Effects of Point Mutations in the Readthrough Domain of the Beet Western Yellows Virus Minor Capsid Protein on Virus Accumulation In Planta and on Transmission by Aphids

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203

144

Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.Most if not all plants possess a defense mechanism against virus infection known as posttranscriptional gene silencing (PTGS; see reference 44 for a recent review). PTGS is a homology-dependent RNA degradation system to recognize and degrade viral RNA (and any homologous transgene mRNA) in the cytoplasm. Mechanistically similar phenomena exist in animals and fungi (reviewed in references 4, 13, and 35). The mechanism by which a virus infection triggers PTGS in plants is not fully understood, but double-stranded RNA is a strong inducer of PTGS (43) and such a form is produced during replication of an RNA virus. Production of small RNAs of ϳ21 to 23 nucleotides, called small interfering RNAs (siRNAs), corresponding to both the plus and minus strands of the target RNA is associated with PTGS (12,13,45). In plants, gene silencing at one site can trigger PTGS in distant tissues and across a graft union (29, 39a). The mobile signal, which must consist at least in part of homologous RNA, is thought to move from cell to cell through plasmodesmata and systemically via the vasculature. Some plant viruses have been shown to encode proteins which can counteract PTGS (8,22,39,40,42). These silencing suppressor proteins may act at different steps in the PTGS pathway. Thus, (i) the potyvirus helper component-proteinase (HCPro) interferes with initiation and maintenance of silencing at a step coincident with or upstream of siRNA production (1,7,16,23,24), (ii) the 2b protein of cucumber mosaic virus (CMV) prevents initiation of PTGS in new growth by inhibiting the long-range PTGS signaling acti...

Section: Methodsmentioning

confidence: 99%

P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing

Pfeffer

Dunoyer

Heim

et al. 2002

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203

144

“…3A). Such variation in the wild-type progeny sequences was observed earlier in a study of mutants in the BWYV RT domain (2) and is presumably due to a combination of natural sequence "drift" during the relatively error-prone viral RNA multiplication process and errors introduced during reverse transcription-PCR amplification of the sequence to be analyzed.…”

Section: Resultsmentioning

confidence: 92%

“…Total RNA from plant tissue was extracted using either ⌻rizol (Invitrogen) or a commercial RNA purification kit (Rneasy Plant Mini-Kit; Qiagen). Viral structural proteins in total-protein extracts of infected protoplasts were detected by Western blotting using antisera specific for the BWYV P3 and RT proteins (2).…”

Section: Construction Of Mutants Of Bwyvmentioning

confidence: 99%

See 1 more Smart Citation

Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

Brault

Bergdoll

Mutterer

et al. 2003

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Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ϳ90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/ N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.Beet western yellows virus (BWYV; genus Polerovirus), like all members of the family Luteoviridae, is transmitted in a circulative, nonpropagative manner by aphids (see reference 7 for a review). Poleroviruses and luteoviruses (the other major genus of the family) are transmitted vector specifically, implying that determinants on the viral capsid selectively interact with specific structures within vector aphids. The particles of poleroand luteoviruses are composed of two types of protein, the major capsid protein (P3) of 22 to 23 kDa and a minor species of ϳ75 kDa known as readthrough (RT) protein (see reference 13 for a review). The RT protein is a C-terminally extended form of P3 produced by episodic suppression of P3 translation termination so that translation continues into the downstream open reading frame (ORF) 5, which encodes the RT domain (12). The RT protein is believed to be incorporated into the capsid via its P3 moiety, with the RT domain protruding from the virion surface (1, 4).Nicotiana clevelandii can be agroinfected with full-length BWYV cDNA (10), and the resulting plants can serve as a virus source in aphid transmission experiments (1). Site-directed mutagenesis of the viral cDNA prior to agroinfection has been used to map sequences in the RT domain important for virus movement in planta and for aphid transmission of BWYV (1-3). In this paper, w...

“…Mutagenesis studies performed with infectious clones have greatly enhanced our understanding of the domains within viral proteins that are responsible for regulating different aspects of the polerovirus life cycle, including virus assembly, systemic movement, phloem retention, and vector transmission (2)(3)(4)(5)(6)(7)(8)(9). However, very few host proteins have been identified as interacting directly with any polerovirus protein (10)(11)(12)(13).…”

mentioning

confidence: 99%

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

DeBlasio

Chavez

Alexander

et al. 2016

Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection-hallmarks of host-pathogen interactions-were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCEThe exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy. P hloem-limited viruses, such as poleroviruses, are among the most devastating, but least understood pathogens infecting plants due to the technical challenges encountered when studying pathogens that naturally replicate and move within intractable cell types (1). Mutagenesis studies performed with infectious clones have greatly enhanced our understanding of the domains within viral proteins that are re...