The Weak Small RNA-Binding Activity of the 2b Proteins of Subgroup II Cucumber Mosaic Virus Strains Is Insufficient for RNA Silencing Suppression

The 2b proteins encoded by cucumber mosaic virus (CMV) subgroup I strains suppress RNA silencing primarily by competitively binding small RNAs (sRNAs) in the host cell cytoplasm. Interestingly, 2b proteins encoded by CMV subgroup II strains accumulate predominantly in nuclei. Here we determined that whereas the 2b protein (Fny2b) of subgroup IA strain Fny-CMV is highly effective in suppressing both sense RNA-induced and inverted repeat-induced posttranscriptional gene silencing, the 2b protein (LS2b) of the subgroup II strain LS-CMV was not as effective. Reducing nuclear accumulation of LS2b by mutating a residue in its nuclear localization sequence had no effect on RNA silencing suppressor activity, while attenuated viral symptoms. Electrophoretic mobility shift assays showed that the sRNA binding of LS2b was weaker and more selective than that of Fny2b. The domain determining the differential sRNA-binding ability was delimited to the putative helix α1 region. Moreover, LS2b mutants that completely lost suppressor activity still retained their weak sRNA-binding ability, suggesting that sRNA binding is not sufficient for LS2b to suppress RNA silencing. Considering the subgroup I strain-encoded 2b proteins that require sRNA-binding ability for the suppression of RNA silencing, we suggest that in addition to binding sRNA, the 2b proteins of subgroup II CMV strains would require extra biological activities to achieve RNA silencing inhibition.

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Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-3-0294 ◽

2010 ◽

Vol 23 (3) ◽

pp. 294-303 ◽

Cited By ~ 125

Author(s):

Inmaculada González ◽

Llucia Martínez ◽

Daria V. Rakitina ◽

Mathew G. Lewsey ◽

Félix A. Atencio ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Small Rnas ◽

Rna Silencing ◽

Silencing Suppressor ◽

Suppressor Activity ◽

Rna Silencing Suppressor ◽

2B Protein

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.

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Symptom induction and RNA silencing suppression by the cucumber mosaic virus 2b protein

Plant Signaling & Behavior ◽

10.4161/psb.5.6.11643 ◽

2010 ◽

Vol 5 (6) ◽

pp. 705-708 ◽

Cited By ~ 17

Author(s):

Mathew G. Lewsey ◽

Inmaculada González ◽

Natalia O. Kalinina ◽

Peter Palukaitis ◽

Tomas Canto ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

2B Protein ◽

Silencing Suppression

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Effects of the 2b Protein of Cucumber mosaic virus Subgroup IB Strain IA on Different Transgene-Induced RNA Silencing Pathways

Plant Molecular Biology Reporter ◽

10.1007/s11105-016-1020-0 ◽

2017 ◽

Vol 35 (2) ◽

pp. 265-272 ◽

Cited By ~ 4

Author(s):

Mayuko Koizumi ◽

Yumi Shimotori ◽

Yuta Saeki ◽

Sayaka Hirai ◽

Shin-ichiro Oka ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

2B Protein ◽

Silencing Pathways

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2b ORFs encoded by subgroup IB strains of cucumber mosaic virus induce differential virulence on Nicotiana species

Journal of General Virology ◽

10.1099/vir.0.82927-0 ◽

2007 ◽

Vol 88 (9) ◽

pp. 2596-2604 ◽

Cited By ~ 28

Author(s):

Zhi-You Du ◽

Fei-Fei Chen ◽

Qian-Sheng Liao ◽

Hua-Rong Zhang ◽

Yan-Fei Chen ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Host Species ◽

Long Distance ◽

Reading Frame ◽

Subgroup Ii ◽

Viral Movement ◽

2B Protein ◽

Rna Accumulation ◽

High Level

Cucumber mosaic virus (CMV)-encoded 2b protein from subgroup IA or subgroup II was shown to be a determinant of virulence in many solanaceous hosts. In this study, the virulence of 2b proteins from subgroup IB strains was analysed using four intraspecies hybrid viruses, which were generated by precise replacement of the 2b open reading frame (ORF) in subgroup IA strain Fny-CMV with the 2b ORFs of four subgroup IB strains, Cb7-CMV, PGs-CMV, Rad35-CMV and Na-CMV, generating FCb72b-CMV, FPGs2b-CMV, FRad352b-CMV and FNa2b-CMV, respectively. FCb72b-CMV was more virulent than Fny-CMV, and was similar in phenotype to its parental virus Cb7-CMV on the three Nicotiana species tested. FNa2b-CMV also was virulent on these host species, equivalent to Fny-CMV or Na-CMV. However, FRad352b-CMV only caused mild mosaic or undetectable symptoms on all the host species tested, and was less virulent than Fny-CMV or Rad35-CMV. FPGs2b-CMV infected all the host species systemically, and induced either mosaic or barely visible symptoms, demonstrating that the inability of PGs-CMV to infect these three Nicotiana species was not due to its 2b protein. The diverse virulence was shown to be mediated by the 2b proteins rather than the C-terminal overlapping parts of the 2a proteins, and was associated with the level of viral progeny RNA accumulation in systemically infected leaves, but not with the rate of long-distance viral movement in host plants. Through analysis of encapsidation of viral RNAs, there was an apparent correlation between the virulence and the high level of encapsidated RNA 2 in virions of Fny-CMV, FCb72b-CMV and FNa2b-CMV.

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Differential Virulence by Strains of Cucumber mosaic virus is Mediated by the 2b Gene

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.9.947 ◽

2002 ◽

Vol 15 (9) ◽

pp. 947-955 ◽

Cited By ~ 55

Author(s):

Bu-Jun Shi ◽

Peter Palukaitis ◽

Robert H. Symons

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Kinetic Studies ◽

Transcriptional Gene Silencing ◽

Long Distance ◽

Post Transcriptional Gene Silencing ◽

Subgroup Ii ◽

2B Protein ◽

Rna Accumulation ◽

Long Distance Movement

The approximately 12-kDa 2b protein, encoded by all cucumoviruses, had been shown to play an important role in viral long-distance movement, hypervirulence, and suppression of post-transcriptional gene silencing. The role of the 2b gene in the hypervirulence of Cucumber mosaic virus (CMV) and whether hypervirulence was linked to movement were analyzed using a hybrid virus (CMV-qw), generated by replacing the 2b gene in a subgroup II strain, Q-CMV, with the 2b gene from a subgroup IA strain, WAII-CMV. CMV-qw was more virulent than Q-CMV or WAII-CMV on most of the host plant species tested. Northern blot and nucleotide sequence analyses demonstrated that CMV-qw was stably maintained during the course of infection and upon passage. Kinetic studies revealed that the hypervirulence induced by the hybrid virus was associated with neither increased viral RNA accumulation nor more rapid viral movement per se, suggesting that other functions of the 2b protein are important in determining the hypervirulence.

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Self-interaction of the cucumber mosaic virus 2b protein plays a vital role in the suppression of RNA silencing and the induction of viral symptoms

Molecular Plant Pathology ◽

10.1111/mpp.12051 ◽

2013 ◽

Vol 14 (8) ◽

pp. 803-812 ◽

Cited By ~ 21

Author(s):

Aixia Xu ◽

Zhijing Zhao ◽

Wenhu Chen ◽

Hengmu Zhang ◽

Qiansheng Liao ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

Vital Role ◽

2B Protein

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The 2b protein of cucumber mosaic virus is essential for viral infection of the shoot apical meristem and for efficient invasion of leaf primordia in infected tobacco plants

Journal of General Virology ◽

10.1099/vir.0.013219-0 ◽

2009 ◽

Vol 90 (12) ◽

pp. 3015-3021 ◽

Cited By ~ 23

Author(s):

Anurag Sunpapao ◽

Takashi Nakai ◽

Fang Dong ◽

Tomofumi Mochizuki ◽

Satoshi T. Ohki

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Shoot Apical Meristem ◽

Rna Silencing ◽

Apical Meristem ◽

Leaf Primordia ◽

Shoot Meristem ◽

Tobacco Plants ◽

2B Protein ◽

Infected Tobacco

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Δ2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Δ2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Δ2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Δ2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Δ2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Δ2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Δ2b, whereas accumulation of Δ2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.

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