A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1 ؊/؊ mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1 ؊/؊ pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1 ؊/؊ animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1 ؊/؊ mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1 ؊/؊ mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.NEPHRIN is a transmembrane protein of the immunoglobulin (Ig) superfamily that is expressed by epithelial podocytes of developing glomeruli (13, 17). Congenital nephrotic syndrome of the Finnish type results from mutations in NPHS1, the human gene encoding NEPHRIN, indicating a role for NEPHRIN in maintaining the filtration barrier that prevents proteins from freely entering the glomerular urinary space (6,17). Recent studies localized NEPHRIN to the slit diaphragms that form the junctions between podocyte foot processes interdigitating along the glomerular basement membrane. This and other studies suggest that NEPHRIN proteins extending toward each other from adjacent podocyte foot processes may interdigitate in a zipper-like structure to form the crucial filtration barrier in the slit diaphragm. The eight Ig-like domains of each NEPHRIN protein, which are of the C2 type of Ig domain known to be involved in cell-cell interactions, are thought to provide the homophilic interactions that bind these NEPHRIN proteins together (13, 17).We use a high-throughput mutagenesis method based on gene trapping in embryonic stem (ES) cells that allows automated production of sequence tags from the trapped and mutated genes (20). These ES cell clones are stored in a library called Omnibank, and the sequence tag from the gene trapped in each clone, referred to as the Omnibank sequence tag or OST, is entered into a searchable database. A protein with Ig domains was identified with...

We have used immunoblotting, immunocytochemical, and gene fusion methods to examine the differential subcellular partitioning of tobacco etch potyvirus proteins that are potentially associated with RNA replication. From the earliest timepoints at which viral proteins could be detected, proteins Nla (49-kilodalton proteinase) and Nlb (58-kilodalton polymerase) were localized primarily in the nucleus, whereas the 71-kilodalton cylindrical inclusion protein was identified in the cytoplasm. The Nla and Nlb coding regions were fused to the 8-glucuronidase (GUS) sequence in a plant expression vector, resulting in synthesis of chimeric proteins in transfected protoplasts and in transgenic plants. In situ localization of GUS activity revealed nuclear localization of the GUS-Nla and GUS-NIb fusion proteins and cytoplasmic localization of nonfused GUS. These results indicate that both Nla and Nlb contain nuclear targeting signals, and that they may serve as useful models for studies of plant cell nuclear transport. A discussion of the general utility of the nuclear transport system described here, as well as the role of nuclear translocation of potyviral proteins, is presented.

We have used immunoblotting, immunocytochemical, and gene fusion methods to examine the differential subcellular partitioning of tobacco etch potyvirus proteins that are potentially associated with RNA replication. From the earliest timepoints at which viral proteins could be detected, proteins Nla (49-kilodalton proteinase) and Nlb (58-kilodalton polymerase) were localized primarily in the nucleus, whereas the 71-kilodalton cylindrical inclusion protein was identified in the cytoplasm. The Nla and Nlb coding regions were fused to the 8-glucuronidase (GUS) sequence in a plant expression vector, resulting in synthesis of chimeric proteins in transfected protoplasts and in transgenic plants. In situ localization of GUS activity revealed nuclear localization of the GUS-Nla and GUS-NIb fusion proteins and cytoplasmic localization of nonfused GUS. These results indicate that both Nla and Nlb contain nuclear targeting signals, and that they may serve as useful models for studies of plant cell nuclear transport. A discussion of the general utility of the nuclear transport system described here, as well as the role of nuclear translocation of potyviral proteins, is presented.

The NIa protein of certain plant potyviruses localizes to the nucleus of infected cells. Previous studies have shown that linkage of NIa to reporter protein beta-glucuronidase (GUS) is sufficient to direct GUS to the nucleus in transfected protoplasts and in cells of transgenic plants. In this study, we mapped sequences in NIa that confer karyophilic properties. A quantitative transport assay using transfected protoplasts, as well as in situ localization technique using epidermal cells from transgenic plants, were employed. Two domains within NIa, one between amino acid residues 1 to 11 (signal domain I) and the other between residues 43 to 72 (signal domain II), were found to function additively for efficient localization of fusion proteins to the nucleus, although either region independently could facilitate a low level of translocation. Like signals from animal cells, both nuclear transport domains of NIa contain a high concentration of basic (arginine and lysine) residues. Nuclear transport signal domain II overlaps or is very near Tyr62, which is the residue that mediates covalent attachment of a subset of NIa molecules to the 5' terminus of viral RNA within infected cells. The nature of the NIa nuclear transport signal and the possibility for regulation of NIa translocation are discussed.

The RNA genome of tobacco etch virus (TEV), a plant potyvirus, functions as an mRNA for synthesis of a 346-kilodalton polyprotein that undergoes extensive proteolytic processing. The RNA lacks a normal 5' cap structure at its terminus, which suggests that the mechanism of translational initiation differs from that of a normal cellular mRNA. We have identified a translation-enhancing activity associated with the 144-nucleotide, 5' nontranslated region (NTR) of the TEV genome. When fused to a reporter gene encoding ,-glucuronidase (GUS), the 5' NTR results in an 8to 21-fold enhancement over a synthetic 5' NTR in a transient-expression assay in protoplasts. A similar effect was observed when the 5' NTR-GUS fusions were expressed in transgenic plants. By using a cell-free translation system, the translation enhancement activity of the TEV 5' NTR was shown to be cap independent, whereas translation of GUS mRNA containing an artificial 5' NTR required the presence of a cap structure. Translation of GUS transcripts containing the TEV 5' NTR was relatively insensitive to the cap analog m7GTP, whereas translation of transcripts containing the artificial 5' NTR was highly sensitive. The 144-nucleotide TEV 5' NTR synthesized in vitro was shown to compete for factors that are required for protein synthesis in the cell-free translation reaction mix. Competition was not observed when a transcript representing the initial 81 nucleotides of the TEV 5' NTR was tested. These results support the hypothesis that the TEV 5' NTR promotes translation in a cap-independent manner that may involve the binding of proteins and/or ribosomes to internal sites within the NTR.

All proteins encoded by the plant potyvirus, tobacco etch virus (TEV), arise by proteolytic processing of a single polyprotein. Two virus‐encoded proteinases (NIa and HC‐Pro) that catalyze most of the proteolytic events have been characterized previously. The two proteins that are derived from the N‐terminal 87 kd region of the viral polyprotein are a 35 kd protein and HC‐Pro (52 kd). It is demonstrated in this study that a third proteolytic activity is required to process the junction between these proteins. Proteolysis at the HC‐Pro N terminus to separate these proteins occurred poorly, if at all, after in vitro synthesis of a 97 kd polyprotein, whereas cleavage of the HC‐Pro C terminus occurred efficiently by an autoprocessing mechanism. Synthesis of the same polyprotein in transgenic tobacco plants, however, resulted in complete and accurate proteolysis at both termini of HC‐Pro. A point mutation affecting an amino acid residue essential for the proteolytic activity of HC‐Pro had no effect on N‐terminal processing. Expression in transgenic plants of a construct with a large deletion in the 35 kd protein coding region resulted in partial inhibition of HC‐Pro N‐terminal cleavage, suggesting that the 35 kd protein may affect the proteolytic event but not in a catalytic role. We speculate that this cleavage event is catalyzed by either a cryptic potyviral proteinase that requires a host factor or subcellular environment for activation, or possibly a host proteinase.

The virus-encoded proteins of tobacco etch virus (TEV), a plant potyvirus, arise by proteolytic processing of a large polyprotein precursor. The TEV genome codes for two proteinases, a 49-kilodalton proteinase and helper component proteinase (HC-Pro), which cleave the polyprotein at specific sites. The only known cleavage event catalyzed by HC-Pro occurs at the HC-Pro carboxyl terminus. The proteolytic activity of HC-Pro was analyzed by expression of the enzyme in bacterial and cell-free systems. The carboxyl-terminal domain of HC-Pro exhibited proteolytic activity in Escherichia coli with a processing half-time of approximately 100 s. The processing kinetics of HC-Pro expressed in vitro by cell-free transcription and translation was variable, depending on the presence or absence of TEV polypeptide sequences at the amino terminus of the proteolytic domain. Cleavage of the HC-Pro carboxyl terminus appeared to proceed exclusively by an autocatalytic mechanism; the proteinase synthesized in vitro exhibited little or no proteolytic activity when reacted with the HC-Pro cleavage site in trans or bimolecular reactions.

The Nla protein of certain plant potyviruses localizes to the nucleus of infected cells. Previous studies have shown that linkage of Nla to reporter protein @-glucuronidase (GUS) is sufficient to direct GUS to the nucleus in transfected protoplasts and in cells of transgenic plants. In this study, we mapped sequences in Nla that confer karyophilic properties. A quantitative transport assay using transfected protoplasts, as well as an in situ localization technique using epidermal cells from transgenic plants, were employed. Two domains within Nla, one between amino acid residues 1 to 11 (signal domain I) and the other between residues 43 to 72 (signal domain ll), were found to function additively for efficient localization of fusion proteins to the nucleus, although either region independently could facilitate a low leve1 of translocation. Like signals from animal cells, both nuclear transport domains of Nla contain a high concentration of basic (arginine and lysine) residues. Nuclear transport signal domain II overlaps or is very near TyP, which is the residue that mediates covalent attachment of a subset of Nla molecules to the 5' terminus of viral RNA within infected cells. The nature of the Nla nuclear transport signal and the possibility for regulation of Nla translocation are discussed.