Identification And Functional Analysis Of RMA Silencing Suppressors From Avian Viruses

RNA silencing is a homologous degradation mechanism against the invasion of virusesand foreign nucleic acids serving as a key component of host defense strategy against virusesin plants, invertebrates, and fungi, as well as higher animals. To counter this defensemechanism, many viruses encode RNA silencing suppressors (RSSs) that are capable ofblocking the generation and dissemination of RNA silencing to guarantee their successfulinvasion. Up to now, more than80RSSs have been found; and most of them are from plantviruses. Most of the identified RSSs are proteins and they are extremely diverse within andacross kingdoms, with no obvious sequence homology, which reflects the complicatedmechanism of RNA silencing and the interaction between virus and host. Therefore, theidentification of more novel RSSs and elucidation of their mode of action are important tounderstand RNA silencing mechanisms, as well as virus-host interactions and work out morespecific anti-viral strategies. In addition, the coexpression of RSSs and objective genes canimprove the expression level of target proteins, which provides a new strategy forheterologous protein expression. In this study, we selected six avian viral proteins as RNAsilencing suppressor candidates and screened potential RNA silencing suppressors using anAgrobacterium coinfiltration assay. And then we studied on the RSS activity and mechanismof the identified RNA silencing suppressors, HVT063and NS1. The main research results areas follows:(1) Identification of RNA silencing suppressors from avian viruses. According to thecharacteristics of known RSSs, most of them were double-stranded RNA (dsRNA) and smallinterfering RNA (siRNA) binding proteins or pathogenesis-related factors, we preliminaryselected6proteins encoded by4avian viruses (including DNA viruses and RNA viruses) asRNA silencing suppressor candidates. The genes of above-mentioned RNA silencingsuppressor candidates were cloned and respectively constructed into the plant expressionvector pBI121. Green fluorescent protein (GFP) transgenic Nicotiana benthamiana line16c asmaterial, and screened potential RNA silencing suppressors using an Agrobacteriumcoinfiltration assay. At3dpi (days post infiltration), the green fluorescence intensity was verystrong in patches coinfiltrated with35S-HVT063or35S-NS1, so HVT063and NS1proteinswere initially identified as RNA silencing suppressors, while the remaining4RNA silencingsuppressor candidates did not have this feature. HVT063protein encoded by Turkey herpesvirus is identified as a new RNA silencing suppressor for the first time; NS1proteinfrom Avian influenza virus H9N2whose RNA silencing suppression activity is also firstlyidentified.(2) HVT063could efficiently suppress both local and systemic RNA silencing. Inpatches coinfiltrated with35S-HVT063and35S-GFP, the green fluorescence intensity wasstrong and the GFP mRNA levels was significantly increased, while the accumulation of GFPsiRNA was drastically reduced to an undetected level at both3and6dpi. An untranslatablemutant of HVT063protein (mHVT063) whose initiating codon ATG of HVT063ORF wasreplaced with GTG could only complete transcription but not translate into protein. Weak orabsent GFP fluorescence was observed in tissues coinoculated with35S-GFP and35S-mHVT063at3dpi, this indicated that the suppression activity of HVT063was obviouslydominated by protein rather than mRNA. HVT063could also suppress the systemic RNAsilencing. GFP silencing occurred in the newly emerging leaves of the plants coinfiltrated with35S-mHVT063, by contrast, there was strong GFP fluorescence in the newly emerging leavesof plants coinfiltrated with35S-HVT063and the level of GFP mRNA accumulation wassignificantly higher than that from leaves coinfiltrated with35S-mHVT063. HVT063couldefficiently suppress RNA silencing induced by either sense RNA or dsRNA. This indicate thatHVT063probably targets a downstream step of the dsRNA formation in the RNA silencingprocess, either by sequestering siRNAs to prevent them integrate into RNA-induced silencingcomplex (RISC) or by interacting with components of RISC to inhibit RNA silencingpathway.(3) Verification of HVT063suppressor activity using PVX vector, while PVX-HVT063could not reverse systemic silencing in newly emerging leaves. HVT063expressed from thePVX vector could enhance the intensity of GFP, significantly increase the GFP mRNA andPVX RNA accumulation in coinfiltrated patches, while PVX-mHVT063did not have thisfeature. The reversal of established silencing by using a PVX vector indicated that HVT063could reverse local silencing but not systemic silencing in newly emerging leaves. Theseresults indicate that HVT063can not block the spread of the systemic RNA silencing signal inplants, which only inhibit a function required for the maintenance of the silenced state.(4) The effect of positively charged amino acid residues on the silencing suppressoractivity of HVT063protein. The siRNAs and/or long dsRNAs binding are the most commonstrategy of animal RSSs for RNA silencing suppression. HVT063protein has two positivelycharged amino acid-rich regions, whether these positively charged amino acids are related toRNA binding and play a key role in the suppression activity of HVT063protein? This two positively charged amino acid-rich regions (region1and region2) of HVT063protein wereanalyzed by single alanine substitutions to test which residues mainly contributed to its RNAsilencing suppressor activity. The results showed that a total of11single alanine substitutionmutants of HVT063in the two regions could decrease the silencing suppressor activity. Threeof four single amino acid mutations (138K,139R and140R) in region1could abolish thesilencing suppressor activity of HVT063, so region1(residues138to141) might be involvedin the formation of key functional domains of HVT063, and the191R and193R mutations inregion2also made HVT063lost the silencing suppressor activity. These amino acids mightdirectly participate in the combination of HVT063and RNA or directly related to thesilencing suppressor function.(5) The suppressor activity of HVT063was related to its concentration and the highconcentration could suppress RNA silencing at the same time induce partial cell deathphenotype in the infiltrated leaf region. In order to further clarified the relationship betweenthe dosage of HVT063, its suppressor activity and death phenotype, we set differentconcentration gradients of the Agrobacterium strain carrying HVT063and tested the RSSactivity of different dilutions using an Agrobacterium coinfiltration assay. These resultsdemonstrated that the suppression activity and the cell death phenotype were both crippled bythe level of HVT063decreasing in the coinfiltrated region. Therefore, these results indicatedthat the death phenotype and the suppressor activity of HVT063were dosage dependent.(6) NS1protein could suppress local and systemic RNA silencing induced by eithersense RNA or dsRNA and the first1-70amino acids of NS1are critical for functional domainof the silencing suppression activity. Comparison of the RNA silencing suppression capacitybetween NS1and the strong RSS p19indicated that the local RNA silencing suppressoractivity of NS1was significantly stronger than p19from7to10dpi, but weaker than that ofp19in the remaining time. NS1could suppress systemic RNA silencing, but it was not able tocompletely prevent the systemic spread of RNA silencing signal in the whole plant. Themutation analysis of NS1suggested that the single alanine substitution mutants of the35Rand the46R residues destroyed the suppression activity of NS1, which were essential aminoacids for NS1silencing suppression function and the mutation of the41K to alanine residuedid not affect the RNA silencing suppressor activity of NS1.â–³71-230mutantion couldefficiently suppress RNA silencing induced by sense GFP RNA, but it was not able to inhibitRNA silencing caused by GFP dsRNA, which suggested that this deletion mutant mightreduce the silencing suppression activity of NS1or change the action stage in RNA silencing. â–³66-230mutantion made NS1lost silencing suppression activity, so the first1-70aminoacids of NS1were necessary for functional domain of the silencing suppression ability,whereas the more stronger silencing suppression activity of NS1required the participation ofthe region after the70residues.