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.

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SUMMARYSDS-PAGE of the 190S virus of Helminthosporium victoriae, using a discontinuous buffer system, revealed two major capsid polypeptides of tool. wt. 88K and 83K (p88 and p83) and a minor polypeptide, p78. Peptide mapping by both limited proteolysis and selective chemical cleavage showed p83 and p78 to be closely related to p88. The origin of p83/p78 could not be explained by proteolysis of p88 during virus preparation and storage. In rabbit reticulocyte lysates, denatured dsRNA directed the synthesis of a single major translation product which was identical to capsid polypeptide p88 on the basis of coelectrophoresis, immunoprecipitation and peptide mapping. No translation products comparable in size to p83 or p78 were detected in vitro. These data indicated that the capsid of the 190S virus is encoded by a single gene and verified the classification of the virus as a member of the family Totiviridae. Radioiodination of intact virus under conditions considered optimum for surface-specific iodination showed p88 to be more readily available for labelling than p83 or p78. Furthermore, when Western blots of capsid polypeptides were reacted with an antiserum to glutaraldehyde-stabilized virus (190S-G), p88 was more reactive to 190S-G antibodies than was p83/p78. These results suggest p88 is external to p83/p78 in the capsid.

The irreversible inactivation of ribonuclease with diethylpyrocarbonate was studied. The products of the reaction were electrophoresed on polyacrylamide gels in sodium dodecyl sulfate and found to contain polymers of ribonuclease. Other proteins reacted similarly with diethylpyrocarbonate. Ribonuclease treated with maleic anhydride was unable to polymerize when treated with diethylpyrocarbonate, but when the N-maleyl group was removed by incubation a t low pH, the capacity of the ribonuclease to polymerize was restored. It was concluded that the diethylpyrocarbonate reaction forms amide bonds between &-amino groups of lysine and carboxylic groups of aspartic or glutamic both intermolecularly or intramolecularly. I n either case the enzyme was inactivated. A mechanism based on the known chemical reactivities of diethylpyrocarbonate is proposed. The conclusions are further substantiated by studies of tryptic peptides and microbiuret positive products obtained from a reaction of diethylpyrocarbonate with assorted amino acids.The interest in specific inhibitors of nucleases is often motivated by their potential importance in the preparation and storage of high molecular weight nucleic acids. Recently, a method for the isolation of high molecular weight RNA which utilized the inhibitory activity of diethylpyrocarbonate was reported [I]. This bactericidal agent was first synthesized by Boehm and Metha [2] whose procedure was subsequently improved and can be found in an extensive patent literature [3]. Extensive chemical studies by Thoma and Rinke [4] suggested that this reagent was useful in esterification reactions under mild conditions and in the conversion of primary and secondary amines into carbonic acid esters (urethanes). More recently the inactivation of a variety of enzymes by diethylpyrocarbonate was studied [5,6]. On the basis of fluorescence measurements, titration curves, and solubility observations, modifications of tryptophan residues, amino groups and conformational changes, respectively, were implicated in the nature of its action. Other investigators suggested in addition a modification of sulfhydryl groups [3,7]. Since pancreatic ribonuclease, which lacks tryptophan residues and sulfhydryl groups, is strongly inactivated by diethylpyrocarbonate [5] we reinvestigated this problem in a search for a more generalized mechanism. Our findings, which are the Unusual Abbreviation. A,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 a t 260 nm when measured in a 1 cm path length cell. subject of this communication, suggest that the general feature of this reaction is a formation of intermolecular peptide-like bonds involving amino and carboxylic groups which yield bizarre polymeric proteins. The inactivation of the monomer is probably achieved by the intramolecular formation of these bonds which results in conformational changes. MATERIALS AND METHODS Inactivation of Ribonuclease with DiethylpyrocarbonateSamples containing a solution of 2 mg of bovine pancreatic ...