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. 2008 Dec;82(24):12304-11.
doi: 10.1128/JVI.01696-08. Epub 2008 Sep 25.

Identification of a movement protein of the tenuivirus rice stripe virus

Ruyi Xiong  1 , Jianxiang Wu, Yijun Zhou, Xueping Zhou
Affiliations

Identification of a movement protein of the tenuivirus rice stripe virus

Ruyi Xiong et al. J Virol. 2008 Dec.

Abstract

Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.

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Figures

FIG. 1.
FIG. 1.
Genome organization of RSV. ORFs are displayed as white boxes on viral-sense RNAs and shaded boxes on viral complementary-sense RNAs. Arrowheads indicate the direction of translation. RdRp, RNA-dependent RNA polymerase; SP, disease-specific protein.
FIG. 2.
FIG. 2.
Histochemical analysis of GUS activity in Nicotiana benthamiana leaves bombarded with pPVX-GUS-BSP (A), 35S-NSvc4 plus pPVX-GUS-BSP (B), or pBI-P25 plus pPVX.GUS-BSP (C). The assay was conducted at 40 hpb. Images were photographed using an Olympus stereomicroscope with enlargement.
FIG. 3.
FIG. 3.
Green fluorescence in bombardment or agroinfiltration-treated onion and Nicotiana benthamiana cells with pCHF3-NSvc4:eGFP or pCHF3-eGFP. Cells were imaged under a confocal microscope at 24 h after bombardment or agroinfiltration. The NSvc4:eGFP fusion protein accumulated in the cytoplasm and nucleus and as punctate spots adjacent to cell walls of bombarded onion (A) and agroinfiltrated tobacco cells (C). GFP alone accumulated evenly in the cytoplasm and nucleus in bombarded onion (B) and agroinfiltrated tobacco cells (D). N. benthamiana leaves bombarded with pCHF3-eGFP showed green fluorescence only in single epidermal cells (E), while N. benthamiana leaves bombarded with pCHF3-NSvc4:eGFP showed green fluorescence in multiple epidermal cells (F).
FIG. 4.
FIG. 4.
Detection of NSvc4 in RSV-infected rice cells using immunogold labeling and electron microscopy. The NSvc4 protein was detected near or in cell walls of infected cells. Arrows in A and B indicate immunogold labeling in cells. No immunogold labeling was seen in RSV-infected rice sections treated with buffer followed by the gold-conjugated secondary antiserum (C). No gold labeling was detected in healthy rice leaf sections probed with the NSvc4 antiserum (D). Bars represent 0.5 μm.
FIG. 5.
FIG. 5.
Electrophoretic mobility shift assay of RNA-NSvc4 complexes. (A) His6-tagged NSvc4 protein before and after purification. (B) Different concentrations of His6-tagged NSvc4 were incubated with [α-33P]UTP-labeled RNA transcripts, and RNA-NSvc4 complexes were resolved individually on native gels. Gels were analyzed by phosphorimaging. [α-33P]UTP-labeled RNA transcripts were a 275-bp fragment representing partial sequences of RSV RNA1, RNA2, RNA3, RNA4, or PVX or a 268-bp fragment representing a partial sequence of the GFP gene. ssRNA, single-stranded RNA.
FIG. 6.
FIG. 6.
Analysis of the interaction between NSvc4 and NCP using a yeast two-hybrid assay. Yeast cells cotransformed with pGADT7-RecT and pGBKT7-53 (row 1) and pGADT7-RecT and pGBKT7-Lam (row 2) were included as positive and negative controls. Row 3 shows yeast cells cotransformed with pGADT7-NSvc4 and pGBKT7-NCP. Row 4 shows yeast cells cotransformed with pGADT7-NCP and pGBKT7-NSvc4. Only yeast cells that cotransformed with pGADT7-RecT and pGBKT7-53 were able to grow on selective medium.
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