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...

Potato yellow dwarf virus (PYDV) and Sonchus yellow net virus (SYNV) belong to the genus Nucleorhabdovirus. These viruses replicate in nuclei of infected cells and mature virions accumulate in the perinuclear space after budding through the inner nuclear membrane. Infection of transgenic Nicotiana benthamiana 16c plants (which constitutively express green fluorescent protein (GFP) targeted to endomembranes) with PYDV or SYNV resulted in virus-specific patterns of accumulation of both GFP and membranes within nuclei. Using immunolocalization and a lipophilic fluorescent dye, we show that the sites of the relocalized membranes were coincident with foci of accumulation of the SYNV nucleocapsid protein. In contrast to the effects of PYDV and SYNV, inoculation of 16c plants with plus-strand RNA viruses did not result in accumulation of intranuclear GFP. Instead, such infections resulted in accumulation of GFP around nuclei, in a manner consistent with proliferation of the endoplasmic reticulum. We propose that the relocalization of GFP in 16c plants can be used to study sites of rhabdovirus accumulation in live cells. This study is the first to use live-cell imaging to characterize the effects of rhabdoviruses on plant nuclear membranes.

No abstract

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.An estimated 95% of human multi-exon genes undergo alternative pre-mRNA splicing (1, 2). Unlike promoter activity that regulates the abundance of transcripts, alternative splicing influences the structure of the mRNAs and their encoded proteins (reviewed in Ref. 3). The high incidence of alternative splicing and its ability to increase the coding capacity of the genome makes it a central element in eukaryotic gene expression.Despite its importance, we do not fully understand how splice sites, especially the alternative ones, are selected. The accurate recognition of splice sites in vivo is the result of a combinatorial mechanism (4 -7) where protein complexes assemble on the nascent pre-mRNA, because of the combination of weak RNA/RNA, RNA/protein, and protein/protein interactions. Transient interaction between the spliceosome and regulatory proteins assembling on the pre-mRNA help in the recognition of splice sites (8). There is increasing evidence that the phosphorylation state of splicing regulatory proteins is a crucial element for this recognition process (reviewed in Ref. 9), because phosphorylation/dephosphorylation often changes the affinity between proteins in these complexes. The spliceosome acts in a stepwise fashion on the pre-mRNA, and four distinct splicing complexes, the H, A, B and C complexes, have been identified (7). The H and E complexes contain the pre-mRNA imbedded with various proteins, among them hnRNPs and SR proteins as well as the U1 snRNP bound to the 5Ј splice site. The entry of U2 snRNP defines the branch point and leads to the formation of the A complex. The entry of the U4/5/6 snRN...

The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERα) and beta (ERβ), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERα)-mediated transcription events with efficacy and potency equal to or greater than that of ERα’s cognate ligand, 17β-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.

In terms of functional genomics research, Nicotiana benthamiana, more so than other model plants, is highly amenable to high-throughput methods, especially those employing virus-induced gene silencing and agroinfiltration. Furthermore, through recent and ongoing sequencing projects, there are now upward of 18,000 unique N. benthamiana ESTs to support functional genomics research. Despite these advances, the cell biology of N. benthamiana itself, and in the context of virus infection, lags behind that of other model systems. Therefore, to meet the challenges of diverse cell biology studies that will be derived from ongoing functional genomics projects, a series of methods relevant to the characterization of membrane and protein dynamics in virus-infected cells are provided here. The data presented here were derived from our studies with plant rhabdoviruses. However, the employed techniques should be broadly applicable within the field of plant virology. We report here on the use of a novel series of binary vectors for the transient or stable expression of autofluorescent protein fusions in plants. Use of these vectors in conjunction with advanced microscopy techniques such as fluorescent recovery after photobleaching and total internal fluorescence microscopy, has revealed novel insight into the membrane and protein dynamics of virus-infected cells.