Abstract
BackgroundThe genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements. However, the interaction networks between viruses and host factors are poorly understood. The genus Potyvirus is the largest group of plus-strand RNA viruses infecting plants. Potyviral nuclear inclusion a (NIa) plays many roles during infection. NIa is a polyprotein consisting of two domains, viral protein genome-linked (VPg) and protease (NIaPro), separated by an inefficiently utilized self-proteolytic site. To gain insights about the interaction between potyviral NIa and the host cell during infection, we constructed Tobacco etch virus (TEV, genus Potyvirus) infectious clones in which the VPg or the NIaPro domains of NIa were tagged with the affinity polypeptide Twin-Strep-tag and identified the host proteins targeted by the viral proteins by affinity purification followed by mass spectrometry analysis (AP-MS).ResultsWe identified 232 different Arabidopsis thaliana proteins forming part of complexes in which TEV NIa products were also involved. VPg and NIaPro specifically targeted 89 and 76 of these proteins, respectively, whereas 67 proteins were targeted by both domains and considered full-length NIa targets. Taking advantage of the currently known A. thaliana interactome, we constructed a protein interaction network between TEV NIa domains and 516 host proteins. The most connected elements specifically targeted by VPg were G-box regulating factor 6 and mitochondrial ATP synthase δ subunit; those specifically targeted by NIaPro were plasma membrane aquaporin PIP2;7 and actin 7, whereas those targeted by full-length NIa were heat shock protein 70–1 and photosystem protein LHCA3. Moreover, a contextualization in the global A. thaliana interactome showed that NIa targets are not more connected with other host proteins than expected by chance, but are in a position that allows them to connect with other host proteins in shorter paths. Further analysis of NIa-targeted host proteins revealed that they are mainly involved in response to stress, metabolism, photosynthesis, and localization. Many of these proteins are connected with the phytohormone ethylene.ConclusionsPotyviral NIa targets many host elements during infection, establishing a network in which information is efficiently transmitted.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2394-y) contains supplementary material, which is available to authorized users.
Highlights
The genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements
The goal of this work was to inquire about the complexity of the interaction network established between an essential protein from a plant RNA virus, the nuclear inclusion a (NIa) protein of potyviruses, and the host proteins by means of a high-throughput proteomics approach, affinity purification coupled to mass spectrometry (AP-MS) [5, 6]
Functional analysis of the host proteins identified in complexes with tobacco etch virus (TEV) NIa From our refined lists of host proteins targeted by NIa products during infection (Additional file 3), we identified the global functional categories of viral protein genome-linked (VPg) and nuclear inclusion a protease domain (NIaPro) specific targets, as well as common targets, using the Panther tool [48] (Fig. 5a)
Summary
The genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements. NIa is a polyprotein consisting of two domains, viral protein genome-linked (VPg) and protease (NIaPro), separated by an inefficiently utilized selfproteolytic site. To gain insights about the interaction between potyviral NIa and the host cell during infection, we constructed Tobacco etch virus (TEV, genus Potyvirus) infectious clones in which the VPg or the NIaPro domains of NIa were tagged with the affinity polypeptide Twin-Strep-tag and identified the host proteins targeted by the viral proteins by affinity purification followed by mass spectrometry analysis (AP-MS). Potyviruses (genus Potyvirus, family Potyviridae) form one of the largest groups of viruses infecting plants and cause important losses in crops worldwide Their genome consists of a single (+)-strand RNA molecule of about 10,000 nucleotides that is translated in two alternative polyproteins (depending on a frameshift in P3 cistron), which are processed by viral proteases in, apparently, a total of eleven mature products [7]. Even more suboptimal, autoproteolytic site exists in some potyviruses close to the carboxy-terminal end of NIaPro producing additional NIa-derived species in infected tissue that may be functionally relevant [17, 18]
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