JGV Papers in Press. Published February 20, 2015 as doi:10.1099/vir.0.000095
Journal of General Virology Mutation analysis of the RNA silencing suppressor NS1 encoded by avian influenza virus H9N2 --Manuscript Draft-Manuscript Number:
VIR-D-15-00079R1
Full Title:
Mutation analysis of the RNA silencing suppressor NS1 encoded by avian influenza virus H9N2
Short Title:
Mutation analysis of AIV H9N2-encoded NS1 protein
Article Type:
Short Communication
Section/Category:
Animal - Negative-strand RNA Viruses
Corresponding Author:
Hongmei Liu Shandong Agricultural University CHINA
First Author:
Xiuli Jing
Order of Authors:
Xiuli Jing Jie Xu Meina Fan Lin Ma Xu Huang Xiaoyun Wang Shuhong Sun Changxiang Zhu Hongmei Liu
Abstract:
Non-structural protein 1 (NS1) binds siRNAs and suppresses RNA silencing in plants, but the underlying mechanism of this suppression is not well understood. Therefore, here we characterized NS1 encoded by the avian influenza virus H9N2. The NS1 protein was able to suppress RNA silencing induced by either sense RNA or doublestranded RNA (dsRNA). Using deletion and point mutants, we discovered that the first 70 residues of NS1 can suppress RNA silencing triggered by sense transgene, but this sequence is not sufficient to block dsRNA-induced silencing. Any mutations of two arginine residues (35R and 46R) of NS1, which contribute to its homodimeric structure, cause the loss of its silencing suppression activity. These results indicate that the region after residue 70 of NS1 is essential for the repression activity on dsRNAinduced RNA silencing, and that the dimeric structure of NS1 plays a critical role in its RNA silencing suppression function.
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Mutation analysis of the RNA silencing suppressor NS1 encoded by avian
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influenza virus H9N2
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Xiuli Jinga,b, Jie Xua, Meina Fana, Lin Maa, Xu Huanga, Xiaoyun Wanga, Shuhong Sunc, Changxiang
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Zhua*, Hongmei Liua*
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Running title: Mutation analysis of AIV H9N2-encoded NS1 protein
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a
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Shandong, China
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b
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c
State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an,
Institute of Immunology, Taishan Medical University, Tai’an, Shandong, China
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai’an, Shandong,
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China
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*
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[email protected] (Hongmei Liu), [email protected] (Changxiang Zhu)
Corresponding authors. Tel.: +86 538 8242656 8440; fax: +86 538 8226399. E-mail addresses:
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The GenBank [/EMBL/DDBJ] accession number for the NS1 gene of avian influenza virus H9N2
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(A/chicken/China/B1-6/2006) is GQ981533.
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Total number of words in the main text: 2491
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Total number of words in the summary: 148
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Total number of figures and tables: 3 figures, 0 tables
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SUMMARY
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Non-structural protein 1 (NS1) binds siRNAs and suppresses RNA silencing in plants, but the underlying
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mechanism of this suppression is not well understood. Therefore, here we characterized NS1 encoded by the
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avian influenza virus H9N2. The NS1 protein was able to suppress RNA silencing induced by either sense
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RNA or double-stranded RNA (dsRNA). Using deletion and point mutants, we discovered that the first 70
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residues of NS1 can suppress RNA silencing triggered by sense transgene, but this sequence is not sufficient
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to block dsRNA-induced silencing. Any mutations of two arginine residues (35R and 46R) of NS1, which
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contribute to its homodimeric structure, cause the loss of its silencing suppression activity. These results
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indicate that the region after residue 70 of NS1 is essential for the repression activity on dsRNA-induced
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RNA silencing, and that the dimeric structure of NS1 plays a critical role in its RNA silencing suppression
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function.
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Keywords: avian influenza virus, NS1, RNA silencing suppressor, H9N2, mutation analysis
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RNA silencing is employed as an antiviral mechanism by both plants and mammalian cells. To counteract
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this defence mechanism, viruses encode specific proteins that are able to block different steps of the RNA
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silencing pathway. These proteins, known as RNA silencing suppressors (RSSs), are essential for the
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replication and systemic infection of the viruses in the host (Deleris et al., 2006). However, different RSSs
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do not always feature an obvious sequence similarity and block different steps of the RNA silencing
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pathway (Bivalkar-Mehla et al., 2011; Burgyan & Havelda, 2011; Chapman et al., 2004). Sequestration of
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siRNAs by siRNA-binding suppressors is a very common mechanism to inhibit the assembly of the
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RNA-induced silencing complex (RISC) (Burgyan & Havelda, 2011; Lakatos et al., 2006). The p19 protein
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of Tomato bushy stunt virus and the P21 protein of Beet yellows virus are reported to target RNA silencing
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in this way (Hsieh et al., 2009; Omarov et al., 2006; Silhavy et al., 2002). In contrast, the P14 protein of
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Pothos latent aureus virus and P38 protein of Turnip crinkle virus have been shown to inhibit the processing
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of dsRNA to siRNA by Dicer (Merai et al., 2006). Other suppressors inhibit RNA silencing through the
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direct or indirect interaction with the protein components of RISC (Zhang et al., 2006).
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Non-structural protein 1 (NS1) was reported to have multiple functions in the modulation of immune
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responses, and NS1 from human influenza virus was first identified as an RNA-silencing suppressor
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(Delgadillo et al., 2004). It contains two functional domains, an N-terminal RNA-binding domain (RBD)
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and a C-terminal effector domain (Murayama et al., 2007) (Fig.1a). Crystal structures of NS1 revealed that
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the NS1 RBD domain (amino acids 1–73) form a homodimeric six-helical fold for dsRNA recognition (Liu
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et al., 1997). A previous study indicated that the dimeric structure and a small number of specific basic
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amino acids are essential for the RNA-binding ability of NS1 (Wang et al., 1999). A comparison of the RSS
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activity of several NS1 subtypes (H1N1, H3N2, H5N1, and H7N7) showed that the RSS activity of NS1
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varied among influenza strains and is likely to contribute to the observed differences in viral replication
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capacity and pathogenicity (de Vries et al., 2009). In the present study, the RNA-silencing suppressor
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activity of NS1 from H9N2 avian influenza virus strain was studied. The results show that NS1/AIV H9N2
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protein can suppress both local and systemic RNA silencing induced by transient infiltration. The mutation
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analysis suggests that the 35R–46R pairs are essential for the silencing suppression function of NS1, and
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that the first 70 amino acids (1–70) of NS1 are critical for its RSS activity on sense RNA-induced RNA 3
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silencing, whereas the suppression activity on dsRNA-induced RNA silencing requires the region beyond
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residue 70.
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Recent studies indicate that the RNA-silencing-suppression ability of NS1 varies among influenza A virus
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strains. For example, the NS1 protein of an H1N1 strain was most potent in suppressing short hairpin
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RNA-mediated gene silencing, while the proteins of the highly pathogenic H5N1 strains were most effective
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in complementing the RSS function of the human immunodeficiency virus type 1 Tat protein (de Vries et al.,
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2009). In order to clarify the RSS activity of the NS1 protein encoded by the low pathogenic AIV H9N2, the
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NS1 gene was amplified by reverse transcription-PCR from the A/chicken/China/B1-6/2006 strain of avian
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influenza virus H9N2, followed by cloning to PMD-18T and sequencing (GQ981533). Then NS1 gene was
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cloned to the binary vector pBI121 to yield construct 35S-NS1. All single alanine mutations were produced
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by reverse PCR from the entire plasmid PMD-NS1 using the primer pairs which containing the
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corresponding nucleotide substitutions (Fig.1b). To investigate the core region of RSS activity, four deletion
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mutants of NS1 were constructed respectively (Fig.1c). The resulting constructs were then inserted
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individually into pBI121 vector and transformed into Agrobacterium tumefaciens GV3101. The
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Agrobacterium clones containing the GFP, p19 and dsGFP expression cassettes have been described
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previously (Jing et al., 2011). The GFP-expressing N. benthamiana 16c plants with four to five leaves were
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infiltrated with the A. tumefaciens strains carrying the above constructs. The RSS activity was monitored
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visually by assessing GFP fluorescence with a hand-held long-wavelength UV lamp.
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RSS activity analysis of NS1 showed that stronger green fluorescence was observed in the leaf patches
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expressing NS1 than in those expressing the empty vector at 3 and 7 days post infiltration (dpi), and the GFP
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fluorescence of NS1-infiltrated leaves was comparable to that of leaves expressing p19, an established
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RNA-silencing suppressor of TBSV (Fig. 2a). GFP mRNA levels were very low in the leaves expressing the
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empty vector at 3 dpi, and strongly decreased at 7 dpi to an undetectable level (Fig. 2b). By contrast, in the
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leaves expressing NS1 or p19, GFP mRNA was abundant throughout 3 to 7 dpi (Fig. 2b). In addition, the
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GFP-specific siRNAs, a hallmark of silencing, accumulated remarkably after infiltration and were
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particularly abundant at 7 dpi in the leaves infiltrated by empty vector. In contrast, GFP siRNA was hardly
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detectable in the leaves expressing NS1 or p19, both at 3 and 7 dpi (Fig. 2b). In order to further investigate 4
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whether NS1 can effectively suppress GFP silencing initiated by a dsGFP (i.e., an inverted repeat generating
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GFP dsRNA) inducer, we infiltrated 16c leaves with 35S-GFP, 35S-dsGFP, and an empty vector, 35S-NS1
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or 35S-p19. Strong GFP fluorescence was observed in NS1 and p19 infiltrations up to 7 dpi (Fig. 3a). The
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results of Northern blot analysis of GFP mRNAs and siRNAs were consistent with the above observations
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(Fig. 3b). The results revealed that the NS1 protein encoded by AIV H9N2 is an efficient RNA-silencing
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suppressor of both sense RNA- and dsRNA-triggered silencing. A possible explanation could be that, in
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plants, NS1 targets the silencing process at the early stages, likely focussing on the initial steps after dsRNA
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formation, because both sense RNA- and dsRNA-induced RNA silencing are suppressed by NS1.
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The mechanism for NS1-mediated suppression of RNA silencing is linked to its direct binding to siRNAs,
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and the positively charged amino acids have been demonstrated to support this RNA binding ability (Cheng
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et al., 2009; de Vries et al., 2009; Bucher et al., 2004). Several previous studies have associated the first 50
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amino acids of NS1 with its RNA binding function. Other studies have indicated that the first 70 amino acids
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are critical for NS1 suppressor activity (Qian et al., 1995; Wang et al., 1999). The positively charged
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residues, including 35R, 37R, 38R, and 41K, are clustered in the middle of the dsRNA-binding surface, and
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their primary role is the formation of hydrogen bonds and electrostatic interactions with both strands of the
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dsRNA (Cheng, et al., 2009). Therefore, four NS1 deletion mutants (Δ51–230, Δ61–230, Δ66–230, and
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Δ71–230) were constructed to determine the core functional region of the protein (Fig.1c). Furthermore,
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three positively charged amino acids (35R, 41K, and 46R) that have not previously been investigated for
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their contribution to the RNA silencing process were selected to test their contribution to the RSS activity of
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NS1 by single alanine substitution analysis (Fig.1b). All mutants were co-infiltrated with 35S-GFP, and NS1
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and the empty vector co-infiltrations served as the positive and negative control, respectively. Strong GFP
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fluorescence was observed in the tissues co-infiltrated with 35S-GFP and K41A or with the Δ71–230
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constructs at both 3 and 7 dpi, similar to the wild type NS1 infiltration. The R35A, R46A, Δ51–230,
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Δ61–230 and Δ66–230 constructs yielded weak fluorescence at 3 dpi, and the fluorescence disappeared
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completely at 7 dpi (Fig.2a). These results were supported by GFP mRNA Northern blot (Fig.2b).
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Correspondingly, GFP siRNA in K41A and Δ71–230 infiltrations accumulated to undetectable levels at 3
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and 7 dpi, and R35A, R46A, Δ51–230, Δ61–230, or Δ66–230 infiltrations featured very high levels of GFP 5
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siRNA (Fig.2b). We also investigated the suppression activity of various NS1 mutants on GFP
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dsRNA-triggered silencing. Most of the mutants showed similar suppression activity on sense RNA or
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dsRNA induced RNA silencing, and only the Δ71–230 mutant could suppress sense RNA- but not
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dsRNA-induced RNA silencing (Fig.3).
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Previous studies revealed both the NS1 crystal structure and the structural basis for dsRNA binding (Cheng
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et al., 2009; de Vries et al., 2009). NS1 was shown to form a homodimer to bind siRNAs and dsRNA, and to
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prevent the assembly of the RISC effector. De Vries et al. (2009) analysed the RSS activity of NS1 from 8
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influenza subtypes, and the different NS1 variants showed distinct RSS activity, which was likely to
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contribute to differences in viral replication capacity and pathogenicity (de Vries et al., 2009). These
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differences could be explained by the fact that although the amino acid sequences of NS1 from different
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virus strains show high homology, only minor difference in the sequences could lead to diverse suppression
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activities. Some studies showed that the 73 N-terminal residues of NS1 formed a symmetric homodimer
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with a unique six-helical chain fold, and specific residues including Thr5, Pro31, Asp34, Arg5, Arg38, Lys41,
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Gly45, Arg46, and Thr49 mediated dsRNA binding (Wang et al., 1999; Yin et al., 2007). Further studies
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revealed that several surface amino acids in helices α2/α2’ played a very important role in dsRNA-binding,
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and mutations of these basic residues always affect dsRNA binding affinity (Cheng et al., 2009). Cheng et al.
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reported that 35R from one monomer and 46R from the symmetric related molecule can form two pairs of
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hydrogen bonds that are involved in dsRNA binding (Cheng et al., 2009). In our study, the single alanine
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substitution mutants of the 35R and the 46R residues reduced the silencing suppression activity. We
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speculated that R35A and the R46A mutants destroyed the homodimer structure of NS1 and thereby reduced
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its siRNA binding ability, leading to the loss of RSS activity. K41A mutation seems not affect the RSS
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activity of NS1 on sense RNA induced silencing (Fig. 2). But K41A slightly decrease the RSS activity of
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NS1 on dsRNA-induced RNA silencing. For the GFP fluorescence signal of K41A infiltration is less than
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that of NS1 (Fig. 3a). And this is confirmed by the Northern blot analysis on the accumulation of GFP
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mRNA (Fig. 3b).
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NS1 is theoretically divided into two distinct functional domains: an N-terminal RNA-binding domain
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(residues 1–73) (Chien et al., 2004; Hatada & Fukuda, 1992; Qian et al., 1995) and a C-terminal ‘effector’ 6
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domain (residues 74–230) (Wang et al., 2002). As mentioned above, the first 73 amino acids of NS1 have
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been determined as critical for its suppressor activity in previous studies, while other reports have associated
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the first 50 amino acids of NS1 with its RNA binding function. Our study suggests that the first 70 amino
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acids of NS1 play a key role in RNA-silencing-suppression activity, because the Δ71–230 mutant could
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efficiently suppress RNA silencing induced by sense GFP RNA, but further deletions such as Δ66–230 did
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not have this effect. Moreover, the Δ71–230 mutant could suppress RNA silencing induced by sense RNA,
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but did not affect the stronger RNA silencing caused by dsRNA. This result suggests that the region beyond
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residue 70 of NS1 is required for the stronger suppressor activity that targets a downstream step of dsRNA
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formation. Previous studies revealed that the C-terminal effector domain of NS1 protein (residues 74–230)
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could functionally stabilize the RNA-binding domain (Wang et al., 2002); therefore, the region beyond
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residue 70 of NS1 was essential to maintain its full suppressor function. In terms of the positively charged
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residues, 35R and 46R residues were critical to the suppression activity of NS1, but 41K was not. Taken
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together, the separate domains of NS1 might allow the protein to inhibit multiple components of RNA
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silencing.
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RSSs interfere with the RNA silencing pathway mainly by sequestering viral siRNAs or by interacting with
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key RNA silencing components (Burgyan & Havelda, 2011). Furthermore, many RSSs are proven to block
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RNA silencing at multiple levels. For example, p19 possesses two independent silencing suppressor
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functions, namely viral siRNA binding and the miR168-mediated AGO1 control, both of which are required
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to efficiently cope with the RNA silencing-based host defence (Varallyay et al., 2014). An RSS B2 encoded
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by Flock house virus (FHV) blocks both the cleavage of the FHV genome by Dicer and the incorporation of
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FHV small interfering RNAs into the RNA-induced silencing complex (Bucher et al., 2004; Chao et al.,
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2005). From an evolutionary perspective, multiple interference points of RSS may ensure its effectiveness
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under different conditions. All of the mutation analysis of NS1 above was bases on the well established plant
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system, however it is of great important to investigate the effects of the deletion and point mutations to the
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RSS activity of NS1 in animal system in the further studies.
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Acknowledgments 7
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We are grateful to David Baulcombe for providing N. benthamiana 16c seeds and p19 gene, and to Zhizhong
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Cui for providing avian virus strains. This study was supported by the Research and Development Projects
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of Shandong Colleges and Universities J13LE17, National Natural Science Foundation of China 31272113
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and 31300730.
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Figure legends
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Fig. 1. Schematic representation of the NS1 protein and its mutagenesis. (a) NS1 protein structure. (b) Single
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alanine mutagenesis of three positively charged amino acids (35R, 41K, and 46R) of the NS1 protein. (c)
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Full length NS1 protein and its four deletion mutants (Δ51–230, Δ61–230, Δ66–230 and Δ71–230).
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Fig. 2. Suppression activity of single alanine substitution mutants and deletion mutants of NS1 on GFP sense
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RNA-induced RNA silencing. (a) Co-infiltrations of GFP transgenic N. benthamiana 16c leaves with
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Agrobacterium carrying 35S-GFP plus either the empty vector (marked as vector) or 35S-p19, 35S-NS1, or
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the NS1 mutant constructs (R35A, K41A, R46A, Δ51–231, Δ61–231, Δ66–231, Δ71–231). The photographs
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were taken at 3 and 7 dpi under UV light. (b) Northern blot analysis of GFP mRNA and siRNA isolated from
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the leaves co-infiltrated with the different strains indicated above each lane at 3 and 7 dpi. 10 μg of total 12
279
RNA and 20 μg low molecular mass RNA were loaded per lane respectively and 32P-labeled full-length GFP
280
probe was used in the hybridization. Ethidium bromide-stained rRNA and tRNA are shown as loading
281
controls for mRNA and siRNA, respectively.
282 283
Fig. 3. Suppression activity of single alanine substitution mutants and deletion mutants of NS1 on GFP
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dsRNA-induced RNA Silencing. (a) Co-infiltration of GFP transgenic N. benthamiana 16c leaves with
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Agrobacterium carrying 35S-GFP, 35S-dsGFP plus either the empty vector (signed as vector) or 35S-p19,
286
35S-NS1, or the NS1 mutant constructs (R35A, K41A, R46A, Δ66–231, Δ71–231). The photographs were
287
taken at 3 and 7 dpi under UV light. (b) Northern blot analysis of GFP mRNA and siRNA isolated from the
288
leaves co-infiltrated with the different strains indicated above each lane at 3 and 7 dpi. 10 μg of total RNA
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and 20 μg low molecular mass RNA were loaded per lane respectively and 32P-labeled full-length GFP probe
290
was used in the hybridization. Ethidium bromide-stained rRNA and tRNA are shown as loading controls for
291
mRNA and siRNA, respectively.
13
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Journal of General Virology Mutation analysis of the RNA silencing suppressor NS1 encoded by avian influenza virus H9N2 --Manuscript Draft-Manuscript Number:
VIR-D-15-00079R1
Full Title:
Mutation analysis of the RNA silencing suppressor NS1 encoded by avian influenza virus H9N2
Short Title:
Mutation analysis of AIV H9N2-encoded NS1 protein
Article Type:
Short Communication
Section/Category:
Animal - Negative-strand RNA Viruses
Corresponding Author:
Hongmei Liu Shandong Agricultural University CHINA
First Author:
Xiuli Jing
Order of Authors:
Xiuli Jing Jie Xu Meina Fan Lin Ma Xu Huang Xiaoyun Wang Shuhong Sun Changxiang Zhu Hongmei Liu
Abstract:
Non-structural protein 1 (NS1) binds siRNAs and suppresses RNA silencing in plants, but the underlying mechanism of this suppression is not well understood. Therefore, here we characterized NS1 encoded by the avian influenza virus H9N2. The NS1 protein was able to suppress RNA silencing induced by either sense RNA or doublestranded RNA (dsRNA). Using deletion and point mutants, we discovered that the first 70 residues of NS1 can suppress RNA silencing triggered by sense transgene, but this sequence is not sufficient to block dsRNA-induced silencing. Any mutations of two arginine residues (35R and 46R) of NS1, which contribute to its homodimeric structure, cause the loss of its silencing suppression activity. These results indicate that the region after residue 70 of NS1 is essential for the repression activity on dsRNAinduced RNA silencing, and that the dimeric structure of NS1 plays a critical role in its RNA silencing suppression function.
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1
Mutation analysis of the RNA silencing suppressor NS1 encoded by avian
2
influenza virus H9N2
3
Xiuli Jinga,b, Jie Xua, Meina Fana, Lin Maa, Xu Huanga, Xiaoyun Wanga, Shuhong Sunc, Changxiang
4
Zhua*, Hongmei Liua*
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Running title: Mutation analysis of AIV H9N2-encoded NS1 protein
6
a
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Shandong, China
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b
9
c
State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an,
Institute of Immunology, Taishan Medical University, Tai’an, Shandong, China
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai’an, Shandong,
10
China
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*
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[email protected] (Hongmei Liu), [email protected] (Changxiang Zhu)
Corresponding authors. Tel.: +86 538 8242656 8440; fax: +86 538 8226399. E-mail addresses:
13 14
The GenBank [/EMBL/DDBJ] accession number for the NS1 gene of avian influenza virus H9N2
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(A/chicken/China/B1-6/2006) is GQ981533.
16 17
Total number of words in the main text: 2491
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Total number of words in the summary: 148
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Total number of figures and tables: 3 figures, 0 tables
20 21 22 23 24 25 26 1
27
SUMMARY
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Non-structural protein 1 (NS1) binds siRNAs and suppresses RNA silencing in plants, but the underlying
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mechanism of this suppression is not well understood. Therefore, here we characterized NS1 encoded by the
30
avian influenza virus H9N2. The NS1 protein was able to suppress RNA silencing induced by either sense
31
RNA or double-stranded RNA (dsRNA). Using deletion and point mutants, we discovered that the first 70
32
residues of NS1 can suppress RNA silencing triggered by sense transgene, but this sequence is not sufficient
33
to block dsRNA-induced silencing. Any mutations of two arginine residues (35R and 46R) of NS1, which
34
contribute to its homodimeric structure, cause the loss of its silencing suppression activity. These results
35
indicate that the region after residue 70 of NS1 is essential for the repression activity on dsRNA-induced
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RNA silencing, and that the dimeric structure of NS1 plays a critical role in its RNA silencing suppression
37
function.
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Keywords: avian influenza virus, NS1, RNA silencing suppressor, H9N2, mutation analysis
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2
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RNA silencing is employed as an antiviral mechanism by both plants and mammalian cells. To counteract
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this defence mechanism, viruses encode specific proteins that are able to block different steps of the RNA
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silencing pathway. These proteins, known as RNA silencing suppressors (RSSs), are essential for the
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replication and systemic infection of the viruses in the host (Deleris et al., 2006). However, different RSSs
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do not always feature an obvious sequence similarity and block different steps of the RNA silencing
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pathway (Bivalkar-Mehla et al., 2011; Burgyan & Havelda, 2011; Chapman et al., 2004). Sequestration of
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siRNAs by siRNA-binding suppressors is a very common mechanism to inhibit the assembly of the
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RNA-induced silencing complex (RISC) (Burgyan & Havelda, 2011; Lakatos et al., 2006). The p19 protein
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of Tomato bushy stunt virus and the P21 protein of Beet yellows virus are reported to target RNA silencing
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in this way (Hsieh et al., 2009; Omarov et al., 2006; Silhavy et al., 2002). In contrast, the P14 protein of
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Pothos latent aureus virus and P38 protein of Turnip crinkle virus have been shown to inhibit the processing
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of dsRNA to siRNA by Dicer (Merai et al., 2006). Other suppressors inhibit RNA silencing through the
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direct or indirect interaction with the protein components of RISC (Zhang et al., 2006).
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Non-structural protein 1 (NS1) was reported to have multiple functions in the modulation of immune
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responses, and NS1 from human influenza virus was first identified as an RNA-silencing suppressor
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(Delgadillo et al., 2004). It contains two functional domains, an N-terminal RNA-binding domain (RBD)
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and a C-terminal effector domain (Murayama et al., 2007) (Fig.1a). Crystal structures of NS1 revealed that
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the NS1 RBD domain (amino acids 1–73) form a homodimeric six-helical fold for dsRNA recognition (Liu
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et al., 1997). A previous study indicated that the dimeric structure and a small number of specific basic
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amino acids are essential for the RNA-binding ability of NS1 (Wang et al., 1999). A comparison of the RSS
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activity of several NS1 subtypes (H1N1, H3N2, H5N1, and H7N7) showed that the RSS activity of NS1
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varied among influenza strains and is likely to contribute to the observed differences in viral replication
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capacity and pathogenicity (de Vries et al., 2009). In the present study, the RNA-silencing suppressor
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activity of NS1 from H9N2 avian influenza virus strain was studied. The results show that NS1/AIV H9N2
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protein can suppress both local and systemic RNA silencing induced by transient infiltration. The mutation
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analysis suggests that the 35R–46R pairs are essential for the silencing suppression function of NS1, and
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that the first 70 amino acids (1–70) of NS1 are critical for its RSS activity on sense RNA-induced RNA 3
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silencing, whereas the suppression activity on dsRNA-induced RNA silencing requires the region beyond
70
residue 70.
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Recent studies indicate that the RNA-silencing-suppression ability of NS1 varies among influenza A virus
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strains. For example, the NS1 protein of an H1N1 strain was most potent in suppressing short hairpin
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RNA-mediated gene silencing, while the proteins of the highly pathogenic H5N1 strains were most effective
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in complementing the RSS function of the human immunodeficiency virus type 1 Tat protein (de Vries et al.,
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2009). In order to clarify the RSS activity of the NS1 protein encoded by the low pathogenic AIV H9N2, the
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NS1 gene was amplified by reverse transcription-PCR from the A/chicken/China/B1-6/2006 strain of avian
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influenza virus H9N2, followed by cloning to PMD-18T and sequencing (GQ981533). Then NS1 gene was
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cloned to the binary vector pBI121 to yield construct 35S-NS1. All single alanine mutations were produced
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by reverse PCR from the entire plasmid PMD-NS1 using the primer pairs which containing the
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corresponding nucleotide substitutions (Fig.1b). To investigate the core region of RSS activity, four deletion
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mutants of NS1 were constructed respectively (Fig.1c). The resulting constructs were then inserted
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individually into pBI121 vector and transformed into Agrobacterium tumefaciens GV3101. The
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Agrobacterium clones containing the GFP, p19 and dsGFP expression cassettes have been described
84
previously (Jing et al., 2011). The GFP-expressing N. benthamiana 16c plants with four to five leaves were
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infiltrated with the A. tumefaciens strains carrying the above constructs. The RSS activity was monitored
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visually by assessing GFP fluorescence with a hand-held long-wavelength UV lamp.
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RSS activity analysis of NS1 showed that stronger green fluorescence was observed in the leaf patches
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expressing NS1 than in those expressing the empty vector at 3 and 7 days post infiltration (dpi), and the GFP
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fluorescence of NS1-infiltrated leaves was comparable to that of leaves expressing p19, an established
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RNA-silencing suppressor of TBSV (Fig. 2a). GFP mRNA levels were very low in the leaves expressing the
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empty vector at 3 dpi, and strongly decreased at 7 dpi to an undetectable level (Fig. 2b). By contrast, in the
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leaves expressing NS1 or p19, GFP mRNA was abundant throughout 3 to 7 dpi (Fig. 2b). In addition, the
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GFP-specific siRNAs, a hallmark of silencing, accumulated remarkably after infiltration and were
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particularly abundant at 7 dpi in the leaves infiltrated by empty vector. In contrast, GFP siRNA was hardly
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detectable in the leaves expressing NS1 or p19, both at 3 and 7 dpi (Fig. 2b). In order to further investigate 4
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whether NS1 can effectively suppress GFP silencing initiated by a dsGFP (i.e., an inverted repeat generating
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GFP dsRNA) inducer, we infiltrated 16c leaves with 35S-GFP, 35S-dsGFP, and an empty vector, 35S-NS1
98
or 35S-p19. Strong GFP fluorescence was observed in NS1 and p19 infiltrations up to 7 dpi (Fig. 3a). The
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results of Northern blot analysis of GFP mRNAs and siRNAs were consistent with the above observations
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(Fig. 3b). The results revealed that the NS1 protein encoded by AIV H9N2 is an efficient RNA-silencing
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suppressor of both sense RNA- and dsRNA-triggered silencing. A possible explanation could be that, in
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plants, NS1 targets the silencing process at the early stages, likely focussing on the initial steps after dsRNA
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formation, because both sense RNA- and dsRNA-induced RNA silencing are suppressed by NS1.
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The mechanism for NS1-mediated suppression of RNA silencing is linked to its direct binding to siRNAs,
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and the positively charged amino acids have been demonstrated to support this RNA binding ability (Cheng
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et al., 2009; de Vries et al., 2009; Bucher et al., 2004). Several previous studies have associated the first 50
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amino acids of NS1 with its RNA binding function. Other studies have indicated that the first 70 amino acids
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are critical for NS1 suppressor activity (Qian et al., 1995; Wang et al., 1999). The positively charged
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residues, including 35R, 37R, 38R, and 41K, are clustered in the middle of the dsRNA-binding surface, and
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their primary role is the formation of hydrogen bonds and electrostatic interactions with both strands of the
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dsRNA (Cheng, et al., 2009). Therefore, four NS1 deletion mutants (Δ51–230, Δ61–230, Δ66–230, and
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Δ71–230) were constructed to determine the core functional region of the protein (Fig.1c). Furthermore,
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three positively charged amino acids (35R, 41K, and 46R) that have not previously been investigated for
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their contribution to the RNA silencing process were selected to test their contribution to the RSS activity of
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NS1 by single alanine substitution analysis (Fig.1b). All mutants were co-infiltrated with 35S-GFP, and NS1
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and the empty vector co-infiltrations served as the positive and negative control, respectively. Strong GFP
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fluorescence was observed in the tissues co-infiltrated with 35S-GFP and K41A or with the Δ71–230
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constructs at both 3 and 7 dpi, similar to the wild type NS1 infiltration. The R35A, R46A, Δ51–230,
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Δ61–230 and Δ66–230 constructs yielded weak fluorescence at 3 dpi, and the fluorescence disappeared
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completely at 7 dpi (Fig.2a). These results were supported by GFP mRNA Northern blot (Fig.2b).
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Correspondingly, GFP siRNA in K41A and Δ71–230 infiltrations accumulated to undetectable levels at 3
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and 7 dpi, and R35A, R46A, Δ51–230, Δ61–230, or Δ66–230 infiltrations featured very high levels of GFP 5
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siRNA (Fig.2b). We also investigated the suppression activity of various NS1 mutants on GFP
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dsRNA-triggered silencing. Most of the mutants showed similar suppression activity on sense RNA or
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dsRNA induced RNA silencing, and only the Δ71–230 mutant could suppress sense RNA- but not
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dsRNA-induced RNA silencing (Fig.3).
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Previous studies revealed both the NS1 crystal structure and the structural basis for dsRNA binding (Cheng
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et al., 2009; de Vries et al., 2009). NS1 was shown to form a homodimer to bind siRNAs and dsRNA, and to
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prevent the assembly of the RISC effector. De Vries et al. (2009) analysed the RSS activity of NS1 from 8
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influenza subtypes, and the different NS1 variants showed distinct RSS activity, which was likely to
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contribute to differences in viral replication capacity and pathogenicity (de Vries et al., 2009). These
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differences could be explained by the fact that although the amino acid sequences of NS1 from different
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virus strains show high homology, only minor difference in the sequences could lead to diverse suppression
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activities. Some studies showed that the 73 N-terminal residues of NS1 formed a symmetric homodimer
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with a unique six-helical chain fold, and specific residues including Thr5, Pro31, Asp34, Arg5, Arg38, Lys41,
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Gly45, Arg46, and Thr49 mediated dsRNA binding (Wang et al., 1999; Yin et al., 2007). Further studies
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revealed that several surface amino acids in helices α2/α2’ played a very important role in dsRNA-binding,
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and mutations of these basic residues always affect dsRNA binding affinity (Cheng et al., 2009). Cheng et al.
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reported that 35R from one monomer and 46R from the symmetric related molecule can form two pairs of
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hydrogen bonds that are involved in dsRNA binding (Cheng et al., 2009). In our study, the single alanine
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substitution mutants of the 35R and the 46R residues reduced the silencing suppression activity. We
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speculated that R35A and the R46A mutants destroyed the homodimer structure of NS1 and thereby reduced
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its siRNA binding ability, leading to the loss of RSS activity. K41A mutation seems not affect the RSS
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activity of NS1 on sense RNA induced silencing (Fig. 2). But K41A slightly decrease the RSS activity of
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NS1 on dsRNA-induced RNA silencing. For the GFP fluorescence signal of K41A infiltration is less than
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that of NS1 (Fig. 3a). And this is confirmed by the Northern blot analysis on the accumulation of GFP
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mRNA (Fig. 3b).
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NS1 is theoretically divided into two distinct functional domains: an N-terminal RNA-binding domain
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(residues 1–73) (Chien et al., 2004; Hatada & Fukuda, 1992; Qian et al., 1995) and a C-terminal ‘effector’ 6
150
domain (residues 74–230) (Wang et al., 2002). As mentioned above, the first 73 amino acids of NS1 have
151
been determined as critical for its suppressor activity in previous studies, while other reports have associated
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the first 50 amino acids of NS1 with its RNA binding function. Our study suggests that the first 70 amino
153
acids of NS1 play a key role in RNA-silencing-suppression activity, because the Δ71–230 mutant could
154
efficiently suppress RNA silencing induced by sense GFP RNA, but further deletions such as Δ66–230 did
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not have this effect. Moreover, the Δ71–230 mutant could suppress RNA silencing induced by sense RNA,
156
but did not affect the stronger RNA silencing caused by dsRNA. This result suggests that the region beyond
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residue 70 of NS1 is required for the stronger suppressor activity that targets a downstream step of dsRNA
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formation. Previous studies revealed that the C-terminal effector domain of NS1 protein (residues 74–230)
159
could functionally stabilize the RNA-binding domain (Wang et al., 2002); therefore, the region beyond
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residue 70 of NS1 was essential to maintain its full suppressor function. In terms of the positively charged
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residues, 35R and 46R residues were critical to the suppression activity of NS1, but 41K was not. Taken
162
together, the separate domains of NS1 might allow the protein to inhibit multiple components of RNA
163
silencing.
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RSSs interfere with the RNA silencing pathway mainly by sequestering viral siRNAs or by interacting with
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key RNA silencing components (Burgyan & Havelda, 2011). Furthermore, many RSSs are proven to block
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RNA silencing at multiple levels. For example, p19 possesses two independent silencing suppressor
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functions, namely viral siRNA binding and the miR168-mediated AGO1 control, both of which are required
168
to efficiently cope with the RNA silencing-based host defence (Varallyay et al., 2014). An RSS B2 encoded
169
by Flock house virus (FHV) blocks both the cleavage of the FHV genome by Dicer and the incorporation of
170
FHV small interfering RNAs into the RNA-induced silencing complex (Bucher et al., 2004; Chao et al.,
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2005). From an evolutionary perspective, multiple interference points of RSS may ensure its effectiveness
172
under different conditions. All of the mutation analysis of NS1 above was bases on the well established plant
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system, however it is of great important to investigate the effects of the deletion and point mutations to the
174
RSS activity of NS1 in animal system in the further studies.
175 176
Acknowledgments 7
177
We are grateful to David Baulcombe for providing N. benthamiana 16c seeds and p19 gene, and to Zhizhong
178
Cui for providing avian virus strains. This study was supported by the Research and Development Projects
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of Shandong Colleges and Universities J13LE17, National Natural Science Foundation of China 31272113
180
and 31300730.
181 182
8
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Figure legends
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Fig. 1. Schematic representation of the NS1 protein and its mutagenesis. (a) NS1 protein structure. (b) Single
271
alanine mutagenesis of three positively charged amino acids (35R, 41K, and 46R) of the NS1 protein. (c)
272
Full length NS1 protein and its four deletion mutants (Δ51–230, Δ61–230, Δ66–230 and Δ71–230).
273
Fig. 2. Suppression activity of single alanine substitution mutants and deletion mutants of NS1 on GFP sense
274
RNA-induced RNA silencing. (a) Co-infiltrations of GFP transgenic N. benthamiana 16c leaves with
275
Agrobacterium carrying 35S-GFP plus either the empty vector (marked as vector) or 35S-p19, 35S-NS1, or
276
the NS1 mutant constructs (R35A, K41A, R46A, Δ51–231, Δ61–231, Δ66–231, Δ71–231). The photographs
277
were taken at 3 and 7 dpi under UV light. (b) Northern blot analysis of GFP mRNA and siRNA isolated from
278
the leaves co-infiltrated with the different strains indicated above each lane at 3 and 7 dpi. 10 μg of total 12
279
RNA and 20 μg low molecular mass RNA were loaded per lane respectively and 32P-labeled full-length GFP
280
probe was used in the hybridization. Ethidium bromide-stained rRNA and tRNA are shown as loading
281
controls for mRNA and siRNA, respectively.
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Fig. 3. Suppression activity of single alanine substitution mutants and deletion mutants of NS1 on GFP
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dsRNA-induced RNA Silencing. (a) Co-infiltration of GFP transgenic N. benthamiana 16c leaves with
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Agrobacterium carrying 35S-GFP, 35S-dsGFP plus either the empty vector (signed as vector) or 35S-p19,
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35S-NS1, or the NS1 mutant constructs (R35A, K41A, R46A, Δ66–231, Δ71–231). The photographs were
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taken at 3 and 7 dpi under UV light. (b) Northern blot analysis of GFP mRNA and siRNA isolated from the
288
leaves co-infiltrated with the different strains indicated above each lane at 3 and 7 dpi. 10 μg of total RNA
289
and 20 μg low molecular mass RNA were loaded per lane respectively and 32P-labeled full-length GFP probe
290
was used in the hybridization. Ethidium bromide-stained rRNA and tRNA are shown as loading controls for
291
mRNA and siRNA, respectively.
13
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
