Begomovirus-host protein-protein interaction network: identifying an intracytoplasmic route for viral DNA intracelular transport

• Ano de defesa: 2019
• Autor(a) principal: Mageste, Bianca Castro Gouveia
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa; Genética e Melhoramento
• Palavras-chave em Português: Begomovírus; Membranas (Biologia) - Transporte; Proteínas SNARE; Sintaxina; Vírus de DNA; Proteínas - Transporte; Biologia Molecular
• Link de acesso: https://locus.ufv.br//handle/123456789/30184

Viruses are obligate intracellular parasites and, once in the host cell cytoplasm, they must move intracellularly to and from the replication site to the plasma membrane for spreading. Understanding how viruses hijack the host intracellular transport system using a limited repertoire of proteins provides an opportunity to uncover the molecular bases of the host transport machinery. Begomovirus, a plant ssDNAvirus, use two movement proteins, MP and NSP, to move intracellularly from the site of replication in the nucleus to the cell surface. However, the host components of vDNA complex competent for intracytoplasmic translocation have not been identified, and the underlying mechanism for intracytoplasmic trafficking of vDNA remains to be elucidated. Here, we used a previously fabricated, in situ synthesized protein microarray containing 4600 ORFs of Arabidopsis to identify NSP- and MPArabidopsis protein-protein interactions (PPIs). Consistent with the movement function of the viral proteins, the identified NSP-MP-PPI network uncovered direct and indirect interactions, over represented under the terms transport activity ontology, protein binding, membrane-bound organelles, intracellular vesicle, and SNARE complex, which may define an intracellular route for vDNA trafficking. These studies thus provided critical framework for future investigationsto elucidate the molecular mechanisms for intracytoplasmic transport of begomoviruses. Based on this assumption, we selected an NSP-specifically interacting syntaxin-6 domaincontaining protein, designated NISP for further characterization. We provided several lines of evidence indicating that NISP may be involved in directing the intracytoplasmic anterograde movement of NSP-vDNA. First, we used coimmunoprecipitation assays and bimolecular fluorescence complementation assay to confirm that NISP interacted with NSP in planta and showed that the complex formation occurred in trafficking vesicles likely associated to trans-Golgi network (TGN)/early endosome. Second, we showed that NISP exhibited a pro-viral function and the begomovirus infection required the NIPS-NSP interaction. The mutant nisp-1 was resistant to begomovirus as the knock lines displayed attenuated symptoms, a delayed course of infection and accumulated much lower viral DNA as compared to Col-0 and overexpressing lines. This nisp-1 resistance phenotype was reversed by NISP complementation, confirming that the nisp-1 phenotype was due to inactivation of NISP gene. In contrast, the overexpressing lines were hypersensitive to begomovirus, as they displayed an accelerated course of infection and lowered load of viral DNA as compared to Col-0. We took advantage of a highly conserved NISP paralog, AT2G18860, to show that NISP-NSP interaction underscored the molecular bases for the NISP pro-viral function. AT2G18860, which shares with NISP a 78.6% identical syntaxin-6 domain, did not interact with NSP and thusdid not interfere with begomovirus infection. Consistent with these data, NISP, but not AT2G18860, was induced by begomovirus infection. Third, NISP was also demonstrated to interact with NIG, which facilitates the release of the NSP-DNA complex from the nuclear pores to the cytosol, and the presence of NSP enhanced the NISP-NIG complex formation. We used ChIP assay to demonstrate that NISP was also associated with vDNA in infected cells, which may be assembled into a NISP-NIG-NSP multiprotein complex. Finally, the NISP interactions relocated the dispersed cytosolic NIG and viral NSP to trafficking vesicle likely associated to TGN/microsomes; thereby, favoring the interaction of NSP-DNA with MP, which has been shown to bind to microsome-associated SYTA for the MP-mediated cell-to-cell movement of vDNA.