Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K 2 protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K 2 GFP and the other expressed 6K 2 mCherry. Green-and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.Positive-strand RNA viruses replicate their genomes on intracellular membranes. Extensive membrane rearrangements leading to cytoplasmic membranous structure production are observed during the infection cycle of many of these viruses (for a review, see reference 32). These virus-induced membrane structures vary greatly in origin, size, and shape. For instance, Flock House virus induces the formation of 50-nm vesicles (spherules), which are outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter (24). On the other hand, poxviruses replicate in 1-to 2-m cytoplasmic foci known as DNA factories (43), which are bounded by rough endoplasmic reticulum (ER). These factories are not only the site of DNA synthesis but also of DNA transcription and RNA translation (21). Similarly, mimiviruses are huge doublestranded DNA viruses that replicate in giant cytoplasmic virus factories (45). Three-dimensional electron microscopic imaging has shown that coronavirus-induced membrane alterations define a reticulovesicular network of modified ER that integrates convoluted membranes, numerous interconnected double-membrane vesicle...

Eukaryotic elongation factor 1-alpha (eEF1A) was identified as an interactor of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp) and VPg-protease (VPg-Pro) using tandem affinity purification and/or in vitro assays. Subcellular fractionation experiments revealed that the level of eEF1A substantially increased in membrane fractions upon TuMV infection. Replication of TuMV occurs in cytoplasmic membrane vesicles, which are induced by 6K-VPg-Pro. Confocal microscopy indicated that eEF1A was included in these vesicles. To confirm that eEF1A was found in replication vesicles, we constructed an infectious recombinant TuMV that contains an additional copy of the 6K protein fused to the green fluorescent protein (GFP). In cells infected with this recombinant TuMV, fluorescence emitted by 6KGFP was associated with cytoplasmic membrane vesicles that contained VPg-Pro, the eukaryotic initiation factor (iso) 4E, the poly(A)-binding protein, the heat shock cognate 70-3 protein, and eEF1A. These results suggest that TuMV-induced membrane vesicles host at least three plant translation factors in addition to the viral replication proteins.

The pharmacokinetics and dosimetry of (86)Y-DOTA(0)- d-Phe(1)-Tyr(3)-octreotide ((86)Y-SMT487) were evaluated in a phase I positron emission tomography (PET) study of 24 patients with somatostatin receptor-positive neuroendocrine tumours. The effect of amino acid (AA) co-infusion on renal and tumour uptake was assessed in a cross-over randomised setting. Five regimens were tested: no infusion, 4-h infusion of 120 g mixed AA (26.4 g l-lysine + l-arginine), 4 h l-lysine (50 g), 10 h 240 g mixed AA (52.8 g l-lysine + l-arginine) and 4 h Lys-Arg (25 g each). Comparisons were performed on an intra-patient basis. Infusions of AA started 0.5 h prior to injection of (86)Y-SMT487 and PET scans were obtained at 4, 24 and 48 h p.i. Absorbed doses to tissues were computed using the MIRD3 method. (86)Y-SMT487 displayed rapid plasma clearance and exclusive renal excretion; uptake was noted in kidneys, tumours, spleen and, to a lesser extent, liver. The 4-h mixed AA co-infusion significantly ( P<0.05) reduced (86)Y-SMT487 renal uptake by a mean of 21%. This protective effect was significant on the dosimetry data (3.3+/-1.3 vs 4.4+/-1.0 mGy/MBq; P<0.05) and was further enhanced upon prolonging the infusion to 10 h (2.1+/-0.4 vs 1.7+/-0.2 mGy/MBq; P<0.05). Infusion of Lys-Arg but not of l-lysine was more effective in reducing renal uptake than mixed AA. Infusion of AA did not result in reduced tumour uptake. The amount of (90)Y-SMT487 (maximum allowed dose: MAD) that would result in a 23-Gy cut-off dose to kidneys was calculated for each study: MAD was higher with mixed AA co-infusion by a mean of 46% (10-114%, P<0.05 vs no infusion). In comparison with 4 h mixed AA, the MAD was higher by a mean of 23% (9-37%; P<0.05) with prolonged infusion and by a mean of 16% (2-28%; P<0.05) with Lys-Arg. We conclude that infusion of large amounts of AA reduces renal exposure during peptide-based radiotherapy and allows higher absorbed doses to tumours. The prolongation of the infusion from 4 to 10 h further enhances the protective effect on the kidneys.

A 3.9 kb BglII-HindIII DNA fragment containing the rubredoxin gene from Clostridium pasteurianum has been cloned using oligonucleotide probes designed from the protein sequence. The 2675 bp SspI-HindIII portion of this fragment has been sequenced and found to contain three open reading frames in addition to the rubredoxin gene. The putative product of one of these open reading frames is similar to various thioredoxin reductases. The rubredoxin gene translates into a sequence that differs from the previously published protein sequence in three positions, D-14, D-22 and E-48 being replaced by the corresponding amides. These changes have been confirmed by partial resequencing of the protein. Promoter-like sequences and a transcription termination signal have been found near the sequence of the rubredoxin gene, which may therefore constitute an independent transcriptional unit. Expression of C. pasteurianum rubredoxin in Escherichia coli strain JM109 has been optimized by subcloning a 476 bp SspI-SspI fragment encompassing the rubredoxin gene. Under these conditions, the latter gene was partly under the control of the lac promoter of pUC18, and the level of rubredoxin production could be increased twofold on addition of a lactose analogue, thus reaching 2-3 mg of pure protein/l of culture. Recombinant rubredoxin was produced in E. coli cells as the holoprotein, and displayed a u.v.-visible-absorption spectrum identical with that of the rubredoxin purified from C. pasteurianum. M.s. and N-terminal sequencing showed that C. pasteurianum rubredoxin expressed in E. coli differs from its native counterpart by having an unblocked N-terminal methionine.

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