SFB796 - Sub project C2

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Functional analysis of the interaction between host factors and viral transport proteins

 

Project summary

Viruses encode specialized proteins to recruit cellular machineries for their transport and replication. In the second phase of the CRC796 we would like to (i) unravel the link between capsid protein binding of the HSP70/HSP40 complex and the ubiquitination / degradation of the viral protein, (ii) to utilize the human cytomegalovirus pUL50 protein to study conserved structural features of plant and vertebrate nuclear envelopes and (iii) to further our understanding of the intracellular trafficking of MP17 and to unravel the role of MP17-interacting RNA helicases during virus infection. Mutant analysis revealed that choline transporter-like 1 (CTL1) is essential for plasmodesmata (PD) targeting of MP17. CTL1 is a predicted PD-associated plasma membrane protein. This opens the possibility that CTL1 is either recruited by MP17 for PD trafficking, binds MP17 within PDs or that the CTL1 mutation leads to an overall altered membrane composition and/or absence of micro-domains. Direct interaction of CTL1 and MP17 will be tested by BIFC analysis or co-localization studies using differentially labeled fusion proteins. The importance of lipid composition and micro-domains for viral spread will further be studied by the biochemical analysis of the CTL1 mutant. In addition PD-targeting of MP17 will be followed in sterol mutants and in Arabidopsis plants treated with inhibitors of sterol biosynthesis. Co-expression of MP17 and WW-domain containing RNA helicases in Arabidopsis mesophyll protoplasts revealed a direct interaction of both proteins and a reallocation of helicases from the nucleus to cytoplasmic bodies. This implicates that MP17 might be involved in the recruitment of host helicases to virus replication and/or transport complexes. The importance of this interaction will be tested by analyzing the effect of helicase knockout or knockdown mutations on viral infections and by designing virus variants with mutations in the proposed interaction domain. Furthermore, attempts will be undertaken to isolate the observed cytosolic bodies by immune precipitation of GFP fused to either helicases or MP17 and MS analysis of the purified protein complexes.

 



Middle: Arabidopsis thaliana plant. Left and right: CLMS pictures of sink (left) and source (right) leaves. The red lines are cell walls of epidermal cells, which are interrupted by green-fluorescent labelled MP-17-GFP, which mark cell-cell connections, the so-called plasmodesmata.

 

Project relevant publications

Lamm C, Link K, Wagner S, Milbradt J, Marschall M, Sonnewald U. (2016).   Human cytomegalovirus nuclear egress proteins ectopically expressed in the heterologous environment of plant cells are strictly targeted to the nuclear envelope.   Viruses 8(3): 73. doi: 10.3390/v8030073.

Link K, Sonnewald U. (2016).   Interaction of movement proteins with host factors, mechanism of viral host cell manipulation and influence of MPs on plant growth and development.   In Kleinow T. (ed) Plant-Virus interactions, Molecular Biology, Intro- and Intercellular Transport. Springer Berlin Heidelberg, pp 1-38. 114: 4-14.

Prasch C, Sonnewald U. (2015).   Signaling events in plants: Stress factors in combination change the picture.   Environ. Exp. Bot. 114: 4-14.

Prasch C, Sonnewald U. (2013).   Simultaneous application of heat, drought and virus to Arabidopsis thaliana plants reveals significant shifts in signaling networks.   Plant Physiol. 162(4): 1849-1866.

Griessl MH, Jungkunz I, Sonnewald U, Muller YA. (2012).   Purification, crystallization and preliminary X-ray diffraction analysis oft he Hsp40 protein CPIP1 from Nicotiana tabacum.   Acta Crystallographica Section F.   [Epub ahead of print].

Thran M, Link K, Sonnewald U. (2012).   The Arabidopsis DCP2 gene is required for propper mRNA turnover and prevents transgene silencing in Arabidopsis.   Plant J.   [Epub ahead of print].

Jungkunz I, Link K, Vogel F, Voll LM, Sonnewald S, Sonnewald U. (2011).   AtHsp70-15 deficient Arabidopsis plants are characterized by reduced growth, a constitutive cytosolic protein response and enhanced resistance to TuMV.   Plant J. 66(6):983-95.

Link K, Vogel F, Sonnewald U. (2011).   PD trafficking of potato leaf roll virus movement protein in Arabidopsis depends on site-specific protein phosphorylation.   Front. Plant Sci. 2:18. doi: 10.3389/fpls.2011.00018.

Vogel F, Hofius D, Paulus KE, Jungkunz I, Sonnewald U. (2011).   The second face of a known player: Arabidopsis silencing suppressor AtXRN4 acts organ-specifically.   New Phytol. 189(2): 484-93.

Hafrén, A., Hofius, D., Rönnholm, G., Sonnewald, U. and Mäkinen, K. (2010).   HSP70 and its co-chaperone CPIP promote potyvirus infection by regulating viral coat protein functions.   The Plant Cell   [Epub ahead of print].

Kronberg, K., Vogel, F., Rutten, T., Hajirezaei, M. R., Sonnewald, U. and Hofius D. (2007).   The silver lining of a viral agent: increasing seed yield and harvest index in Arabidopsis by ectopic expression of the potato leaf roll virus movement protein.   Plant Physiol 145, 905-918.

Hofius, D., Maier, A., Dietrich, C., Jungkunz, I., Börnke, F., Maiss, E. and Sonnewald U. (2007).   Capsid protein-mediated recruitment of host DnaJ-like proteins is required for Potato virus Y infection in tobacco plants.   J Virology 81, 11870-11880.

Vogel, F., Hofius, D. and Sonnewald, U. (2007).   Intracellular trafficking of potato leaf roll virus movement protein in transgenic Arabidopsis.   Traffic 9, 1205-1214.

Hofius, D., Herbers, K., Melzer, M., Omid, A., Tacke, E., Wolf, S. and Sonnewald, U. (2001).   Evidence for expression level-dependent modulation of carbohydrate status and viral resistance by the potato leafroll virus movement protein in transgenic tobacco plants.   Plant J. 28(5), 529-544.