Judith A. Lesnaw

Cell-free translation of tobacco vein mottling virus RNA.

Tobacco vein mottling virus (TVMV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system. The RNA was approximately 5% as active as rabbit globin mRNA in directing protein synthesis. Translation was stimulated approximately 15% by the cap analog pppm7G, a phenomenon which has also been observed with uncapped viral RNAs. Treatment with NaIO4 and NaB(3H]4 under conditions which label the caps of globin and ovalbumin mRNA failed to produce labeled cap structures in TVMV RNA. Approximately 20 polypeptides, ranging from 20,000 to 100,000 daltons, were produced in the reticulocyte system programmed with TVMV RNA. The predominant species (P75) had a molecular weight of 75,000. The same major polypeptides were produced in the wheat germ system, but there was relatively greater synthesis of the smaller polypeptides. Incubation of the reticulocyte system in the presence of cycloheximide following an initial period of synthesis failed to cause breakdown of larger polypeptides into smaller polypeptides. Preincubation of TVMV RNA with the reticulocyte system before addition of labeled amino acids caused greater synthesis of the smaller polypeptides, suggesting that they are derived from fragments of the RNA produced during the incubation. Translation of TVMV RNA which had been bound to oligo(dT)-cellulose yielded nearly the same spectrum of polypeptides, but synthesis of polypeptides smaller than 52,000 daltons was reduced. At low ionic strength, only polypeptides of 52,000 daltons and smaller were synthesized. At high ionic strength, P75 was essentially the only product synthesized. Antibody to whole virus precipitated many of the polypeptides, including P75, suggesting that they contain the coat protein sequence. No polypeptide with the electrophoretic mobility of authentic coat protein, however, was precipitated. Comparison of partial proteolytic digests of P75 and authentic coat protein provided further evidence for sequence homology.

Live-cell imaging of rhabdovirus-induced morphological changes in plant nuclear membranes.

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.

Nucleotide sequence of the L gene of vesicular stomatitis virus (New Jersey): Identification of conserved domains in the New Jersey and Indiana L proteins

The nucleotide sequence of the L gene of vesicular stomatitis virus, New Jersey serotype (Hazelhurst subtype), was determined. Primer extension dideoxy sequencing of genomic RNA using reverse transcriptase initiated within the adjacent G gene provided a consensus sequence of 6522 nucleotides. The G/L intergenic junction spanned 21 nucleotides and contained a pseudo transcription start signal as well as two sequences (10 and 6 nucleotides in length) which are reiterated within the L coding region. The predicted L mRNA was 6398 nucleotides long and contained a single open reading frame corresponding to an L protein encompassing 2109 amino acids with a MW of 241,546. Comparison of the amino acid sequence of this New Jersey serotype L protein to that previously reported for the L protein of the serologically and genetically distinct Indiana serotype (M. Schubert, G. G. Harmison, and E. Meier (1984). J. Virol. 51, 505-514.) revealed a high degree of functional homology. In addition, six regions (43 to 103 amino acids in length) which displayed a high percentage of identical amino acids (85 to 96%) were identified. Five of these regions were clustered within the amino-terminal half of the L protein. Two of these regions contained sequences, 41 amino acids in length, which were significantly similar to corresponding regions of the L proteins of the paramyxoviruses Sendai and Newcastle disease virus. These structurally conserved regions may correspond to functional domains of the multifunctional L protein.

Expression of potyvirus proteins in insect cells infected with a recombinant baculovirus

The N-terminal portion (P1-HC-Pro-P3) of the tobacco vein mottling virus (TVMV) polyprotein was expressed in insect cells and larvae by a recombinant baculovirus. The proteases necessary to process this TVMV polyprotein fragment were active in insect cells, since mature P1, HC-Pro and P3 proteins were detected by specific antisera in Western blots. Antisera to P1, HC-Pro and P3 also recognized polypeptides with apparent M(r) values predicted for the intermediate processing products of the polyprotein fragment. The results of this study indicate that the autocatalytic processing of TVMV HC-Pro from the polyprotein is supported by insect cells. Helper component activity in extracts of cells infected with recombinant baculovirus was not detected by aphid transmission assay.

Tulip Breaking: Past, Present, and Future

This article focuses on the oldest recorded plant virus disease, tulip breaking, and reflects the authors’ interests in molecular virology, the history of virology, and the broad influence of viruses upon societies and their culture. The potyvirus Tulip breaking virus (TBV) induces in the petals of its host tulips beautiful variegated color patterns that break the solid color of the uninfected tulips, hence the name “tulip breaking.” The human passions of possession that these “broken tulips” induced in seventeenth century Holland generated an economic and social disorder with lasting cultural ramifications referred to as “tulipomania.” Although the lure of the broken tulip persists in the twenty-first century, the molecular mechanisms governing the virus-induced color breaking in tulips remain little understood. Here, we review aspects of the historical impact of tulipomania, the biology of TBV, the pathways and regulation of plant pigment formation, and the potential mechanisms underlying virus-induced color breaking. The reader is cautioned that tulipomania, like tulip breaking, is still contagious.

Infection control in gene therapy.

Gene therapy is now being studied for the treatment of a wide variety of acquired and inherited diseases. Viruses used as vectors for gene transfer include retroviruses, adenoviruses, vaccinia viruses, adeno-associated viruses, and herpesviruses. These vectors, developed in the laboratory and in animal studies, are now being introduced into the clinical arena Infection control practitioners will be involved invariably in reviewing the use of these agents in their clinics and hospitals. This review summarizes key aspects of the more common vectors and makes recommendations for infection control.

In vitro transcription of wound tumor virus RNA by virion-associated RNA transcriptase

The results of this communication indicate that wound tumor virus (WTV) has a virion-bound polymerase which can synthesize all 12 single-stranded RNA copies of the genome segments in vitro . Chymotrypsin treatment of the purified virus did not affect its transcriptase activity. Previous observations have shown that virus treated with chymotrypsin lacks the 131,000- and 96,000-dalton polypeptides. Since we have shown in the present study that the enzyme activity was retained following chymotrypsin treatment, we conclude that neither of these polypeptides are the polymerase. In addition, all cations tested, with the exception of Mg 2+ and Mn 2+ , did not enhance the enzyme activity. Phosphoenolpyruvate (PEP) together with PEP-kinase substantially increased in vitro transcription, and the transcribed products, when annealed with denatured WTV genome RNA, gave an electrophoretic profile identical to that of the native viral genome.

In vitro functional analysis of a temperature-sensitive mutant of vesicular stomatitis virus, New Jersey serotype, defective in transcription.

The temperature sensitive mutant B1 of vesicular stomatitis virus. New Jersey serotype, previously shown to be defective in primary transcription at the restrictive temperature in vivo (Lesnaw, J. A., and Reichmann, M. E. (1975). Virology 63, 492–504), has been analyzed in vitro in order to locate the site of the temperature-sensitive lesion. Transcribing cores lacking the matrix and glycoproteins were prepared from purified wild-type and B1 virions. In vitro transcription reaction sprimed with the B1 cores were more temperature-sensitive than the corresponding wild-type reactions. In addition, the B1 cores were shown to be more thermolabile than the wild-type cores. Therefore, the B1 temperature-sensitive defect in transcription is amenable to in vitro analysis. The transcribing cores were dissociated into enzyme fractions containing the L and NS proteins, and template fractions containing the N protein and the RNA. Neither fraction contained transcriptase activity when assayed alone, but both homologous and heterologous mixtures of mutant and wild-type enzyme and template fractions were active when assayed at 29°. However, complexes containing the B1 enzyme were totally inactive at 42°, while complexes involving the wild-type enzyme were active at 42°. These results suggested that the B1 enzyme fraction was defective. An alternate method was also employed in which thermal inactivation of the dissociated enzyme and template fractions was carried out prior to reconstitution. Transcriptase activity of the reconstituted complexes was then assayed at 29°. The results revealed that the B1 enzyme fraction was more thermolabile than the wild-type enzyme. Thus, two different in vitro functional analyses place the B1 temperature-sensitive lesion in the enzyme fraction.

Infection Control for Gene Therapy: A Busy Physician's Primer

Gene therapy is being studied for the treatment of a wide variety of acquired and inherited disorders. Retroviruses, adenoviruses, poxviruses, adeno-associated virus, herpesviruses, and others are being engineered to serve as gene therapy vectors and are being administered to patients in a clinical setting. Infection control professionals will be asked to evaluate the use and safety of these agents in their clinics and hospitals. This review summarizes key aspects of the biotechnology and the vectors involved in gene therapy and makes recommendations for infection control.

A simple and efficient procedure for the oral inoculation of Trichoplusia ni larvae with polyhedrin-negative recombinant baculovirus

Current procedures for inoculating lepidopteran larvae with polyhedrin-negative recombinant baculovirus, i.e. intracoelomic injection or coinfection with wild type virus, are laborious and can compromise final yields of recombinant protein. Herein is described a simple and efficient method for oral inoculation. Up to 100% infection was obtained when individual early fifth instar Trichoplusia ni larvae were fed a small piece of a formaldehyde-free insect diet to which 4.2 x 10(5) PFU of a polyhedrin-negative recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) containing the gene for beta-galactosidase was applied. Infected larvae were identified by assaying hemolymph for beta-galactosidase activity. The maximum levels of beta-galactosidase detected in these hemolymph samples were identical to those obtained for larvae infected by intracoelomic injection. The dose of polyhedrin-negative recombinant virus recommended for intracoelomic injection of T. ni was efficacious for the oral route of inoculation.