The Genetics Underlying Natural Variation in the Biotic Interactions of Arabidopsis thaliana: The Challenges of Linking Evolutionary Genetics and Community Ecology.

In the context of global change, predicting the responses of plant communities in an ever-changing biotic environment calls for a multipronged approach at the interface of evolutionary genetics and community ecology. However, our understanding of the genetic basis of natural variation involved in mediating biotic interactions, and associated adaptive dynamics of focal plants in their natural communities, is still in its infancy. Here, we review the genetic and molecular bases of natural variation in the response to biotic interactions (viruses, bacteria, fungi, oomycetes, herbivores, and plants) in the model plant Arabidopsis thaliana as well as the adaptive value of these bases. Among the 60 identified genes are a number that encode nucleotide-binding site leucine-rich repeat (NBS-LRR)-type proteins, consistent with early examples of plant defense genes. However, recent studies have revealed an extensive diversity in the molecular mechanisms of defense. Many types of genetic variants associate with phenotypic variation in biotic interactions, even among the genes of large effect that tend to be identified. In general, we found that (i) balancing selection rather than directional selection explains the observed patterns of genetic diversity within A. thaliana and (ii) the cost/benefit tradeoffs of adaptive alleles can be strongly dependent on both genomic and environmental contexts. Finally, because A. thaliana rarely interacts with only one biotic partner in nature, we highlight the benefit of exploring diffuse biotic interactions rather than tightly associated host-enemy pairs. This challenge would help to improve our understanding of coevolutionary quantitative genetics within the context of realistic community complexity.

Diet creates metabolic niches in the “inmature gut” that shapemicrobial communities

Although diet composition has been implicated as a major factor in the etiology of various gastrointestinal diseases, conclusive evidence remains elusive. This is particularly true in diseases such as necrotizing enterocolitis where breast milk as opposed to commercial formula appears to confer a ‘protective effect’ to the ‘immature gut’. Yet the mechanism by which this occurs continues to remain speculative. In the present study we hypothesize that the basic chemical composition of diet fundamentally selects for specific intestinal microbiota which may help explain disparate disease outcome and therapeutic direction. Complimentary animal and human studies were conducted on young piglets (21 d.)(n = 8)(IACUC protocols 08070 and 08015) and premature infants (adjusted gestational age 34-36 weeks) (n = 11)(IRB Protocol 15895A). In each study, cecal or stool contents from two groups (Breast milk-fed (BF) vs. Formulafed (FF)) were analyzed by gas chromatography/masss pectrometry (GC/MS) and comprehensive metabolic profiles generated and compared. Concurrently, bacterial community structure was assayed and respective representative microbiota of the groups determined by 16SrRNA gene amplicon pyrosequencing. Statistical modeling and analysis was done using SIMCA-P+ and R software. GC/MS metabolomics identified clear differences between BF and FF groups in the intestinal environment of piglets and humans. Sugars, amino-sugars, fatty acids, especially unsaturated fatty acids, and sterols were identified as being among the most important metabolites for distinguishing between BF and FF groups. Joint analysis (AU)

Aunque se ha implicado a la composición de la dieta como un factor principal en la etiología de varias enfermedades gastrointestinales, la evidencia concluyente sigue siendo esquiva. Esto es particularmente cierto en enfermedades como la enterocolitis necrosante en la que la leche materna, en contraposición de las fórmulas comerciales, parece conferir un ‘efecto protector’ para el ‘intestino inmaduro’ o el ecosistema intestinal juvenil del ‘intestino inmaduro’, si bien el mecanismo por el que esto ocurre sigue siendo una especulación. La hipótesis de nuestro estudio es que la composición química básica de la dieta selecciona fundamentalmente microbióticos intestinales específicos que pueden explicar los resultados dispares de la enfermedad y tener implicaciones terapéuticas. Se realizaron estudios adicionales en animales y humanos en lechones (21 d.) (n = 8) (protocolos IACUC08070 y 08015) y lactantes prematuros (edad gestacional ajustada de 34-36 semanas) (n = 11) (Protocolo IRB15895A). En cada estudio, se analizaron los contenidos cecales y fecales de ambos grupos (alimentación materna(AM) y alimentación con fórmula (AF)) mediante cromatografía de gases/espectrometría de masas (CG/EM) y se generaron y compararon perfiles metabólicos completos. De forma concurrente, se probó la estructura de la comunidad bacteriana y se determinaron los representantes (AU)

Diet creates metabolic niches in the "immature gut" that shape microbial communities.

Although diet composition has been implicated as a major factor in the etiology of various gastrointestinal diseases, conclusive evidence remains elusive. This is particularly true in diseases such as necrotizing enterocolitis where breast milk as opposed to commercial formula appears to confer a "protective effect" to the "immature gut." Yet the mechanism by which this occurs continues to remain speculative. In the present study we hypothesize that the basic chemical composition of diet fundamentally selects for specific intestinal microbiota which may help explain disparate disease outcome and therapeutic direction. Complimentary animal and human studies were conducted on young piglets (21 d.)(n = 8) (IACUC protocols 08070 and 08015) and premature infants (adjusted gestational age 34-36 weeks) (n = 11) (IRB Protocol 15895A). In each study, cecal or stool contents from two groups (Breast milk-fed (BF) vs. Formula-fed (FF)) were analyzed by gas chromatography/mass spectrometry (GC/MS) and comprehensive metabolic profiles generated and compared. Concurrently, bacterial community structure was assayed and respective representative microbiota of the groups determined by 16S rRNA gene amplicon pyrosequencing. Statistical modeling and analysis was done using SIMCA-P+ and R software. GC/MS metabolomics identified clear differences between BF and FF groups in the intestinal environment of piglets and humans. Sugars, amino-sugars, fatty acids, especially unsaturated fatty acids, and sterols were identified as being among the most important metabolites for distinguishing between BF and FF groups. Joint analysis of microbiota and metabolomics pinpointed specific sets of metabolites (p < 0.05) associated with the dominant bacterial taxa. The chemical composition of diet appears to have a significant role in defining the microbiota of the immature gut. Tandem analysis of intestinal microbial and metabolic profiles is potentially a powerful tool leading to better understanding of the role of diet in disease perhaps even leading to specific strategies to alter microbial behavior to improve clinical outcome.

Distribution of genetic variation within and among local populations of Arabidopsis thaliana over its species range.

A detailed description of local population structure in Arabidopsis thaliana is presented, including an assessment of the genetic relatedness of individuals collected from the same field. A hierarchical sample of four individuals from 37 local populations, including North America, England, Eastern and Western Europe, and Asia, and a selection of ecotypes, were analysed for variation in Adh, ChiA, FAH1, F3H, Rpm1, Rps5, and five microsatellite loci. Twenty-eight of the 37 population samples contained individuals with identical multilocus haplotypes, 12 of which were fixed for a single haplotype. These monomorphic populations were evenly distributed over the species range. Only in North America did we find a single multilocus haplotype shared among different populations, perhaps indicating a continental founder event. Despite the occurrence of local inbreeding, a considerable amount of genetic variation was found segregating within and among local populations. A novel analysis of haplotype differences reveals that genetic differentiation occurs at every geographic scale in A. thaliana, where we find a surprising under-representation of recent migrants between local populations. This leads us to hypothesize that most dispersal between A. thaliana populations is by pollen rather than seed. Based on the structure of A. thaliana populations, it appears that regional groups of local populations may provide the most appropriate genetic material for linkage disequilibrium mapping of adaptive traits.

Salicylic acid inhibits jasmonic acid-induced resistance of Arabidopsis thaliana to Spodoptera exigua.

The role of salicylic acid (SA) in plant responses to pathogens has been well documented, but its direct and indirect effects on plant responses to insects are not so well understood. We examined the effects of SA, alone and in combination with jasmonic acid (JA), on the performance of the generalist herbivore, Spodoptera exigua, in wild-type and mutant Arabidopsis thaliana genotypes that varied genetically in their ability to mount SA- and JA-mediated defence responses. In one experiment, growth of S. exigua larvae was highest on the Wassilewskija wild-type, intermediate on the Columbia wild-type and the JA-deficient fad mutant, and lowest on the nim1-1 and jar1-mutants, which are defective in the SA and JA pathways, respectively. Activity of guaiacol peroxidase, polyphenoloxidase, n-acetylglucosaminidase, and trypsin inhibitor varied by genotype but did not correlate with insect performance. SA treatment increased growth of S. exigua larvae by approximately 35% over all genotypes, but had no discernable effect on activities of the four defence proteins. In a second experiment, growth of S. exigua was highest across treatments on the cep1 mutant, a constitutive expressor of high SA levels and systemic acquired resistance, and lowest on the fad mutant, which is JA-deficient. JA treatment generally increased activity of all four defence proteins, increased total glucosinolate levels and reduced insect growth by approximately 25% over all genotypes. SA generally inhibited expression of JA-induced resistance to S. exigua when both hormones were applied simultaneously. Across genotypes and treatments, larval mass was negatively correlated with the activity of trypsin inhibitor and polyphenoloxidase and with total glucosinolate levels, and insect damage was negatively correlated with the activity of polyphenoloxidase. SA had little effect on the induction of defence protein activity by JA. However, SA attenuated the induction of glucosinolates by JA and therefore may explain better the interactive effects of SA and JA on insect performance. This study illustrates that direct and indirect cross-effects of SA on resistance to S. exigua can occur in A. thaliana. Effects of SA may be mediated through effects on plant defence chemistry or other aspects of the suitability of foliage for insect feeding and growth.

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

Fitness costs of R-gene-mediated resistance in Arabidopsis thaliana.

Resistance genes (R-genes) act as an immune system in plants by recognizing pathogens and inducing defensive pathways. Many R-gene loci are present in plant genomes, presumably reflecting the need to maintain a large repertoire of resistance alleles. These loci also often segregate for resistance and susceptibility alleles that natural selection has maintained as polymorphisms within a species for millions of years. Given the obvious advantage to an individual of being disease resistant, what prevents these resistance alleles from being driven to fixation by natural selection? A cost of resistance is one potential explanation; most models require a lower fitness of resistant individuals in the absence of pathogens for long-term persistence of susceptibility alleles. Here we test for the presence of a cost of resistance at the RPM1 locus of Arabidopsis thaliana. Results of a field experiment comparing the fitness of isogenic strains that differ in the presence or absence of RPM1 and its natural promoter reveal a large cost of RPM1, providing the first evidence that costs contribute to the maintenance of an ancient R-gene polymorphism.

Negative cross-talk between salicylate- and jasmonate-mediated pathways in the Wassilewskija ecotype of Arabidopsis thaliana.

Plants often respond to attack by insect herbivores and necrotrophic pathogens with induction of jasmonate-dependent resistance traits, but respond to attack by biotrophic pathogens with induction of salicylate-dependent resistance traits. To assess the degree to which the jasmonate- and salicylate-dependent pathways interact, we compared pathogenesis-related protein activity and bacterial performance in four mutant Arabidopsis thaliana lines relative to their wild-type backgrounds. We found that two salicylate-dependent pathway mutants (cep1, nim1-1) exhibited strong effects on the growth of the generalist biotrophic pathogen, Pseudomonas syringae pv. tomato, whereas two jasmonate-dependent pathway mutants (fad3-2fad7-2fad8, jar1-1) did not. Leaf peroxidase and exochitinase activity were negatively correlated with bacterial growth, whereas leaf polyphenol oxidase activity and trypsin inhibitor concentration were not. Interestingly, leaf total glucosinolate concentration was positively correlated with bacterial growth. In the same experiment, we also found that application of jasmonic acid generally increased leaf peroxidase activity and trypsin inhibitor concentration in the mutant lines. However, the cep1 mutant, shown previously to overexpress salicylic acid, exhibited no detectable biological or chemical responses to jasmonic acid, suggesting that high levels of salicylic acid may have inhibited a plant response. In a second experiment, we compared the effect of jasmonic acid and/or salicylic acid on two ecotypes of A. thaliana. Application of salicylic acid to the Wassilewskija ecotype decreased bacterial growth. However, this effect was not observed when both salicylic acid and jasmonic acid were applied, suggesting that jasmonic acid negated the beneficial effect of salicylic acid. Collectively, our results confirm that the salicylate-dependent pathway is more important than the jasmonate-dependent pathway in determining growth of P. syringae pv. tomato in A. thaliana, and suggest important negative interactions between these two major defensive pathways in the Wassilewskija ecotype. In contrast, the Columbia ecotype exhibited little evidence of negative interactions between the two pathways, suggesting intraspecific variability in how these pathways interact in A. thaliana.

Reduction of chromatin assembly factor 1 p60 and C21orf2 protein, encoded on chromosome 21, in Down syndrome brain.

Trisomy 21 (Down syndrome, DS) is the most common genetic cause of mental retardation, resulting from triplication of the whole or distal part of human chromosome 21. Overexpression of genes located on chromosome 21, as a result of extra gene load, has been considered a central hypothesis for the explanation of the DS phenotype. This gene dosage hypothesis has been challenged, however. We have therefore decided to study proteins whose genes are encoded on chromosome 21 in brain of patients with DS and Alzheimer's disease (AD), as all patients with DS from the fourth decade show Alzheimer-related neuropathology. Using immunoblotting we determined Coxsackievirus and adenovirus receptor (CAR), Claudin-8, C21orf2, Chromatin assembly factor 1 p60 subunit (CAF-1 p60) in frontal cortex from DS, AD and control patients. Significant reduction of C21orf2 and CAF-1 p60, but comparable expression of CAR and claudin-8 was observed in DS but all proteins were comparable to controls in AD, even when related to NSE levels to rule out neuronal cell loss or actin to normalise versus a housekeeping protein. Reduced CAF-1 p60 may reflect impaired DNA repair most probably due to oxidative stress found as early as in fetal life continuing into adulthood. The decrease of C21orf2 may represent mitochondrial dysfunction that has been reported repeatedly and also data on CAR and claudin-8 are not supporting the gene-dosage hypothesis at the protein level. As aberrant expression of the four proteins was not found in brains of patients with AD, decreased CAF and C21orf2 can be considered specific for DS.

Determination of molecules regulating gene delivery using adenoviral vectors in ovarian carcinomas.

Gene therapeutic approaches currently favor adenoviral vectors over alternatively available vector systems. Ovarian cancer represents an attractive model for an intraperitoneal adenovirus-based gene therapy, which is now under intensive clinical investigation. Adenovirus-mediated gene transfer depends on adequate virus uptake and thus on the presence of sufficient amounts of high-affinity coxsackie-adenovirus receptor (CAR) and alphavbeta3- and alphavbeta5 integrins on target cells. This fact has been ignored in most ongoing clinical trials. This investigation, therefore, determined expression of CAR by immunohistochemistry in 37 ovarian carcinomas and compared it with that of alphavbeta3 and alphavbeta5 integrins. In all samples, except one undifferentiated carcinoma, CAR was immunohistochemically demonstrable. Grade 1 tumors exhibited stronger CAR immunostaining as compared with higher-grade cancers (P < 0.03). Integrins alphavbeta3 and alphavbeta5 were detectable in 62% and 65% of carcinomas, respectively, and staining for both classes correlated positively (P < 0.005). Cancers classified as undifferentiated completely lacked alphavbeta3 expression. Furthermore, in undifferentiated and grade 3 carcinomas the three molecules studied exhibited marked distributional heterogeneity with regard to focal positivity and negativity within the same tumor. Either the absence of CAR, alphavbeta3 and alphavbeta5 or the pronounced heterogeneity in their expression might seriously compromise the efficiency of adenovirus-based gene therapy in ovarian cancer.

The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction.

The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic cell aggregation, and recruited the tight junction protein ZO-1 to sites of cell-cell contact. In polarized epithelial cells, CAR and ZO-1 colocalized to tight junctions and could be coprecipitated from cell lysates. CAR expression led to reduced passage of macromolecules and ions across cell monolayers, and soluble CAR inhibited the formation of functional tight junctions. Virus entry into polarized epithelium required disruption of tight junctions. These results indicate that CAR is a component of the tight junction and of the functional barrier to paracellular solute movement. Sequestration of CAR in tight junctions may limit virus infection across epithelial surfaces.

Models and data on plant-enemy coevolution.

Although coevolution is complicated, in that the interacting species evolve in response to each other, such evolutionary dynamics are amenable to mathematical modeling. In this article, we briefly review models and data on coevolution between plants and the pathogens and herbivores that attack them. We focus on "arms races," in which trait values in the plant and its enemies escalate to more and more extreme values. Untested key assumptions in many of the models are the relationships between costs and benefits of resistance in the plant and the level of resistance, as well as how costs of virulence or detoxification ability in the enemy change with levels of these traits. A preliminary assessment of these assumptions finds only mixed support for the models. What is needed are models that are more closely tailored to particular plant-enemy interactions, as well as experiments that are expressly designed to test existing models.

The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure.

The coxsackie and adenovirus receptor (CAR) is identified as a high-affinity receptor for adenovirus type 5. We observed that invasive bladder cancer specimens had significantly reduced CAR mRNA levels compared with superficial bladder cancer specimens, which suggests that CAR may play a role in the progression of bladder cancer. Elevated CAR expression in the T24 cell line (CAR-negative cells) increased its sensitivity to adenovirus infection and significantly inhibited its in vitro growth, accompanied by p21 and hypophosphorylated retinoblastoma accumulation. Conversely, decreased CAR levels in both RT4 and 253J cell lines (CAR-positive cells) promoted their in vitro growth. To unveil the mechanism of action of CAR, we showed that the extracellular domain of CAR facilitated intercellular adhesion. Furthermore, interrupting intercellular adhesion of CAR by a specific antibody alleviates the growth-inhibitory effect of CAR. We also demonstrated that both the transmembrane and intracellular domains of CAR were critical for its growth-inhibitory activity. These data indicate that the cell-cell contact initiated by membrane-bound CAR can elicit a negative signal cascade to modulate cell cycle regulators inside the nucleus of bladder cancer cells. Therefore, the presence of CAR cannot only facilitate viral uptake of adenovirus but also inhibit cell growth. These results can be integrated to formulate a new strategy for bladder cancer therapy.

Plant density and nutrient availability constrain constitutive and wound-induced expression of trypsin inhibitors in Brassica napus.

We investigated the effects of plant density on plant size, leaf total soluble protein content, and constitutive and wound-induced levels of proteinaceous trypsin inhibitors in pot-grown Brassica napus seedlings in two greenhouse studies. We manipulated plant density by varying the number of intraspecific neighbors surrounding a target plant in the center of each pot. In general, constitutive and induced levels of trypsin inhibitors were significantly reduced by competition in a density-dependent manner, to the extent that induction was greatly reduced or abolished in target plants surrounded by six neighbors. To investigate whether the effects of plant density on inhibitor production were mediated by nutrient availability, we manipulated the concentration of a complete fertilizer applied to target plants surrounded by six neighbors in two greenhouse studies. In general, constitutive and wound-induced levels of inhibitors in plants surrounded by six neighbors were increased by nutrient addition in a dose-dependent manner, such that wound-induction was completely restored in competing plants under conditions of high nutrient availability. Leaf total soluble protein content, measured only in the second trial of each experiment, was not affected by any of the treatments. The effects of plant density, nutrient addition, and wounding on inhibitor levels in all experiments were independent of their effects on above-ground plant size at the time of wounding. Overall, our results suggest that decreasing nutrient availability mediates the density-dependent reductions in inhibitor levels in B. napus seedings.

Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: implications for cardiotropic viral infections.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) was identified as a common cellular receptor for both viruses, but its biological and pathogenic relevance is uncertain. Knowledge of CAR localization in the human cardiovascular system is limited but important with respect to CAR-dependent viral infections and gene transfer using CAR-dependent viral vectors. METHODS AND RESULTS: Explanted failing hearts from 13 patients (8 with dilated cardiomyopathy [DCM] and 5 with other heart diseases [non-DCM]) and normal donor hearts (n=7) were investigated for the expression levels and subcellular localization of CAR and the adenovirus coreceptors alpha(v)beta(3) and alpha(v)beta(5) integrins. CAR immunoreactivity was very low in normal and non-DCM hearts, whereas strong CAR signals occurred at the intercalated discs and sarcolemma in 5 of the 8 DCM hearts (62.5%); these strong signals colocalized with both integrins. In all hearts, CAR was detectable in subendothelial layers of the vessel wall, but not on the luminal endothelial surface, and on interstitial cells. Human CAR (hCAR) expressed in rat cardiomyocytes was targeted to cell-cell contacts, which resembled CAR localization in DCM hearts and resulted in 15-fold increased adenovirus uptake. CONCLUSIONS: Low hCAR abundance may render normal human myocardium resistant to CAR-dependent viruses, whereas re-expression of hCAR, such as that observed in DCM, may be a key determinant of cardiac susceptibility to viral infections. Asymmetric expression of hCAR in the vessel wall may be an important determinant of adenovirus tropism in humans. hCAR subcellular localization in human myocardium and hCAR targeting to cell-cell contacts in cardiomyocyte cultures suggest that hCAR may play a role in cell-cell contact formation.

Evolutionary dynamics of plant R-genes.

Plant R-genes involved in gene-for-gene interactions with pathogens are expected to undergo coevolutionary arms races in which plant specificity and pathogen virulence continually adapt in response to each other. Lending support to this idea, the solvent-exposed amino acid residues of leucine-rich repeats, a region of R-genes involved in recognizing pathogens, often evolve at unusually fast rates. But within-species polymorphism is also common in R-genes, implying that the adaptive substitution process is not simply one of successive selective sweeps. Here we document these features in available data and discuss them in light of the evolutionary dynamics they likely reflect.

Diamondback moth compensatory consumption of protease inhibitor-transformed plants.

Prior study of the effect of protease inhibitors (PIs) on diamondback moths suggests that moths are resistant to them, so PIs represent an ineffective defence against moths. However, our data suggest that diamondback moths do suffer lower growth rates when they consume plants transformed with potato protease inhibitor (PI2), but that effect is hidden by compensatory consumption. Plants, instead of gaining an advantage by lowering the insect growth rate, suffer a disadvantage as moths consume more tissue to mitigate the effect. Furthermore, PI2, when used in conjunction with another transgenic pesticidal protein, Bt (from Bacillus thuringiensis) counteracts the effectiveness of Bt at protecting plant tissue. Thus, transgenic PIs are not only less effective than previously thought in protecting Brassica plants from diamondback moths, they may actually lead to increased plant damage by the moths.

Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

Multiple regions within the coxsackievirus and adenovirus receptor cytoplasmic domain are required for basolateral sorting.

The coxsackievirus and adenovirus receptor (CAR) mediates attachment and infection by coxsackie B viruses and many adenoviruses. In human airway epithelia, as well as in transfected Madin-Darby canine kidney cells, CAR is expressed exclusively on the basolateral surface. Variants of CAR that lack the cytoplasmic domain or are attached to the cell membrane by a glycosylphosphatidylinositol anchor are expressed on both the apical and basolateral surfaces. We have examined the localization of CAR variants with progressive truncations of the cytoplasmic domain, as well as with mutations that ablate a potential PDZ (PSD95/dlg/ZO-1) interaction motif and a putative tyrosine-based sorting signal. In addition, we have examined the targeting of two murine CAR isoforms, with different C-terminal sequences. The results suggest that multiple regions within the CAR cytoplasmic domain contain information that is necessary for basolateral targeting.

Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells.

Exploiting the lytic life cycle of viruses has gained recent attention as an anticancer strategy (oncolysis). To explore the utility of adenovirus (Ad)-mediated oncolysis for rhabdomyosarcoma (RMS), we tested RMS cell lines for Ad gene transduction and infection. RMS cells were variably transduced by Ad. Compared with control cells, RMS cells were less sensitive or even resistant to oncolysis by wild-type virus. RMS cells expressed the Ad internalization receptors, alpha(v) integrins, but had low or undetectable expression of the major attachment receptor, coxsackievirus-Ad receptor (CAR). Mutant Ads with ablated CAR binding exhibited only 5-20% of transgene expression in RMS cells seen with a wild-type vector, suggesting that residual or heterogeneous CAR expression mediated the little transduction that was detectable. Immunohistochemical analysis of archived clinical specimens showed little detectable CAR expression in five embryonal and eight alveolar RMS tumors. Stable transduction of the cDNA for CAR enabled both efficient Ad gene transfer and oncolysis for otherwise resistant RMS cells, suggesting that poor CAR expression is the limiting feature. Gene transfer to RMS cells was increased >2 logs using Ads engineered with modified fiber knobs containing either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop. The RGD modification enabled increased oncolysis for RMS cells by a conditionally replicative Ad, Ad delta24RGD, harboring a retinoblastoma-binding mutation in the E1A gene. Thus, the development of replication-competent vectors targeted to cell surface receptors other than CAR is critical to advance the use of Ad for treating RMS.