Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro

[EN] Our knowledge on the mode of evolution of the multifunctional viral proteins remains incomplete. To tackle this problem, here, we have investigated the evolutionary dynamics of the potyvirus multifunctional protein HC-Pro, with particular focus on its functional domains. The protein was partitioned into the three previously described functional domains, and each domain was analyzed separately and assembled. We searched for signatures of adaptive evolution and evolutionary dependencies of amino acid sites within and between the three domains using the entire set of available potyvirus sequences in GenBank. Interestingly, we identified strongly significant patterns of co-occurrence of adaptive events along the phylogenetic tree in the three domains. These patterns suggest that Domain I, whose main function is to mediate aphid transmission, has likely been coevolving with the other two domains, which are involved in different functions but all requiring the capacity to bind RNA. By contrast, episodes of positive selection on Domains II and III did not correlate, reflecting a trade-off between their evolvability and their evolutionary dependency likely resulting from their functional overlap. Covariation analyses have identified several groups of amino acids with evidence of concerted variation within each domain, but interdomain significant covariations were only found for Domains II and III, further reflecting their functional overlappingThis work was supported by grants BFU2012-30805 (SFE) and BFU2012-36346 (MAF) from the Spanish Direccio´n General de Investigacio´n Cientı´fica y Te´cnica and by an EMBO Short-Term Fellowship and the Mentoring Program from the Foundation for Polish Science (BHJ).Hasiów-Jaroszewska, B.; Fares Riaño, MA.; Elena Fito, SF. (2014). Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro. 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Role of the Yeast Gin4p Protein Kinase in Septin Assembly and the Relationship between Septin Assembly and Septin Function

To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother–bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Δ vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Δ cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605–616)

Reduced Selective Constraint in Endosymbionts: Elevation in Radical Amino Acid Replacements Occurs Genome-Wide

As predicted by the nearly neutral model of evolution, numerous studies have shown that reduced Ne accelerates the accumulation of slightly deleterious changes under genetic drift. While such studies have mostly focused on eukaryotes, bacteria also offer excellent models to explore the effects of Ne. Most notably, the genomes of host-dependent bacteria with small Ne show signatures of genetic drift, including elevated Ka/Ks. Here, I explore the utility of an alternative measure of selective constraint: the per-site rate of radical and conservative amino acid substitutions (Dr/Dc). I test the hypothesis that purifying selection against radical amino acid changes is less effective in two insect endosymbiont groups (Blochmannia of ants and Buchnera of aphids), compared to related gamma-Proteobacteria. Genome comparisons demonstrate a significant elevation in Dr/Dc in endosymbionts that affects the majority (66–79%) of shared orthologs examined. The elevation of Dr/Dc in endosymbionts affects all functional categories examined. Simulations indicate that Dr/Dc estimates are sensitive to codon frequencies and mutational parameters; however, estimation biases occur in the opposite direction as the patterns observed in genome comparisons, thereby making the inference of elevated Dr/Dc more conservative. Increased Dr/Dc and other signatures of genome degradation in endosymbionts are consistent with strong effects of genetic drift in their small populations, as well as linkage to selected sites in these asexual bacteria. While relaxed selection against radical substitutions may contribute, genome-wide processes such as genetic drift and linkage best explain the pervasive elevation in Dr/Dc across diverse functional categories that include basic cellular processes. Although the current study focuses on a few bacterial lineages, it suggests Dr/Dc is a useful gauge of selective constraint and may provide a valuable alternative to Ka/Ks when high sequence divergences preclude estimates of Ks. Broader application of Dr/Dc will benefit from approaches less prone to estimation biases

Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

Background:Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals.Methodology/Principal findings:We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2β, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells.Conclusions/Significance:Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Atienza, Augusto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Perez Prados, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Buying, Alcinette. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Santos, Radleigh. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Tasso, Laura Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bonato, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Chiale, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Pinilla, Clemencia. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Judkowski, Valeria A.. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Phylogenomics: Gene Duplication, Unrecognized Paralogy and Outgroup Choice

Comparative genomics has revealed the ubiquity of gene and genome duplication and subsequent gene loss. In the case of gene duplication and subsequent loss, gene trees can differ from species trees, thus frequent gene duplication poses a challenge for reconstruction of species relationships. Here I address the case of multi-gene sets of putative orthologs that include some unrecognized paralogs due to ancestral gene duplication, and ask how outgroups should best be chosen to reduce the degree of non-species tree (NST) signal. Consideration of expected internal branch lengths supports several conclusions: (i) when a single outgroup is used, the degree of NST signal arising from gene duplication is either independent of outgroup choice, or is minimized by use of a maximally closely related post-duplication (MCRPD) outgroup; (ii) when two outgroups are used, NST signal is minimized by using one MCRPD outgroup, while the position of the second outgroup is of lesser importance; and (iii) when two outgroups are used, the ability to detect gene trees that are inconsistent with known aspects of the species tree is maximized by use of one MCRPD, and is either independent of the position of the second outgroup, or is maximized for a more distantly related second outgroup. Overall, these results generalize the utility of closely-related outgroups for phylogenetic analysis

Chance and necessity in the genome evolution of endosymbiotic bacteria of insects

[EN] An open question in evolutionary biology is how does the selection¿drift balance determine the fates of biological interactions. We searched for signatures of selection and drift in genomes of five endosymbiotic bacterial groups known to evolve under strong genetic drift. Although most genes in endosymbiotic bacteria showed evidence of relaxed purifying selection, many genes in these bacteria exhibited stronger selective constraints than their orthologs in free-living bacterial relatives. Remarkably, most of these highly constrained genes had no role in the host¿symbiont interactions but were involved in either buffering the deleterious consequences of drift or other host-unrelated functions, suggesting that they have either acquired new roles or their role became more central in endosymbiotic bacteria. Experimental evolution of Escherichia coli under strong genetic drift revealed remarkable similarities in the mutational spectrum, genome reduction patterns and gene losses to endosymbiotic bacteria of insects. Interestingly, the transcriptome of the experimentally evolved lines showed a generalized deregulation of the genome that affected genes encoding proteins involved in mutational buffering, regulation and amino acid biosynthesis, patterns identical to those found in endosymbiotic bacteria. Our results indicate that drift has shaped endosymbiotic associations through a change in the functional landscape of bacterial genes and that the host had only a small role in such a shiftThis work was supported by Science Foundation Ireland (12/IP/1637) and grants from the Spanish Ministerio de Economia y Competitividad (MINECO-FEDER; BFU2012-36346 and BFU2015-66073-P) to MAF. 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Genome-Wide Functional Divergence after the Symbiosis of Proteobacteria with Insects Unraveled through a Novel Computational Approach

Symbiosis has been among the most important evolutionary steps to generate biological complexity. The establishment of symbiosis required an intimate metabolic link between biological systems with different complexity levels. The strict endo-cellular symbiotic bacteria of insects are beautiful examples of the metabolic coupling between organisms belonging to different kingdoms, a eukaryote and a prokaryote. The host (eukaryote) provides the endosymbiont (prokaryote) with a stable cellular environment while the endosymbiont supplements the host's diet with essential metabolites. For such communication to take place, endosymbionts' genomes have suffered dramatic modifications and reconfigurations of proteins' functions. Two of the main modifications, loss of genes redundant for endosymbiotic bacteria or the host and bacterial genome streamlining, have been extensively studied. However, no studies have accounted for possible functional shifts in the endosymbiotic proteomes. Here, we develop a simple method to screen genomes for evidence of functional divergence between two species clusters, and we apply it to identify functional shifts in the endosymbiotic proteomes. Despite the strong effects of genetic drift in the endosymbiotic systems, we unexpectedly identified genes to be under stronger selective constraints in endosymbionts of aphids and ants than in their free-living bacterial relatives. These genes are directly involved in supplementing the host's diet with essential metabolites. A test of functional divergence supports a strong relationship between the endosymbiosis and the functional shifts of proteins involved in the metabolic communication with the insect host. The correlation between functional divergence in the endosymbiotic bacterium and the ecological requirements of the host uncovers their intimate biochemical and metabolic communication and provides insights on the role of symbiosis in generating species diversity

Molecular evolution of the vertebrate TLR1 gene family - a complex history of gene duplication, gene conversion, positive selection and co-evolution

<p>Abstract</p> <p>Background</p> <p>The Toll-like receptors represent a large superfamily of type I transmembrane glycoproteins, some common to a wide range of species and others are more restricted in their distribution. Most members of the Toll-like receptor superfamily have few paralogues; the exception is the TLR1 gene family with four closely related genes in mammals TLR1, TLR2, TLR6 and TLR10, and four in birds TLR1A, TLR1B, TLR2A and TLR2B. These genes were previously thought to have arisen by a series of independent gene duplications. To understand the evolutionary pattern of the TLR1 gene family in vertebrates further, we cloned the sequences of TLR1A, TLR1B, TLR2A and TLR2B in duck and turkey, constructed phylogenetic trees, predicted codons under positive selection and identified co-evolutionary amino acid pairs within the TLR1 gene family using sequences from 4 birds, 28 mammals, an amphibian and a fish.</p> <p>Results</p> <p>This detailed phylogenetic analysis not only clarifies the gene gains and losses within the TLR1 gene family of birds and mammals, but also defines orthologues between these vertebrates. In mammals, we predict amino acid sites under positive selection in TLR1, TLR2 and TLR6 but not TLR10. We detect co-evolution between amino acid residues in TLR2 and the other members of this gene family predicted to maintain their ability to form functional heterodimers. In birds, we predict positive selection in the TLR2A and TLR2B genes at functionally significant amino acid residues. We demonstrate that the TLR1 gene family has mostly been subject to purifying selection but has also responded to directional selection at a few sites, possibly in response to pathogen challenge.</p> <p>Conclusions</p> <p>Our phylogenetic and structural analyses of the vertebrate TLR1 family have clarified their evolutionary origins and predict amino acid residues likely to be important in the host's defense against invading pathogens.</p