Amplification of Citrus tristeza virus from a cDNA clone and infection of citrus trees.

Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the approximately 20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts ( approximately 0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population.

An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication.

Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of approximately 20 kilobases that expresses the replicase-associated genes as an approximately 400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.

Transcriptional strategy of closteroviruses: mapping the 5' termini of the citrus tristeza virus subgenomic RNAs.

Citrus tristeza virus (CTV) induces formation of a nested set of at least nine 3' coterminal subgenomic RNAs (sgRNAs) in infected tissue. The organization and expression of the 19,296-nucleotide (nt) CTV genome resembles that of coronaviruses, with polyprotein processing, translational frameshifting, and multiple sgRNA formation, but phylogenetically the CTV polymerase, like polymerases of other closteroviruses, belongs to the Sindbis virus-like lineage of RNA virus polymerases. Both positive-strand RNA virus supergroups, coronaviruses and Sindbis-like viruses, utilize different mechanisms of transcription. To address the mechanism of CTV transcription, 5' termini for the two most abundant sgRNAs, 1.5 and 0.9 kb, respectively, were mapped by runoff reverse transcription. The two sgRNAs were demonstrated to have 48- and 38-nt 5' untranslated regions (5'-UTRs), respectively. The 5'-UTR for the 1.5-kb RNA was cloned, sequenced, and demonstrated to be colinear with the 48-nt genomic sequence upstream of the initiator codon of the respective open reading frame 10, i.e., to be of continuous template origin. The data obtained suggest that the sgRNA transcription of CTV is dissimilar from the coronavirus transcription and consistent with the transcriptional mechanism of other Sindbis-like viruses. Thus, the Sindbis virus-like mechanism of transcription of the positive-strand RNA genomes might be successfully utilized by the closterovirus genome of up to 19.3 kb with multiple sgRNAs.

Kinetics of accumulation of citrus tristeza virus RNAs.

Citrus tristeza virus (CTV), a member of the closterovirus group, is one of the more complex single-stranded RNA viruses. The 5' portion of its 19,296-nt, single-stranded RNA genome is expressed as an approximately 400-kDa polyprotein that is proteolytically processed, while the 10 3' open reading frames are expressed from 3'-coterminal subgenomic RNAs (sg RNAs). As an initial examination of the gene expression of this virus, we found that the kinetics of accumulation of genomic and sg RNAs and coat protein of the T36 isolate of CTV were similar in protoplasts of the natural host, citrus, and the experimental nonhost Nicotiana benthamiana. Newly synthesized genomic RNA was detected 2 days postinoculation and increased to a maximum at 3-5 days. The RNA complementary to the full-length virion RNA increased with similar kinetics, but at approximately one-tenth the concentration of genomic plus strands. Most of the abundant sg RNAs also accumulated in parallel to that of the genomic RNA. However, the smallest sg RNA, which corresponds to the p23 gene, increased earlier. The different sg RNAs accumulated in greatly differing amounts, in general with 3'-most sg RNAs accumulating to higher levels than 5' sg RNAs. However, some 3' sg RNAs (p13 and p18) accumulated to low levels. The two 3'-most sg RNAs (p23 and p20) accumulated to high levels approximately equal to that of the genomic RNA. The accumulation curve for coat protein paralleled that of its mRNA, suggesting that its regulation was transcriptional. Progeny virions from protoplasts were used to sequentially infect new protoplasts, serving as a potential source of virus that could evolve free from the genetic selection in intact plants for aphid transmission and movement.

Complete sequence of the citrus tristeza virus RNA genome.

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells.

Two forms of herpes simplex virus glycoprotein gD were recombined into Autographa californica nuclear polyhedrosis virus (baculovirus) and expressed in infected Spodoptera frugiperda (Sf9) cells. Each protein was truncated at residue 306 of mature gD. One form, gD-1(306t), contains the coding sequence of Patton strain herpes simplex virus type 1 gD; the other, gD-1(QAAt), contains three mutations which eliminate all signals for addition of N-linked oligosaccharides. Prior to recombination, each gene was cloned into the baculovirus transfer vector pVT-Bac, which permits insertion of the gene minus its natural signal peptide in frame with the signal peptide of honeybee melittin. As in the case with many other baculovirus transfer vectors, pVT-Bac also contains the promoter for the baculovirus polyhedrin gene and flanking sequences to permit recombination into the polyhedrin site of baculovirus. Each gD gene was engineered to contain codons for five additional histidine residues following histidine at residue 306, to facilitate purification of the secreted protein on nickel-containing resins. Both forms of gD-1 were abundantly expressed and secreted from infected Sf9 cells, reaching a maximum at 96 h postinfection for gD-1(306t) and 72 h postinfection for gD-1(QAAt). Secretion of the latter protein was less efficient than gD-1(306t), possibly because of the absence of N-linked oligosaccharides from gD-1(QAAt). Purification of the two proteins by a combination of immunoaffinity chromatography, nickel-agarose chromatography, and gel filtration yielded products that were > 99% pure, with excellent recovery. We are able to obtain 20 mg of purified gD-1(306t) and 1 to 5 mg of purified gD-1(QAAt) per liter of infected insect cells grown in suspension. Both proteins reacted with monoclonal antibodies to discontinuous epitopes, indicating that they retain native structure. Use of this system for gD expression makes crystallization trials feasible.

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.

The tobamovirus capsid protein functions as a host-specific determinant of long-distance movement.

The tobamoviruses, tobacco mosaic virus (TMV) and Odontoglossum ringspot virus (ORSV), differ in the range of plant species that each can systemically infect. Both viruses systemically infect Nicotiana benthamiana similarly, but differ in the ability to systemically infect tobacco (N. tabacum). ORSV is confined to the inoculated leaves of N. tabacum, whereas TMV causes a rapid systemic infection. Genetic chimeras were created to identify viral genes involved in specific interactions in long-distance movement in N. tabacum. In N. tabacum, a chimera expressing the ORSV 30-kDa protein exhibited a reduced rate of cell-to-cell spread, similar to that of ORSV, and, also like ORSV, was deficient in systemic invasion, implying a relationship between the rates of cell-to-cell and long-distance movement. However, a TMV chimera expressing the ORSV capsid protein gene spread cell to cell similarly to TMV, but was deficient in long-distance movement and systemic infection. These data suggest that tobamovirus capsid protein(s) may be required to interact with host components in a specific manner to allow efficient long-distance movement, and the ORSV capsid protein did not function in this manner in tobacco plants.

Rapid, high-level expression of biologically active alpha-trichosanthin in transfected plants by an RNA viral vector.

alpha-Trichosanthin, a eukaryotic ribosome-inactivating protein from Trichosanthes kirilowii, inhibits the replication of the human immunodeficiency virus (HIV) in vitro. The alpha-trichosanthin gene was placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant RNA viral vector. Two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated alpha-trichosanthin to levels of at least 2% of total soluble protein. The recombinant alpha-trichosanthin was purified and its structural and biological properties were analyzed. The 23-amino acid signal peptide was recognized by N. benthamiana and the processed enzyme caused a concentration-dependent inhibition of protein synthesis in vitro. The high level of heterologous gene expression observed in these studies is due to the unique features of the RNA viral-based transfection system.

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Tobacco mosaic virus (TMV) produces large quantities of RNA and protein on infection of plant cells. This and other features, attributable to its autonomous replication, make TMV an attractive candidate for expression of foreign sequences in plants. However, previous attempts to construct expression vectors based on plant RNA viruses, such as TMV, have been unsuccessful in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. A hybrid viral RNA (TB2) was constructed, containing sequences from two tobamoviruses (TMV-U1 and odontoglossum ringspot virus). Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamiana, although they differed in their stability on serial passage. Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein, facilitating their transmission and amplification on passaging to subsequent plants. The vector TB2 provides a rapid means of expressing genes and gene variants in plants.

Antibiotic therapy for Pseudomonas aeruginosa bacteremia: outcome correlations in a prospective study of 200 patients.

PURPOSE AND PATIENTS AND METHODS: We performed a prospective clinical study of 200 consecutive patients with Pseudomonas aeruginosa bacteremias to analyze in vitro susceptibility and synergistic testing of antibiotics the patients received and clinical parameters to assess their relationship to survival. RESULTS: No significant correlation between in vitro susceptibility testing (minimal inhibitory concentrations/minimal bactericidal concentrations) and outcome could be demonstrated. Similarly, improved outcome could not be demonstrated for patients receiving antibiotic combinations that were synergistic in vitro (either time-kill or checker-board) versus those combinations that were not. There was also no correlation between results obtained by time-kill curve and checkerboard synergistic testing, i.e., combinations found to be synergistic by one method were not necessarily synergistic by the other method. Clinical parameters associated with improved survival were a urinary portal of entry and absence of neutropenia. Conversely, survival was significantly decreased when the portal was the respiratory tract. The mortality rate between patients receiving combination therapy (27%) and monotherapy (47%) was significant (p less than 0.02); this significant relationship held true for most subgroups including malignancy, nosocomial infection, and infection site. CONCLUSION: Increasing effort should be placed on ensuring timely administration of combination therapy to patients with P. aeruginosa bacteremia since the use of combination therapy was even more important in determining outcome than was underlying disease.

A tobacco mosaic virus-hybrid expresses and loses an added gene.

An additional open reading frame from the chloramphenicol acetyltransferase (CAT) gene was fused behind a tobacco mosaic virus (TMV) subgenomic RNA promoter and inserted into different positions in the complete TMV genome to examine how much this viral genome can be altered with continued replication. One hybrid virus, CAT-CP, with the insertion between the 30K and coat protein genes, replicated efficiently, produced an additional subgenomic RNA and CAT activity, and assembled into 350-nm virions, compared to 300-nm virions of wild-type TMV. However, during systemic infection of plants, the inserted sequences were deleted. This deletion was exact, resulting in progeny wild-type TMV. Another hybrid virus examined was CP-CAT, which had the insertion between the coat protein gene and the nontranslated 3' region. This virus replicated poorly, produced only minimal levels of CAT activity, and did not systemically invade infected plants. These data show that some extensive modifications of the TMV genome still allow efficient virus replication.

Effects of midazolam on directly stimulated muscle biopsies from control and malignant hyperthermia positive patients.

Midazolam is a water soluble benzodiazepine of interest to the anaesthetist for use as a premedicant and for induction of anaesthesia. The effects of midazolam were observed on the resting tension of directly stimulated muscle biopsied from control and malignant hyperthermia (MH) susceptible patients. In addition, interactions between midazolam and the two most commonly used MH diagnostic agents (halothane and caffeine) were examined. Midazolam, at maximum therapeutic concentrations (ca. 0.5 microgram X ml-1), had no detectable effects on muscle contraction in preparations from control or MH positive patients. Midazolam did elicit a contracture from control and MH positive preparations when used in a concentration range of 160-1280 micrograms X ml-1. There was no significant difference between control and MH positive patients in minimum concentration of midazolam causing contracture or the strength of contracture at the respective eliciting concentration. There appears to be no interaction between midazolam and either halothane or caffeine on the resting tension of the directly stimulated muscle twitch preparation.