Metadata-driven comparative analysis tool for sequences (meta-CATS): an automated process for identifying significant sequence variations that correlate with virus attributes.

The Virus Pathogen Resource (ViPR; www.viprbrc.org) and Influenza Research Database (IRD; www.fludb.org) have developed a metadata-driven Comparative Analysis Tool for Sequences (meta-CATS), which performs statistical comparative analyses of nucleotide and amino acid sequence data to identify correlations between sequence variations and virus attributes (metadata). Meta-CATS guides users through: selecting a set of nucleotide or protein sequences; dividing them into multiple groups based on any associated metadata attribute (e.g. isolation location, host species); performing a statistical test at each aligned position; and identifying all residues that significantly differ between the groups. As proofs of concept, we have used meta-CATS to identify sequence biomarkers associated with dengue viruses isolated from different hemispheres, and to identify variations in the NS1 protein that are unique to each of the 4 dengue serotypes. Meta-CATS is made freely available to virology researchers to identify genotype-phenotype correlations for development of improved vaccines, diagnostics, and therapeutics.

A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.

Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level.

New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models.

During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolyloxazoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral dosing. This compound, and others of its structural class, may prove to be useful in the development of new chemotherapeutic agents to treat human cancers.

Novel sulfonate derivatives: potent antimitotic agents.

The synthesis and biological evaluation of novel sulfonate analogues of E-7010 are reported. Several of the compounds are potent inhibitors of cell proliferation and tubulin polymerization. Importantly, these compounds are also active against P-glycoprotein positive (+) cancer cells, which are resistant to many other antitumor agents.

Novel sulfonate analogues of combretastatin A-4: potent antimitotic agents.

Sulfonate analogues of combretastatin A-4 have been prepared. These compounds compete with colchicine and combretastatin A-4 for the colchicine binding site on tubulin and are potent inhibitors of tubulin polymerization and cell proliferation. Importantly, these compounds also inhibit the proliferation of P-glycoprotein positive (+) cancer cells, which are resistant to many other antitumor agents.

Modulation of drug resistance by alpha-tubulin in paclitaxel-resistant human lung cancer cell lines.

Beta(beta)-tubulin isotype variation has recently been implicated in the modulation of resistance to paclitaxel in human lung cancer cells and in primary human ovarian tumour samples. Whether alpha-tubulin is involved in drug resistance has not been reported. We have generated a paclitaxel-resistant cell line (H460/T800) from the sensitive human lung carcinoma parental cell line NCI-H460. The resistant cells are more than 1000-fold resistant to taxol and overexpress P-glycoprotein. Interestingly, H460/T800 cells also overexpress alpha- and beta-tubulin as detected by Western blot analysis. From Northern blot analysis, the mechanism of tubulin overexpression appears to be post-transcriptional. To understand whether alpha-tubulin plays a role in drug resistance, we transfected antisense human kalpha1 cDNA construct into the H460/T800 paclitaxel-resistant cells. The antisense clones displayed a reduced alpha-tubulin expression, and the cells were 45-51% more sensitive to paclitaxel and other known antimitotic drugs, compared with vector transfected controls. Complementary experiments of transfecting the sense kalpha1 cDNA into H460 cells conferred a 1.8- to 3.3-fold increase in the IC(50) of several antimitotic agents. Our study suggests that alpha-tubulin is one of the factors that contributes to drug resistance.

RNA determinants of a specific RNA-coat protein peptide interaction in alfalfa mosaic virus: conservation of homologous features in ilarvirus RNAs.

Alfalfa mosaic virus (AMV) coat protein and tobacco streak virus (TSV) coat protein bind specifically to the 3' untranslated regions of the viral RNAs and are required with the genomic RNAs to initiate virus replication. A combination of nucleotide substitutions, hydroxyl radical footprinting, and ethylation and chemical modification interference analysis has been used to define the RNA determinants important for the specific binding of the 3'-terminal 39 nucleotides of AMV RNA 3/4 (AMV843-881) to an amino-terminal coat protein peptide (CP26). The results demonstrate that potential phosphate and base-specific contacts as well as ribose moieties protected upon peptide binding cluster in lower hairpin stems and flanking AUGC sequences of the viral RNA, without direct involvement of loop nucleotides. Nucleotides identified in the modification-interference analyses as important for RNA-protein interactions are highly conserved among AMV and the ilarvirus RNAs. This RNA sequence homology, coupled with the recent identification of an RNA binding consensus sequence for AMV and ilarvirus coat proteins, provides a framework for understanding the functional equivalence of AMV and TSV coat proteins in binding RNA and activating virus replication and may explain why heterologous AMV and ilarvirus coat protein-RNA mixtures are infectious.

Viral coat protein peptides with limited sequence homology bind similar domains of alfalfa mosaic virus and tobacco streak virus RNAs.

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077-5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3'-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3'-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.

Cleavage site mapping and substrate-specificity of Leishmaniavirus 2-1 capsid endoribonuclease activity.

The Leishmaniavirus capsid protein possesses an RNA endoribonuclease activity that cleaves viral positive-sense RNA at a specific, single site within the 5' untranslated region. The site of cleavage in LRV1-4 RNA was previously mapped to nucleotide 320 of the LRV1-4 genome. Here we show that an LRV2-1-derived substrate RNA transcript is also cleaved at a single site in an in vitro cleavage assay with LRV2-1 virions. Precise RNA cleavage site mapping in this divergent Old World virus, LRV2-1, confirms that cleavage is occurring within a region of homology to the LRV1 isolates. Substrate RNA transcripts possessing viral sequences from LRV1-4 or LRV2-1 genomes were assayed for susceptibility to cleavage by the cognate and noncognate capsid endoribonucleases to determine the level of substrate specificity.

Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.

In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs.

The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis.

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious.

Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist.

Endothelins (ET) are potent vasoactive peptides implicated in the pathogenesis of a number of vascular diseases. The effects of ET on mammalian organs and cells are initiated by binding to ETA or ETB receptors. In this report, we document the pharmacology of A-127722, a novel ETA-selective receptor antagonist. A-127722 inhibits [125I]ET-1 binding to cloned human ETA and ETB receptors competitively with Ki values of 69 pM and 139 nM, respectively. A-127722 exhibits a dose-dependent inhibition of ET-1-induced arachidonic acid release in human pericardium smooth muscle cells with a pA2 value of 10.5 and inhibits ET-1-induced vasoconstriction in isolated rat aorta with a pA2 value of 9.2. In vivo, A-127722 dose-dependently blocks the pressor response to ET-1 (0.3 nmol/kg i.v.) in conscious rats. Statistically significant (P < .05) antagonism is seen at doses greater than 0.1 mg/kg p.o. Maximal inhibition, at 10 mg/kg, remains constant for at least 8 hr after dosing. No effect is seen on the ETB-mediated transient vasodepressor effect of exogenous ET-1. In conclusion, A-127722 is ETA-selective, orally bioavailable and efficacious for inhibiting the effects of ET in the rat, and A-127722 is the most potent ET receptor antagonist yet reported.

Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation.

Magnetization transfer in cartilage and its constituent macromolecules.

The goal of this work was to investigate magnetization transfer (MT) in cartilage by measuring water proton signals Ms/Mo, as an indicator of MT, in (i) single-component systems of the tissue's constituent macromolecules and (ii) intact cartilage under control conditions and after two pathomimetic interventions. Ms/Mo was quantified with a 12-microT saturation pulse applied 6 kHz off resonance. Both glycosaminoglycans (GAG) and collagen exhibited concentration dependent effects on Ms/Mo, being approximately linear for GAG solutions (Ms/Mo = -0.0137[% GAG] + 1.02) and exponential for collagen suspensions (Ms/Mo = 0.80 x exp[-(%collagen)/6.66] + 0.20); the direct saturation of water could not account for the measured Ms/Mo. Although the effect of collagen on Ms/Mo is much stronger than for a corresponding concentration of GAG, Ms/Mo is not very sensitive to changes in collagen concentration in the physiological range. Tissue degradation with 25 mg/ml trypsin led to an increase in Ms/Mo from the baseline value of 0.2 (final/initial values = 1.15 +/- 0.13, n = 11, P < 0.001). In contrast, a 10-day treatment of cartilage with 100 ng/ml of interleukin-1 beta (IL-1 beta) caused a 19% decrease in Ms/Mo (final/initial values = 0.81 +/- 0.08, n = 3, P = 0.085). The changes in hydration and macromolecular content for the two treatments were comparable, suggesting that Ms/Mo is sensitive to macromolecular structure as well as concentration. In conclusion, whereas the baseline Ms/Mo value in cartilage may be primarily due to the tissue collagen concentration, changes in Ms/Mo may be due to physiological or pathophysiological changes in GAG concentration and tissue structure, and the measured Ms/Mo may differentiate between various pathomimetic degradative procedures.

Eukaryotic translation initiation factor 4E regulates expression of cyclin D1 at transcriptional and post-transcriptional levels.

Regulation of the cell cycle is orchestrated by cyclins and cyclin-dependent kinases. We have demonstrated previously that overexpression of eukaryotic translation initiation factor 4E (eIF-4E) in NIH 3T3 cells growing in 10% fetal calf serum leads to highly elevated levels of cyclin D1 protein without significant increase in cyclin D1 mRNA levels, suggesting that a post-transcriptional mechanism is involved. (Rosenwald, I. B., Lazaris-Karatzas, A., Sonenberg, N., and Schmidt, E. V. (1993) Mol. Cell. Biol. 13, 7358-7363). In the present research, we did not find any significant effect of eIF-4E on polysomal distribution of cyclin D1 mRNA. However, the total amount of cyclin D1 mRNA associated with polysomes was significantly increased by eIF-4E overexpression. Further, we determined that the levels of both cyclin D1 protein and mRNA are increased in serum-deprived cells overexpressing eIF-4E. Nuclear run-on experiments demonstrated that the rate of the cyclin D1 transcription is not down-regulated in serum-deprived cells overexpressing eIF-4E. Thus, elevated levels of eIF-4E may lead to increased transcription of the cyclin D1 gene, and this effect becomes visible when serum deprivation down-regulates the rate of cyclin D1 mRNA synthesis in control cells. However, artificial overexpression of cyclin D1 mRNA in serum-deprived cells in the absence of eIF-4E overexpression did not cause the elevation of cyclin D1 protein, and this overexpressed cyclin D1 mRNA accumulated in the nucleus, suggesting that one post-transcriptional role of eIF-4E is to transport cyclin D1 mRNA from the nucleus to cytoplasmic polysomes.

mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nucleotide elements permitting selective AIMV CP expression, we tested capped mRNAs containing structured or unstructured 5' leader sequences in addition to an mRNA containing the poliovirus internal ribosome entry site (IRES). Translations were performed with PI-extracts and extracts prepared from mock-infected HeLa cells (MI-extracts). A number of control criteria demonstrated that the HeLa cells were infected by poliovirus and that the extracts were translationally active. The data strongly indicate that translation of RNAs lacking an internal ribosome entry site, including AIMV CP RNA, was severely compromised in PI-extracts, and we find no evidence that the unstructured AIMV CP RNA 5' leader sequence acts in cis to bypass the poliovirus translational control. Nevertheless, cotranslation assays in the MI-extracts demonstrate that mRNAs containing the unstructured AIMV CP RNA 5' untranslated region have a competitive advantage over those containing the rabbit alpha-globin 5' leader. Previous reports of AIMV CP RNA translation in PI-extracts likely describe inefficient expression that can be explained by residual cap-dependent initiation events, where AIMV CP RNA translation is competitive because of a diminished quantitative requirement for initiation factors.

Inhibition of protein synthesis in insect cells by baculovirus-expressed heme-regulated eIF-2 alpha kinase.

To study further the regulation of the heme-regulated eIF-2 alpha kinase (HRI), we have produced functional wild type HRI using the baculovirus expression system. The amount of recombinant HRI protein expressed in insect cells is approximately 10 times higher than levels in reticulocytes. Baculovirus-expressed HRI (BV-HRI) is indistinguishable from HRI purified from rabbit reticulocytes. It is active both as an autokinase and an eIF-2 alpha kinase. BV-HRI is regulated by heme in vitro as well as in intact insect cells. Coexpression of the wild type HRI with the inactive K199R HRI, S51A eIF-2 alpha, or interleukin-1 beta (IL-1 beta) results in diminished expression of these proteins. Expression of wild type HRI also results in severe inhibition of general protein synthesis in Sf9 cells when compared with cells expressing K199R HRI or IL-beta. In addition, the guanine nucleotide exchange activity of eIF-2B is suppressed in Sf9 cells expressing wild type HRI but not in cells expressing the K199R HRI or IL-1 beta. Furthermore, expression of wild type HRI is increased by coexpression with the nonphosphorylatable S51A eIF-2 alpha or by the addition of hemin, which inhibits HRI activity. These results provide evidence that translational regulation by phosphorylation of eIF-2 alpha and sequestration of eIF-2B can operate in insect cells.