Membrane and protein dynamics in live plant nuclei infected with Sonchus yellow net virus, a plant-adapted rhabdovirus

Abstract

Sonchus yellow net virus (SYNV) serves as the paradigm for the cell biology of plant-adapted rhabdoviruses. Fluorescence recovery after photobleaching (FRAP) demonstrated that SYNV-induced intranuclear membranes are contiguous with the endomembrane system. Fluorescence intensity measurements of a green fluorescent protein-tagged nuclear envelope marker were consistent with electron microscopy studies, which suggest that infection by SYNV results in invagination of the inner nuclear membrane. Fusions of a red fluorescent protein to five SYNV-encoded proteins were used to determine the relationship between virus-induced intranuclear membranes and the localization of viral proteins. These data establish definitively that localization in the context of infected cells provides a superior means to predict protein function compared with localization studies conducted in mock-inoculated cells. Substructure has been identified within the viroplasm, the putative site of virus replication, which suggests that the nucleocapsid (N) protein occupies a region at the junction between the viroplasm and intranuclear membranes that largely excludes the phosphoprotein. Within virus-infected nuclei, the SYNV matrix (M) protein and glycoprotein (G) were associated predominantly with membranes, whereas sc4, the predicted movement protein, accumulated primarily at punctate loci on the periphery of cells. Coexpression of differently tagged SYNV protein fusions in combination with FRAP analyses suggest a model whereby the replication and morphogenesis of SYNV are spatially separated events. Finally, an M protein-containing complex was discovered that appears to bud from the nucleus and that moves on ER membranes. Taken together, these data represent the most comprehensive analyses of rhabdoviral protein localization conducted in the context of infected cells. INTRODUCTIONPlant-adapted rhabdoviruses are classified into two genera. Members of the genus Nucleorhabdovirus differ from members of the genus Cytorhabdovirus and their mammal-, fishand insect-infecting relatives in that they replicate and undergo morphogenesis in nuclei of infected cells Reed et al., 2005;Revill et al., 2005;Tsai et al., 2005;Dietzgen et al., 2006).Nucleorhabdoviruses share many of the structural features of animal rhabdoviruses such as vesicular stomatitis virus (VSV; Jackson et al., 2005). They are consequently composed of an infectious nucleocapsid 'core' surrounded by a phospholipid membrane. The core can be purified by density-gradient centrifugation of non-ionic detergenttreated virions (Wagner et al., 1996). In the case of Sonchus yellow net virus (SYNV), the core is a ribonucleoprotein (RNP) complex that consists of the negative-strand genomic RNA (Jackson & Christie, 1977) encapsidated by three associated proteins, namely the nucleocapsid (N), phospho-(P) and polymerase (L) proteins Zuidema et al., 1987;Choi et al., 1992). The membrane fraction of mature virions contains a glycoprotein (G) that protrudes from the surface of the virion (Goldberg et al., 1...

Intranuclear membranes induced by lipidated proteins are derived from the nuclear envelope

“…ER marker proteins did not induce intranuclear membranes on their own when expressed NCS-related structures, the R-rings, induced by overexpression of the nucleolar chaperon Nopp140, 6 for ODVs produced in insect cells during baculovirus infection, 17 for membranes formed after overexpression of particular nuclear proteins [22][23][24][25] as well as for those membranes formed in nuclei of several pathological cells. 13 In the above-mentioned cases the membrane budding from the INM has been either followed directly by live cell imaging 6,15,22 or membrane continuity between the intranuclear membranes and the INM has been demonstrated by TEM analysis. 6,10,13 In addition, the presence of ER marker proteins within the intranuclear membrane structures has been taken as conclusive evidence for their NE/ER origin.…”

“…This leads to hyperproliferation of the INM and eventually to the formation of intranuclear membranes. [15][16][17] The intranuclear membrane vesicles induced by the nucleopolyhedrovirus Autographa californica (baculovirus) ultimately become the envelope of the occlusion-derived virus (ODV). 17 Proliferation of the INM can be induced in non-infected cells by expression of particular viral proteins.…”

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis

“…All interactions were confirmed using both combinations of reciprocal nEYFP/cEYFP fusion proteins in two separate experiments (three replicates per experiment). Protein localization assays were done using agroinfiltration of C-terminal YFP/CFP tagged proteins in transgenic N. benthamiana plants expressing RFP-H2B or RFP tagged with the amino-terminal signal sequence of Arabidopsis thaliana basic chitinase and a carboxy-terminal HDEL ER-retention signal (Goodin et al, 2007).…”

New Gateways to Discovery

“…Next, we will compare protein localization data produced using transient assays versus transgenic plants. We will also discuss recent results from our lab supporting that protein localization data obtained using transient assays is comparable to that obtained from transgenic plants Goodin et al, 2007). Finally, we discuss how advances in AFP-vector development will realize their greatest utility when used in conjunction with state-of-the-art imaging systems.…”

“…To begin, we will review some of the most recently published vector systems for the expression of AFPs in plant cells, with particular emphasis on the pSAT (modular satellite plasmid) and pSITE (stable integration and transient expression plasmid) vectors Chakrabarty et al, 2007;Goodin et al, 2007). Using the pSITE vectors, we have developed several transgenic lines to support cell biology studies conducted with Nicotiana benthamiana, a model host essential for the study of plant-pathogen interactions, which is being utilized increasingly in plant biology research, primarily due to the facile manner by which large populations of cells can be transfected.…”