SFB796 - Associated project AP2

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AP2 : Björn Krenz
Analysis of the Begomoviral nuclear export

 

Project summary

Geminiviruses infect crops, ornamentals and weeds and cause substantial agro-economical losses around the world. The incidence and severity of geminivirus diseases has greatly increased in the past 20 years. The family Geminiviridae is one of the largest and most important families of plant viruses. The DNA genomes of geminiviruses encode only for 5-7 proteins that redirect host machineries and processes to establish a productive infection. These interactions reprogram plant cell cycle and transcriptional controls, inhibit cell death pathways, interfere with cell signaling and protein turnover, and suppress defense.

The genome of bipartite begomoviruses (family Geminiviridae) consists of two circular ssDNA molecules: DNA A and DNA B. Both genome components are relatively small (2.5 - 3.0 kb per single-stranded DNA circle) and replicate within the plant nucleus. Thus, systemic infection requires the permeation across two cellular barriers, the nuclear envelope and plasmodesmata. The two DNA B-encoded proteins, the nuclear-shuttle protein (NSP) and movement protein (MP), mediate the viral transport processes. Several studies provide evidence that NSP facilitates trafficking of viral DNA (vDNA) into and out of the nucleus, and that MP serves as a membrane adaptor and mediates cell-to-cell transfer via plasmodesmata as well as long-distance spread through the phloem. The begomoviral transport complex and the mechanism of intra- and intercellular movement have yet not been fully elucidated. The nature of the viral transport complex is unknown, but as the initial step in systemic spread, it needs to cross the nuclear envelope. It is therefore necessary to: (1) identify the specific NSP-interacting host factors and (2) analyze the nucleus export mechanism and intracellular movement complex of begomovirus.

Earlier studies have shown that geminiviral transport proteins of Abutilon mosaic virus (AbMV, a bipartite begomovirus) induce characteristic changes in the plant nuclear architecture. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when NSP and MP were simultaneously expressed in leaves. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. To visualize the impact of MP and NSP on the plant nuclear envelope (PNE) the LBR:GFP marker was used in transient expression experiments. The LBR:GFP marker consists of the first 238 amino acids of the human lamin B receptor (LBR), fused to the green fluorescent protein (GFP). When expressed in tobacco plants, fluorescence accumulated only at the PNE of leaf epidermal cells. The association of the LBR-marker with the plant NE is evident, but the actual binding partner(s) is/are unknown, because plants do not possess lamins. Thus, we want to identify the binding partner(s) of the LBR:GFP marker in plant nuclei to gain further insight into the plant nucleus inner membrane architecture. Furthermore, the results will also broaden our understanding in the plant nucleus interactome compared to the human one.

Plants do not have identifiable molecular homologues of LBR. This may suggest that plant NE has a unique composition, which could have arisen differently from yeast and animals. However, targeting the amino-terminal 238 amino acids of the human LBR to the higher plant NE suggests that at least one NE protein targeting and anchoring mechanism, as yet unknown, may be common in plants, animals and yeast. Preliminary results have shown that MP and NSP transiently expressed in HeLa cells localized to the same compartments as in plant cells. AbMV induced invaginations in infected plant nuclei, later on necking and budding as monitored by the LBR:GFP marker. A re-localization of the LBR:GFP marker can be observed in a time dependent manner, which suggests a rearrangement of the PNE and re-location of LBR. Microscope images suggest that vesicles are budding from the nucleus and are directed to the cell periphery. Transient expression experiments then showed that co-expression of MP and NSP induce this vesicle formation. Based on these observations, we postulate a remodeling character of the begomoviral transport proteins on human nuclei and therefore we want to investigate if vesicle formation is also induced in human cells.

If the begomoviral transport proteins affect the lamin layer, then we want to investigate the potential of MP and NSP to interfere with the nuclear egress machinery of HCMV. The nuclear egress complex of HCMV is composed of several viral and host proteins, e.g. LBR. The p32-pUL97 complex binds to the lamin B receptor, which is necessary for the destabilization process of the nuclear lamin layer. The begomoviral proteins might prevent formation of the p32-pUL97-LBR and therefore interfere with the nuclear egress of HCMV. On the other hand, begomoviral movement proteins might have a positive, synergistic effect on the nuclear egress of HCMV and might be able to complement a dominant-negative mutant for nuclear egress. In both cases, these analyses will provide more insight into the nuclear egress mechanism of HCMV.

The project will not only broaden our understanding in the plant nucleus inner membrane architecture and interactome, but also reveal conserved structural features of the plant and human nucleus. Despite the fact that plant NE has a unique composition, we address the question if NE protein targeting, retention and anchoring mechanism may be common in animal and plant cells. In addition, we will analyze the nature and mechanism of the begomoviral nuclear export complex, which might lead to an applied approach in virus resistance in the future. The proposed research will also clarify the observed remodeling effect of begomoviral movement proteins on the plant, as well as on the human nucleus.

 

Project relevant publications

Krenz, B., Jeske H, Kleinow T.   (2012).   The induction of stromule formation by a plant DNA-virus in epidermal leaf tissues suggests a novel intra- and intercellular macromolecular trafficking route.   Front Plant Sci. 3:291 .

Krenz, B., Neugart F, Kleinow T, Jeske H.   (2011).   Self-interaction of Abutilon mosaic virus replication initiator protein (Rep) in plant cell nuclei.   Virus Res. 161, 194-197 .

Krenz, B., Wege C, Jeske H, Kleinow T.   (2010).   A plastidal heat shock cognate 70 kDa protein interacts with the Abutilon mosaic virus movement protein.   Virology. Feb 27 .

Kleinow, T., Tanwir, F., Kocher, C., Krenz, B., Wege, C., and Jeske, H.   (2009).   Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells.   Virology 391, 212-220 .

Morilla G, Krenz, B, Jeske H, Bejarano ER, Wege C.   (2004).   Tete a tete of tomato yellow leaf curl virus and tomato yellow leaf curl sardinia virus in single nuclei.   J Virol. 78(19):10715-23 .