Selection on Glycine beta-1,3-endoglucanase genes differentially inhibited by a Phytophthora glucanase inhibitor protein.

Plant endo-beta-1,3-glucanases (EGases) degrade the cell wall polysaccharides of attacking pathogens and release elicitors of additional plant defenses. Isozymes EGaseA and EGaseB of soybean differ in susceptibility to a glucanase inhibitor protein (GIP1) produced by Phytophthora sojae, a major soybean pathogen. EGaseA, the major elicitor-releasing isozyme, is a high-affinity ligand for GIP1, which completely inhibits it, whereas EGaseB is unaffected by GIP1. We tested for departures from neutral evolution on the basis of partial sequences of EGaseA and EGaseB from 20 widespread accessions of Glycine soja (the wild progenitor of soybean), from 4 other Glycine species, and across dicotyledonous plants. G. soja exhibited little intraspecific variation at either locus. Phylogeny-based codon evolution models detected strong evidence of positive selection on Glycine EGaseA and weaker evidence for selection on dicot EGases and Glycine EGaseB. Positively selected peptide sites were identified and located on a structural model of EGase bound to GIP1. Positively selected sites and highly variable sites were found disproportionately within 4.5 angstroms of bound GIP1. Low variation within G. soja EGases, coupled with positive selection in both Glycine and dicot lineages and the proximity of rapidly evolving sites to GIP1, suggests an arms race involving repeated adaptation to pathogen attack and inhibition.

Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls.

The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall. One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules. As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5). In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin. Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT. LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments. Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment. LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media. Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species. The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.

Replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (RSV) antigens protect mice against RSV challenge.

Foreign glycoproteins expressed in recombinant vesicular stomatitis virus (VSV) can elicit specific and protective immunity in the mouse model. We have previously demonstrated the expression of respiratory syncytial virus (RSV) G (attachment) and F (fusion) glycoprotein genes in recombinant VSV. In this study, we demonstrate the expression of RSV F and G glycoproteins in attenuated, nonpropagating VSVs which lack the VSV G gene (VSVDeltaG) and the incorporation of these RSV proteins into recombinant virions. We also show that intranasal vaccination of mice with nondefective VSV recombinants expressing RSV G (VSV-RSV G) or RSV F (VSV-RSV F) elicited RSV-specific antibodies in serum (by enzyme-linked immunosorbent assay [ELISA]) as well as neutralizing antibodies to RSV and afford complete protection against RSV challenge. In contrast, VSVDeltaG-RSV F induced detectable serum antibodies to RSV by ELISA, but no detectable neutralizing antibodies, yet it still protected from RSV challenge. VSVDeltaG-RSV G failed to induce any detectable serum (by ELISA) or neutralizing antibodies and failed to protect from RSV challenge. The attenuated, nonpropagating VSVDeltaG-RSV F is a particularly attractive candidate for a live attenuated recombinant RSV vaccine.

Optimization of cottontail rabbit papilloma virus challenge technique.

Disease induced by Cottontail Rabbit Papilloma Virus (CRPV) scarification in domestic rabbits shares many attributes with disease induced by human papilloma virus (HPV). CRPV induces squamous papillomas in domestic rabbits, of which approximately 70% transform into invasive carcinomas. In advanced tumors, virus is often undetectable, and occasionally, some rabbits undergo spontaneous regression of papillomas. Techniques utilized to scarify rabbit skin are diverse, often labor intensive and time consuming with the possibility for significant variability. Using four unique infection techniques, resultant papilloma incidence, time to onset, and total papilloma volumes were compared to determine an optimal challenge method. Five rabbits were each infected with CRPV via a tattoo gun with and without ink, an intradermal injection, manual use of a tattoo needle, or a sterile blade followed by manual use of a tattoo needle. Papilloma formation was monitored weekly after inoculation for 6 weeks. CRPV papillomas began as pinpoint foci at 3 weeks post challenge and grew exponentially throughout the course of measurement. Individual foci coalesced rapidly to form larger papilloma aggregates. Although intradermal injection was well tolerated and easily performed, it was the worst method of papilloma production (2.2 mm(3) at 6 weeks). The best method, a sterile blade followed by manual use of a tattoo needle, produced significantly larger papillomas over all time periods (>1100 mm(3) at 6 weeks, P<0.01). Inoculation of CRPV using this method produces highly repeatable papillomas beginning 3 weeks post-infection.

Mechanisms of loss of foreign gene expression in recombinant vesicular stomatitis viruses.

We investigated the stability and mechanisms of loss of foreign gene expression in two recombinant vesicular stomatitis viruses (VSVs). A recombinant expressing the cellular CD4 protein exhibited remarkable stability of foreign gene expression. However, after 26 sequential passages, a mutant no longer expressing CD4 was recovered from the virus stock. Sequencing of the CD4 coding region in this mutant revealed a single nucleotide deletion causing a frameshift and termination of protein synthesis. A second VSV recombinant expressing the measles virus F protein grew poorly and exhibited extreme instability of expression of the F protein. Expression of F protein was lost rapidly through mutations of the upstream transcription termination site from (3')AUAC(5') to (3')AUAU(5'), as well as lengthening of the subsequent U(7) tract that is the template for poly(A) addition to VSV G mRNA. Such mutations resulted in fusion of the F mRNA to the 3' end of the G mRNA, making the F protein translation initiation codon inaccessible. We suggest that the VSV polymerase is error prone during replication of the U(7) tract, providing a rapid means for complete elimination of expression of proteins that are toxic to the virus life cycle.

An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants.

We developed an AIDS vaccine based on attenuated VSV vectors expressing env and gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.

Analyses of habituation in Caenorhabditis elegans.

Although the nonassociative form of learning, habituation, is often described as the simplest form of learning, remarkably little is known about the cellular processes underlying its behavioral expression. Here, we review research on habituation in the nematode Caenorhabditis elegans that addresses habituation at behavioral, neural circuit, and genetic levels. This work highlights the need to understand the dynamics of a behavior before attempting to determine its underlying mechanism. In many cases knowing the characteristics of a behavior can direct or guide a search for underlying cellular mechanisms. We have highlighted the importance of interstimulus interval (ISI) in both short- and long-term habituation and suggested that different cellular mechanisms might underlie habituation at different ISIs. Like other organisms, C. elegans shows both accumulation of habituation with repeated training blocks and long-term retention of spaced or distributed training, but not for massed training. Exposure to heat shock during the interblock intervals eliminates the long-term memory for habituation but not the accumulation of short-term habituation over blocks of training. Analyses using laser ablation of identified neurons, and of identified mutants have shown that there are multiple sites of plasticity for the response and that glutamate plays a role in long-term retention of habituation training.

Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles.

The envelope glycoprotein (G) of vesicular stomatitis virus (VSV) contains a short cytoplasmic domain of 29 amino acids. To determine whether VSV particle assembly could accommodate a G protein with a large cytoplasmic domain, we constructed a gene called G/GFP encoding the VSV G protein with the 27-kDa green fluorescent protein linked to its cytoplasmic domain. This gene was inserted into the infectious clone of VSV and we recovered a recombinant virus expressing G/GFP from this extra gene. This VSV-G/GFP virus grew to titers equivalent to that of wild-type virus and was stable upon passaging. The G/GFP protein formed mixed trimers containing an average of two wild-type G proteins and one G/GFP protein. This heterotrimeric protein was expressed on the cell surface, and was incorporated into virus particles with almost the same efficiency as wild-type VSV G protein. These results indicate that there is substantial space available between the viral membrane and the nucleocapsid that can accommodate such a large cytoplasmic domain. The green fluorescent virus particles were readily visualized by fluorescence microscopy and had a normal morphology by electron microscopy. To determine whether virus assembly could occur efficiently when all G proteins contained the GFP cytoplasmic domain, a VSV recombinant in which the G gene was completely replaced by the VSV-G/GFP gene was recovered. This virus rapidly lost expression of the GFP protein sequence through introduction of a stop codon within the sequence encoding the G cytoplasmic domain, indicating strong selection against homotrimeric G protein bearing such a large cytoplasmic domain.

Optimization of transfection.

The single most important factor in optimizing transfection efficiency is selecting the proper transfection protocol. This overview discusses the most commonly used procedures: calcium phosphate-mediated gene transfer, DEAE-dextran-mediated gene transfer, electroporation, and liposome-mediated transfection. Fusion techniques such as protoplast fusion and microinjection may also be considered.

Glycoprotein exchange vectors based on vesicular stomatitis virus allow effective boosting and generation of neutralizing antibodies to a primary isolate of human immunodeficiency virus type 1.

Live recombinant vesicular stomatitis viruses (VSVs) expressing foreign antigens are highly effective vaccine vectors. However, these vectors induce high-titer neutralizing antibody directed at the single VSV glycoprotein (G), and this antibody alone can prevent reinfection and boosting with the same vector. To determine if efficient boosting could be achieved by changing the G protein of the vector, we have developed two new recombinant VSV vectors based on the VSV Indiana serotype but with the G protein gene replaced with G genes from two other VSV serotypes, New Jersey and Chandipura. These G protein exchange vectors grew to titers equivalent to wild-type VSV and induced similar neutralizing titers to themselves but no cross-neutralizing antibodies to the other two serotypes. The effectiveness of these recombinant VSV vectors was illustrated in experiments in which sequential boosting of mice with the three vectors, all encoding the same primary human immunodeficiency virus (HIV) envelope protein, gave a fourfold increase in antibody titer to an oligomeric HIV envelope compared with the response in animals receiving the same vector three times. In addition, only the animals boosted with the exchange vectors produced antibodies neutralizing the autologous HIV primary isolate. These VSV envelope exchange vectors have potential as vaccines in immunizations when boosting of immune responses may be essential.