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

8/9/2019 Expression of a viral polymerase-bound host factor turns human cell lines permissive to a plant- and insect-infecting virus

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Expression of a viral polymerase-bound host factor

turns human cell lines permissive to a plant- andinsect-infecting virus

Ricardo B. de Medeiros, Juliana Figueiredo,

Renato de O. Resende, and Antonio C. De Avila

PNAS (2005):102, pp1175


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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.

RdRp is multifunctional : replication, transcription, genomic

strand selection, cap snatching mechanism

Far western blotting to search the interacting partners:


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(B) Immunoprecipitation (IP) and Western blotting (WB) usinganti-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, pGEXFoTF, pGEX-FoTFN, and pGEX-FoTFC. GST

beads were applied to whole-cell lysates ofTSWV-infectedF.

occidentalis larvae.

N FoTF binds to the C of the RdRp


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(D) Yeast two-hybrid X-Gal filter staining assay after

cotransformation with TSWV RdRp fragments (pTL1, pTL1797,

pTL2249, and pTL908) orTSWV N (pN) with F. occidentalis

FoTF (pFoTF); yeast growth (and -gal expression) indicates

direct interaction; negative controls were transformations with

pFoTF and pTL2249 only; positive control was pLC1 (which

codes for GAL4, and triggers-gal expression without the need of

any interaction).

(E) FoTF map and position of deletion mutants

used in this study.


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RNA gel shift assay. Autoradiographof RNA-binding reactions after addition of radiolabeledTSWV S RNA transcripts to

purified FoTFonly (A), TSWV N protein as positive control (B), FoTFN (C), BSA as a negative control (D), orFoTF plus

nonlabeled TSWV S RNA (E). Protein concentrations varied from 0 toQ20 M; , no protein added.

N FoTF is important for the TSWV RNA binding


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In vitro RNA synthesis assay. (A) Gel electrophoresisof radiolabeled TSWV RNA products after amendments to the standard

reaction, as indicated in the table. After decay of the radioactivity, gels as shown inA 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, isvisualized, 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 inA, the sizes, in kb, are indicated in BF. Results shown are representative of six

independent experiments.

FoTF improves TSWV RNA replication in vitro in the presence of Reticulocyte lysate


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TSWV infectionofF. occidentalis FO1 cells. (A) Graphic representation of NSm mRNA accumulationover time, afterTSWV infection,of nontransfected (solid

line and squares) and FoTF-transfectedFO1 (dashed lines and diamonds). (B) Northern hybridizations showing NSm mRNA accumulation over time of

nontransfected and FoTF-transfectedFO1, as indicated, and actin mRNA as the loading control from one of the blots. (C) Graphic representation of NSm mRNA

accumulation over time ofFoTF-, FoTFC-, and TL2249-transfectedFO1. (D) Northern hybridizations showing NSmmRNAaccumulation over time ofFoTFC- and

TL2249-transfectedFO1, 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.

FoTFC and TL2249 block the infecttion of F.occidentalis Cells


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TSWV replication in HeLa cells. (A) Graphic representation of

NSm RNA accumulationover time ofTSWV-transfected HeLa

cells, previously nontransfected (dashed line and squares) or

FoTF-transfected (dashed line and triangles), compared withTSWV-infectedFoTF-non-transfectedFO1 cells (solid line and

diamonds). (B) Northern hybridization showing NSm RNA

accumulation over time ofTSWV-transfected HeLa cells

previously nontransfected orFoTF-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), FoTFC- (dashed line and triangles), or

FoTFN-transfected (dashed line and squares). (D) Northern

hybridization showing NSm RNA accumulation over time of

TSWV-transfected He

La cells previ

ously

FoTFC-orFoTF

N-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 nontransfectedorFoTF-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-, orFoTFC-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.

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