A Multifunctional Protein Encoded by Turkey Herpesvirus Suppresses RNA Silencing in Nicotiana benthamiana

Abstract

Many plant and animal viruses counteract RNA silencing-mediated defense by encoding diverse RNA silencing suppressors. We characterized HVT063, a multifunctional protein encoded by turkey herpesvirus (HVT), as a silencing suppressor in coinfiltration assays with green fluorescent protein 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 (dsRNA). 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 the HVT063 protein showed that each selected single amino acid contributed to the suppression activity of HVT063 and region 1 (residues 138 to 141) was more important, because three of four single amino acid mutations in this region could abolish the silencing suppressor activity of HVT063. Moreover, HVT063 seemed to induce a cell death phenotype in the infiltrated leaf region, and the HVT063 dilutions could decrease the silencing suppressor activity and alleviate the cell death phenotype. Collectively, these results suggest that HVT063 functions as a viral suppressor of RNA silencing that targets a downstream step of the dsRNA formation in the RNA silencing process. 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, since HVT063 has been demonstrated to be an RNA binding protein.Small interfering RNA (siRNA)-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 proteins into small RNA duplexes of 21 to 24 nucleotides (nt) (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 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 king...

RNA helicase domains of viral origin in proteins of insect retrotransposons: possible source for evolutionary advantages

“…Our experimental data demonstrated that the predicted P. xylostella LINE VSR domain exhibits a weak, compared to the potent plant virus VSR p19, but detectable ability to suppress RNA silencing in the Nicotiana benthamiana leaves ( Lazareva et al, 2015 ). In this context, it is important that plant and insect VSRs can substitute for each other in different eukaryotic model systems ( Jing et al, 2011 ; Maliogka et al, 2012 ; Zhu et al, 2012 ). Moreover, the plant VSRs were shown to suppress retrotransposon silencing in heads and ovaries of insects by endogenous siRNAs ( Berry et al, 2009 ).…”

The VP3 Factor from Viruses of Birnaviridae Family Suppresses RNA Silencing by Binding Both Long and Small RNA Duplexes

“…The key relevance of these proteins for the infection life cycle has been established in multiple virus/host combinations, showing that the absence of an active VSR results in greatly reduced viral infection [19,[20][21][22][23][24][25]. Interestingly, it has been found that several vertebrate-infecting viruses also encode proteins with the ability to counteract RNA silencing, such as NS1 from Influenza A virus [26][27][28][29], VP30, VP35 and VP40 from Ebola virus [28,30,31], NSs from La Crosse virus [32], s3 from Mammalian orthoreovirus type 3 [33], E3L from Vaccinia virus [28,29], Tas from Primate foamy virus type 1 [34], Tat from Human immunodeficiency virus [35], Rex from Human T-cell lymphotropic virus [36], C and E2 proteins from Hepatitis C virus [37][38][39], 7a from Severe acute respiratory syndrome virus [40], and HVT063 from Turkey herpesvirus [41], presumably as part of the attack-defense-counterdefense arms race between viruses and their hosts (some of these RSSs were recently reviewed [42,[43][44][45][46]).…”

Thylakoid membrane oxidoreductase LTO1/AtVKOR is involved in ABA‐mediated response to osmotic stress in Arabidopsis

“…The harvested leaves were used for total RNA isolation according to a previous study (Jing et al ). The cDNA was synthesized according to the standard procedures of the RevertAid Fist Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA).…”