A multifunctional protein encoded by Turkey herpesvirus suppresses RNA silencing in Nicotiana benthamiana Running head: silencing suppression by an avian viral protein

Many of plant and animal viruses counteract the RNA silencing-mediated defense by encoding diverse RNA silencing suppressors (RSSs). Here, we characterized HVT063, a multifunctional protein encoded by Turkey herpesvirus (HVT), as a silencing suppressor in co-infiltration assays with GFP transgenic Nicotiana benthamiana line 16c. Our results indicated that HVT063 could strongly suppress both local and systemic RNA silencing induced by either sense RNA or double-stranded RNA. HVT063 could reverse local silencing, but not systemic silencing in newly emerging leaves. The local silencing suppression activity of HVT063 was also verified using the heterologous vector PVX. Further, single alanine substitution of arginine or lysine residues of HVT063 protein showed that each selected single amino acid contributed to suppression activity of HVT063 and the region1 (138-141) was more important, because three out of four single amino acid mutations in this region could abolish the silencing suppressor activity of HVT063. Moreover, HVT063 seemed induce a cell death phenotype in infiltrated leaf region, and the HVT063 dilutions could decrease silencing suppressor activity and alleviate cell death phenotype. Collectively, these results suggest that HVT063 function as a viral suppressor of RNA silencing that targets a downstream step of the dsRNA formation in RNA silencing process. The positively charged amino acids in HVT063, such as arginine and lysine, might contribute to the suppressor activity by boosting the interaction between HVT063 and RNA, as HVT063 was demonstrated as an RNA on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom binding protein. INTRODUCTION Small RNA-guided gene silencing serves as a key component of host defense strategy against viruses in plants, invertebrates, and fungi as well as higher animals (2, 5, 9, 20). RNA silencing refers to the suppression of gene expression through homology-dependent mRNA degradation and it is thought to be initiated by double stranded RNA (dsRNA) molecules (21). The dsRNA is recognized and processed by Dicer or Dicer-like (DCL) proteins into small RNA duplexes of 21-24nt (5), and one strand of the duplex is subsequently incorporated into a multisubunit endonuclease called the RNA-induced silencing complex (RISC) that initiates the sequence-specific degradation of target RNAs (48). To counteract host antiviral defenses, viruses have evolved sophisticated mechanisms including encoding proteins that are capable of suppressing the RNA silencing process (52). The first silencing suppressor was discovered in plant virus, and then the list for different suppressor proteins were identified in many plant viruses as well as animal viruses (3). The discovery of RNA silencing suppressor (RSS) functions in animal viruses provided evidence of conserved RNA silencing pathway in the plant and animal kingdoms (45, 59, 61). Up to now, more than 70 RSSs have been found; however, less than 15 animal proteins have been identified as RSSs. The RSSs are extremely diverse within and across kingdoms, with no obvious sequence homology, appearing to have evolved independently to overcome silencing-mediated defense. The various RSSs target at distinct silencing stages during the RNA silencing on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom process, such as viral RNA recognition, dicing, RISC assembly, RNA targeting and amplification (7, 28, 29, 36, 56). For example, P14 protein of Pothos latent aureusvirus (PoLV), P38 protein of Turnip crinkle virus (TCV), 2b protein of Tomato aspermy cucumovirus (TAV) and B2 protein of the insect–infecting Flock house virus (FHV) have been shown to bind dsRNA in a size-independent way and inhibit the processing of dsRNA to siRNAs (11, 12, 37, 38); p19 protein of Tombusviruses (TBSV) and Influenza A virus NS1 protein prevent RNA silencing by siRNA sequestration through binding siRNA in a size-specific manner (17, 47); 2b protein of Cucumber Mosaic virus (CMV) and P0 protein of Potato leaf roll virus (PLRV) target the AGO protein to prevent RISC assembly (9); P38 protein of Turnip crinkle virus (TCV) and P1 protein of Sweet potato mild mottle ipomovirus (SPMMV) interact with AGO proteins by the glycine/tryptophan (GW/WG) residues (4, 22); Human immunodeficiency virus type 1 (HIV-1) Tat protein and the core protein of Hepatitis C virus (HCV) interact with Dicer to ablate the effect of RNAi (6, 42, 60). So, the continuing studies on the functions of various viral suppressors should contribute significantly to our understanding of the specific steps of RNA silencing. RNA silencing is non-cell autonomous, which initially was induced at the single-cell level then transmitted to remote cells or tissues to cause systemic RNA silencing. In plants, the silencing signal moves from cell to cell through plasmodesmata and over long distance via the vascular system (24, 40, 53, 54, 57). Recent studies suggest that the short distances spread of RNA silencing is mediated by 21-nt siRNAs, however, the role of small RNAs in long distance signaling remains on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom to be elucidated (19). Roth et al. reported that several suppressors, such as CMV 2b, TBSV p19, TSWV NSs and PVX P25 could prevent gene silencing in systemic tissue (44). Among many of the known mammalian viral suppressors, B2 protein of Flock house virus (30) and influenza A virus NS1 protein (8, 18) first exhibit RNA silencing suppressor activity in plant cells. For this reason, RNA silencing represented as an important antiviral defence mechanism in plants most likely also plays an antiviral role in animal cells (31). The identification of novel viral suppressors and elucidation of their mode of action are important to understand RNA silencing mechanisms as well as virus-host interactions. Here, we present HVT063 encoded by Turkey herpesvirus (HVT) is a strong RSS. HVT is a double-stranded DNA avian virus, and it is classified as the third serotype of the Marek’s disease virus (MDV) group (1). The complete genome of HVT is 159,160 bp, which encodes an estimated 99 putative proteins (1). HVT063, one of these proteins, located at the ends of the unique long (UL) region of HVT (1), a multifunctional expression regulator, holds some features that make it a good RSS candidate such as binding RNA and shuttling between nucleus and cytoplasm (refer to its sequence information from NCBI). The results showed that HVT063 suppressed both local and systemic RNA silencing induced by sense RNA or dsRNA. And HVT063 protein could reverse local silencing with a preexisting silenced transgene, and its RSS activities were dosage-dependent. Moreover, the single alanine substitutions of arginine or lysine residues indicated that the RSS activity of HVT063 was likely relational to RNA or siRNA binding ability. on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom MATERIALS AND METHODS Plant materials and plasmid constructs. GFP transgenic N. benthamiana line 16c plants were grown at 24 ± 2°C under a 16h illumination (34W fluorescent bulbs) and 8h dark regimen. The HVT063 gene (NP_073349) used in this experiment was PCR amplified from Turkey herpesvirus genome using specific primers HVT063-F/HVT063-R (Table S1). Furthermore, pp24 gene (ABF72222), pp38 gene (ABF72309) and MEQ gene (ABF72204) were PCR amplified from Marek's Disease Virus genome with specific primers pp24-F/pp24-R, pp38-F/pp38-R, MEQ-F/MEQ-R and N gene (AC037573) was amplified by reverse transcription-PCR (RT-PCR) from total RNA extracted from Avian infectious bronchitis virus with specific primers N-F/N-R (Table S1). The resulting PCR products were cloned into PMD-18T vector (TaKaRa, DaLian, China) to produce PMD-HVT063, PMD-pp24, PMD-MEQ and PMD-N. After that, the above constructs were digested with specific enzymes and inserted into the binary vector pBI121 between the 35S promoter and the Nos terminator to yield constructs 35S-HVT063, 35S-pp24, 35S-pp38, 35S-MEQ and 35S-N. All single alanine point mutations of the HVT063 protein were produced by reverse PCR from the entire plasmid PMD-HVT063 using specific primers (i.e. HVT063K138A-F/HVT063K138A-R, HVT063R139A-F/ HVT063R139A-R, HVT063R140A-F/HVT063R140A-R, HVT063R141A-F/ HVT063R141A-R, respectively, Table S1). The PCR products were circularized by blunt end-ligation and on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom digested with DpnI to remove any residual parental plasmid before transformation into E. coli. mHVT063, an untranslatable mutant of HVT063 protein, was made by replacing the initiating codon ATG with GTG using PCR and primer mHVT063-F (Table S1). These constructs were digested with XbaI and BamHI restriction enzymes and cloned into the pBI121 vector. The GFP gene (792 nucleotides) was amplified from total DNA of N. benthamiana line 16c using the GFP primes GFP-F/GFP-R (Table S1) and incorporated into pBI121 vector to construct 35S-GFP and the p19 gene of Tomato bushy stunyvirus (TBSV) was amplified from pBIN61-p19 plasmid with primes p19-F/p19-R (Table S1) and incorporated into pBI121 vector to generate 35S-p19 as a positive control in various experiments. An inverted repeat sequence of GFP amplified from the plasmid 35S-GFP with primers GFP-Xba-F/GFP-Bam-R, and GFP-Sac-F/GFP-Kpn-R (Table S1) was introduced into the binary vector pBI121 to generate 35S-dsGFP. To produce PVX-HVT063, the HVT063 sequence was amplified from plasmid PMD-HVT063 with primes PVX-HVT063-F/PVX-HVT063-R (Table S1), and then inserted into the ClaI/SalI site in the PVX vector pGR106. All constructs generated by PCR were confirmed correct by nucleotide sequencing, and all plasmids described above were verified by restriction site analysis. Each of the constructs was transformed into A. tumefaciens strain GV3101 containing the helper plasmid pJIC SA_Rep by the freeze-thaw method (27). Co-infiltration and GFP imaging. The GFP expressing Nicotiana benthamiana 16c plants with four to five leaves were infiltrated with the A. tumefaciens GV3101 on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom strain carrying the above constructs according to Voinnet et al. (57). Prior to co-infiltration, each culture OD600 was adjusted to 1.0. Co-expreesion of GFP with different viral proteins, the corresponding bacterial cultures were mixed in 1:1 (v:v) ratio before co-infiltration which was achieved by pressure infiltration. The cells of A.tumefaciens cultures were incubated at room temperature for at least 3h before infiltration. Local and systemic RNA silencing were determined through observing the GFP fluorescence in both infiltrated and the newly emerging leaves under long-wavelength UV (365nm) light (Spectroline model SB-100P/A, UV products, New York, USA), plants were photographed with a Coolpix 5400 Nikon digital camera. Laser-scanning microscope (LSM) photographs were taken using a Zeiss LSM510 microscope. Total RNA and siRNA isolation for Northern blot analysis. Approximately 1g of frozen fresh samples of leaf infiltrated zones or systemic silencing leaves were ground into powder in liquid nitrogen, then transferred to 15mL tubes containing 2 to 3mL hot phenol buffer (Extraction buffer: 0.1M LiCl, 100mM Tris-Cl pH8.0, 10mM EDTA and 1% SDS. Phenol buffer: extraction buffer was mixed with an equal volume of phenol water, preheated at 80°C for 5min). 1/4 volume of chloroform was added and vortexed for 20s. The tubes were centrifuged at 10, 000g for 10min at room temperature and the aqueous phase was transferred to new tubes, then an equal volume of 4M LiCl was added. The contents were gently mixed and left at –20°C overnight. The total RNA pellets were obtained by centrifugation at 10, 000g for 15min at 4°C (keep the supernatant for siRNAs isolation). The pellets were on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom resuspended in 300 L DEPC treated TE buffer (10mM Tris-Cl, 1mM EDTA pH8.0), about 1/10 volume of 3M sodium acetate (pH 5.2) and 2 volumes of ethanol absolute were added, incubated for at least 3h at 20°C. The RNA was precipitated by centrifugation at 10, 000g for 15min at 4°C, washed with 70% ethanol and resuspended in 50 to 100 L DEPC treated H2O. Low-molecular-weight RNAs were enriched from isolation of mRNA. Firstly, add 1/10 volume of 3M sodium acetate (pH5.2) and 2 volumes of ethanol to the supernatant, incubated for at least 3h at 20°C. The RNA pellets were precipitated by centrifugation at 10, 000g for 15min at 4°C, then washed with 70% ethanol and resuspended in 100 L DEPC treated H2O. Add 900 L of Trizol to the supernatant followed by vortexing for 20s, then add 200 L chloroform and vortex for 20s. Then, tubes were centrifuged at 12,000g for 15min at 4°C, transfered the aqueous phase into a fresh eppendorf tube and added 500 L isopropanol and vortex for 15s. The RNA was precipitated by centrifugation at 12, 000g for 10min at 4°C and washed with 75 % ethanol twice. The pellets were air dried and dissolved in 50-60 L DEPC treated H2O. Total RNA Northern blot analysis. For northern blot analysis of high-molecular-weight RNA, 10 g of total RNA was separated on a 1% agarose/MOPS/formaldehyde gel in 1×MOPS buffer, transferred to Hybond-N membranes (Amersham) for Northern blot analysis as previously described (43). Thereafter, the membrane UV crosslinked and treated at 80°C for at least 2h, then stored at 4°C until used. The membrane was briefly washed in 6×SSC for 10min at on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom room temperature. Then, the wash solution was discarded and enough pre-hybridization solution was added to prehybridize the membrane at 42°C for 6–12h. The membrane was hybridized with [ -P] dCTP-labelled full-length GFP probe or PVX MP probe, which synthesized using the primer-a-gene labelling kit (Promega). The probe was added to the membrane and then incubated for 12-24h at 42°C. The membranes were washed three times as follows: 2×SSC+0.2% SDS at 42°C for 20min, 0.2×SSC+0.2% SDS at 42°C for 10min twice. The membrane was covered with a filter paper and excess wash buffer removed before exposure to X-ray film. The membranes were reprobed once after stripping them in 1% SDS at 85°C for