Eran Bacharach

Selecton 2007: advanced models for detecting positive and purifying selection using a Bayesian inference approach

Biologically significant sites in a protein may be identified by contrasting the rates of synonymous (Ks) and non-synonymous (Ka) substitutions. This enables the inference of site-specific positive Darwinian selection and purifying selection. We present here Selecton version 2.2 (http://selecton.bioinfo.tau.ac.il), a web server which automatically calculates the ratio between Ka and Ks (ω) at each site of the protein. This ratio is graphically displayed on each site using a color-coding scheme, indicating either positive selection, purifying selection or lack of selection. Selecton implements an assembly of different evolutionary models, which allow for statistical testing of the hypothesis that a protein has undergone positive selection. Specifically, the recently developed mechanistic-empirical model is introduced, which takes into account the physicochemical properties of amino acids. Advanced options were introduced to allow maximal fine tuning of the server to the users specific needs, including calculation of statistical support of the ω values, an advanced graphic display of the proteins 3-dimensional structure, use of different genetic codes and inputting of a pre-built phylogenetic tree. Selecton version 2.2 is an effective, user-friendly and freely available web server which implements up-to-date methods for computing site-specific selection forces, and the visualization of these forces on the proteins sequence and structure.

Infectivity of Moloney Murine Leukemia Virus Defective in Late Assembly Events Is Restored by Late Assembly Domains of Other Retroviruses

ABSTRACT The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutantgag gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.

Selecton: a server for detecting evolutionary forces at a single amino-acid site

UNLABELLED We present an algorithmic tool for the identification of biologically significant amino acids in proteins of known three dimensional structure. We estimate the degree of purifying selection and positive Darwinian selection at each site and project these estimates onto the molecular surface of the protein. Thus, patches of functional residues (undergoing either positive or purifying selection), which may be discontinuous in the linear sequence, are revealed. We test for the statistical significance of the site-specific scores in order to obtain reliable and valid estimates. AVAILABILITY The Selecton web server is available at: http://selecton.bioinfo.tau.ac.il SUPPLEMENTARY INFORMATION More information is available at http://selecton.bioinfo.tau.ac.il/overview.html. A set of examples is available at http://selecton.bioinfo.tau.ac.il/gallery.html.

Phage therapy of coral disease

At present there are no known procedures for preventing or treating infectious diseases of corals. Toward this end, the use of phage therapy has been investigated. Lytic bacteriophages (phages) were isolated for two bacterial pathogens that are responsible for coral diseases, Vibrio coralliilyticus, which is the causative agent of bleaching and tissue lysis of Pocillopora damicornis, and Thalosomonas loyaeana, which causes the white plague-like disease of Favia favus. By using these phages in controlled aquaria experiments, it was demonstrated that each of these diseases could be controlled by the pathogen-specific phage. The data indicate that initially the phages bind to the pathogen in seawater and are then brought to the coral surface where they multiply and lyse the pathogen. The phages remained associated with the coral and could prevent subsequent infections. These data suggest that phage therapy has the potential to control the spread of infectious coral diseases.

The conserved carboxy terminus of the capsid domain of human immunodeficiency virus type 1 gag protein is important for virion assembly and release.

ABSTRACT The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an α-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.

Towards realistic codon models

Codon evolutionary models are widely used to infer the selection forces acting on a protein. The non-synonymous to synonymous rate ratio (denoted by Ka/Ks) is used to infer specific positions that are under purifying or positive selection. Current evolutionary models usually assume that only the non-synonymous rates vary among sites while the synonymous substitution rates are constant. This assumption ignores the possibility of selection forces acting at the DNA or mRNA levels. Towards a more realistic description of sequence evolution, we present a model that accounts for among-site-variation of both synonymous and non-synonymous substitution rates. Furthermore, we alleviate the widespread assumption that positions evolve independently of each other. Thus, possible sources of bias caused by random fluctuations in either the synonymous or non-synonymous rate estimations at a single site is removed. Our model is based on two hidden Markov models that operate on the spatial dimension: one describes the dependency between adjacent non-synonymous rates while the other describes the dependency between adjacent synonymous rates. The presented model is applied to study the selection pressure across the HIV-1 genome. The new model better describes the evolution of all HIV-1 genes, as compared to current codon models. Using both simulations and real data analyses, we illustrate that accounting for synonymous rate variability and dependency greatly increases the accuracy of Ka/Ks estimation and in particular of positively selected sites. Finally, we discuss the applicability of the developed model to infer the selection forces in regulatory and overlapping regions of the HIV-1 genome.

Tumor-Microenvironment Interactions The Fucose-Generating FX Enzyme Controls Adhesive Properties of Colorectal Cancer Cells

Extravasation of tumor cells is a pivotal step in metastasis formation. This step is initiated by an interaction of extravasating tumor cells with endothelial cells. Among the molecules mediating tumor-endothelium interactions are selectins and their fucosylated ligands. In a previous study, we demonstrated that the fucose-generating FX enzyme regulates the expression of selectin ligands by B and T lymphocytes and by head and neck squamous cell carcinoma cells. It was also shown that the FX enzyme regulated important interaction parameters between these cancer cells and endothelial cells. The present study was aimed to determine whether the FX enzyme controls adhesive interactions between colorectal cancer cells and endothelial cells. The results clearly indicate that this is indeed the case. Overexpressing the FX enzyme by the transfer of FX cDNA to low FX-expressing colorectal cancer cells resulted in an increased adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. Down-regulating FX levels in colorectal cancer cells expressing high levels of endogenous FX by transfection with small-interfering RNA resulted in a down-regulated expression of the selectin ligand sialyl Lewis-a and a decrease in the adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. These transfection experiments also indicated that manipulating the levels of the FX enzyme affected global cellular fucosylation and altered the interaction of colorectal cancer cells with some extracellular matrix components such as fibronectin. We also found that highly metastatic colorectal cancer variants express higher levels of FX and of sialyl Lewis-a than low metastatic variants originating in the same tumors. These results lead us to hypothesize that the FX enzyme controls the capacity of colorectal cancer to extravasate and form metastasis. If this hypothesis will be confirmed the FX enzyme could become a target molecule for metastasis prevention.

The Carboxy-Terminal Fragment of Nucleolin Interacts with the Nucleocapsid Domain of Retroviral Gag Proteins and Inhibits Virion Assembly

ABSTRACT A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

p12 Tethers the Murine Leukemia Virus Pre-integration Complex to Mitotic Chromosomes

The p12 protein of the murine leukemia virus (MLV) is a constituent of the pre-integration complex (PIC) but its function in this complex remains unknown. We developed an imaging system to monitor MLV PIC trafficking in live cells. This allowed the visualization of PIC docking to mitotic chromosomes and its release upon exit from mitosis. Docking occurred concomitantly with nuclear envelope breakdown and was impaired for PICs of viruses with lethal p12 mutations. Insertion of a heterologous chromatin binding module into p12 of one of these mutants restored PICs attachment to the chromosomes and partially rescued virus replication. Capsid dissociated from wild type PICs in mitotic cells but remained associated with PICs harboring tethering-negative p12 mutants. Altogether, these results explain, in part, MLV restriction to dividing cells and reveal a role for p12 as a factor that tethers MLV PIC to mitotic chromosomes.

Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia

ABSTRACT Tilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5′ and 3′ termini, consistent with the genome organization found in other orthomyxoviruses. In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease. IMPORTANCE The economic impact of worldwide trade in tilapia is estimated at