). (F) Probe specific to the NSs gene, the NSs mRNA, could be visualized because it could be differentiated by the size (1.7 kb); h-i, heat-inactivated virus (lane 1); a molecular weight marker (lane M) shows relative sizes of detected RNA species in A, the sizes, in kb, are indicated in B–F. Results shown are representative of six independent experiments.

Fig. 4.

TSWV infection of F. occidentalis FO1 cells. (A) Graphic representation of NSm mRNA accumulation over time, after TSWV infection, of nontransfected (solid line and squares) and FoTF-transfected FO1 (dashed lines and diamonds). (B) Northern hybridizations showing NSm mRNA accumulation over time of nontransfected and FoTF-transfected FO1, as indicated, and actin mRNA as the loading control from one of the blots. (C) Graphic representation of NSm mRNA accumulation over time of FoTF-, FoTFΔC-, and TL2249-transfected FO1. (D) Northern hybridizations showing NSm mRNA accumulation over time of FoTFΔC- and TL2249-transfected FO1, as indicated, and actin mRNA as the loading control from one of the blots. Results shown are representative of three independent experiments. The highest RNA signal of all three experiments, as measured by the Storm860 PhosphorImager, was set to represent the 100% level (total) of NSm mRNA, and percentages were extracted from that total at each time point. Average and SD numbers were calculated from the data collected in three experiments.

Fig. 5.

TSWV replication in HeLa cells. (A) Graphic representation of NSm RNA accumulation over time of TSWV-transfected HeLa cells, previously nontransfected (dashed line and squares) or FoTF-transfected (dashed line and triangles), compared with TSWV-infected/FoTF-non-transfected FO1 cells (solid line and diamonds). (B) Northern hybridization showing NSm RNA accumulation over time of TSWV-transfected HeLa cells previously nontransfected or FoTF-transfected, as indicated; actin mRNA is shown as a loading control from one of the blots. (C) Graphic representation of NSm RNA accumulation over time of TSWV-transfected HeLa cells, previously FoTF-transfected (solid line and diamonds), FoTFΔC- (dashed line and triangles), or FoTFΔN-transfected (dashed line and squares). (D) Northern hybridization showing NSm RNA accumulation over time of TSWV-transfected HeLa cells previously FoTFΔC- or FoTFΔN-transfected, as indicated; actin mRNA is shown as a loading control from one of the blots. (E) Western blotting of HeLa cells with anti-FoTF with nontransfected or FoTF-transfected samples, as indicated; samples collected at the indicated time points after virus transfection. (F) Western blotting of HeLa cells with anti-N with nontransfected, FoTF-, or FoTFΔC-transfected samples, as indicated; samples collected at the indicated time points after virus transfection. Results shown are representative of three independent experiments. The highest RNA signal of all three experiments, measured by the Storm860 PhosphorImager, was set to represent the 100% level (total) of NSm mRNA, and percentages were extracted from that total at each time point. Average and SD numbers were calculated from the data collected in three experiments.