History of Smallpox: Timeline of a Serial Killer.

<p>History of Smallpox: Timeline of a Serial Killer.</p

Different Strategies for Inhibiting TNF by Pathogens

<p>Pathogens have evolved diverse strategies to target almost every step of TNF biology. Virus-encoded proteins inhibit TNF-mediated responses by directly binding to TNF with secreted soluble decoy TNFR (vTNFRs) and vTNFBPs, downregulating the cellular death receptors, interacting with the TNFR-associated factors, blocking caspase activation, and regulating the apoptotic checkpoint function of mitochondria. Viruses also regulate the pathways leading to TNF-mediated activation of NF-κB. Bacteria and other pathogens can express proteins that regulate TNF-mediated responses by activating or inhibiting NF-κB at different levels of signaling that range from the death receptor to nuclear localization of NF-κB.</p

TNF-Mediated Death and Survival Pathways

<p>TNF-mediated death and survival pathways are activated following interaction with the TNFRs. The apoptotic pathway is activated through TNFR1 by forming the DISC, which activates caspase-8. Activated caspase-8 or −10 then activates the proapoptotic Bcl-2 family members, which leads to cell death by releasing cytochrome c from mitochondria and loss of MMP. The NF-κB-mediated survival pathway is activated by both TNFR1 and TNFR2. Association of TRAFs with these receptors activate signaling proteins like NIK (NF-κB inhibitor kinase) and MEKK1 (MAPK kinase 1), which activate the inhibitor of NF-κB (IkB) kinase (IKK) signalosome complex. IKK phosphorylates IkB, resulting in the degradation of the inhibitor. The free NF-κB than translocates to nucleus to induce the expression of inflammatory or antiapoptotic genes.</p

Positive Evolutionary Selection On the RIG-I-Like Receptor Genes in Mammals

<div><p>The mammalian RIG-I-like receptors, RIG-I, MDA5 and LGP2, are a family of DExD/H box RNA helicases responsible for the cytoplasmic detection of viral RNA. These receptors detect a variety of RNA viruses, or DNA viruses that express unusual RNA species, many of which are responsible for a great number of severe and lethal diseases. Host innate sentinel proteins involved in pathogen recognition must rapidly evolve in a dynamic arms race with pathogens, and thus are subjected to long-term positive selection pressures to avoid potential infections. Using six codon-based Maximum Likelihood methods, we were able to identify specific codons under positive selection in each of these three genes. The highest number of positively selected codons was detected in MDA5, but a great percentage of these codons were located outside of the currently defined protein domains for MDA5, which likely reflects the imposition of both functional and structural constraints. Additionally, our results support LGP2 as being the least prone to evolutionary change, since the lowest number of codons under selection was observed for this gene. On the other hand, the preponderance of positively selected codons for RIG-I were detected in known protein functional domains, suggesting that pressure has been imposed by the vast number of viruses that are recognized by this RNA helicase. Furthermore, the RIG-I repressor domain, the region responsible for recognizing and binding to its RNA substrates, exhibited the strongest evidence of selective pressures. Branch-site analyses were performed and several species branches on the three receptor gene trees showed evidence of episodic positive selection. In conclusion, by looking for evidence of positive evolutionary selection on mammalian RIG-I-like receptor genes, we propose that a multitude of viruses have crafted the receptors biological function in host defense, specifically for the RIG-I gene, contributing to the innate species-specific resistance/susceptibility to diverse viral pathogens. </p> </div

Maximum likelihood (ML) phylogenetic tree of RIG-I gene used for codon-based ML analysis.

<p>The GTR+G nucleotide substitution model was employed in mammalian RIG-I gene phylogenetic reconstruction. Bootstrap values >50 are indicated on the branches.</p

Maximum likelihood (ML) phylogenetic tree of mammalian LGP2 gene used for codon-based ML.

<p>The TPM2uf+I+G nucleotide substitution model was employed in mammalian LGP2 gene phylogenetic reconstruction. Bootstrap values >50 are indicated on the branches. </p

Structural representation and identification of positively-selected branches and codons in mammalian RIG-I.

<p>(A) Based on human protein structure, the key domains of RIG-I (<a href="http://www.uniprot.org/uniprot/o95786" target="_blank">http://www.uniprot.org/uniprot/O95786</a>) and the corresponding boundaries are schematically represented. Also, the human domain boundaries while in the mammalian RIG-I deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s004" target="_blank">Figure S4</a>) are shown in brackets. (B) Cladogram of 26 mammalian RIG-I genes collected from Ensembl and NCBI databases. Branch-site analyses were performed to identify specific branches under episodic positive selection. Branches with statistically significant likelihood ratio tests (LRTs) when performing PAML branch-site model A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t002" target="_blank">Table 2</a>) are colored in green; branches simultaneously identified by PAML branch-site model A and Hyphy branch-site REL method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t005" target="_blank">Table 5</a>) are colored in blue. (C) Positively-selected codons are exhibited in the table and numbered according to the mammalian RIG-I deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s004" target="_blank">Figure S4</a>). Symbol “-” represents a deletion. Colors on the codon numbering row correspond to the RIG-I domain with the same color in the protein structural representation (A). The background colors on the identified sites match different amino acid properties: polar positive (yellow), polar negative (orange), polar neutral (green), non-polar neutral (purple), non-polar aliphatic (blue) and non-polar aromatic (pink). The used abbreviations correspond, by order of appearance, to the following species: Hosa – Human; Patr – Chimpanzee; Papa – Bonobo; Gogo – Gorilla; Poab – Orangutan; Paan – Olive baboon; Mamu – Rhesus macaque; Sabo – Black-capped squirrel monkey; Caja – Marmoset; Mimu – Mouse lemur; Otga – Bushbaby; Bota – Cow; Ovar – Sheep; Susc – Pig; Mylu – Little brown myotis; Ptva – Large flying fox; Ptal – Black flying fox; Aime – Giant panda; Calu – Dog; Feca – Cat; Eqca – Horse; Loaf – Elephant; Ictr – Squirrel; Capo – Guinea pig; Mumu – Mouse; Orcu – European rabbit. </p

Structural representation and identification of positively-selected branches and codons in mammalian LGP2.

<p>(A) Based on human protein structure, the key domains of LGP2 (<a href="http://www.uniprot.org/uniprot/q96c10" target="_blank">http://www.uniprot.org/uniprot/Q96C10</a>) and the corresponding boundaries are schematically represented. Also, the human domain boundaries while in the mammalian LGP2 deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s006" target="_blank">Figure S6</a>) are shown in brackets. (B) Cladogram of 30 mammalian LGP2 genes collected from Ensembl and NCBI databases. Branch-site analyses were performed to identify specific branches under episodic positive selection. Branches with statistically significant likelihood ratio tests (LRTs) when performing PAML branch-site model A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t004" target="_blank">Table 4</a>) are colored in green; branch colored in blue has been simultaneously identified by PAML branch-site model A and Hyphy branch-site REL method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t005" target="_blank">Table 5</a>). (C) Positively-selected codons are exhibited in the table and numbered according to the mammalian LGP2 deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s006" target="_blank">Figure S6</a>). Colors on the codon numbering row correspond to the LGP2 domain with the same color in the protein structural representation (A). The background colors on the identified codons match different amino acid properties: polar positive (yellow), polar negative (orange), polar neutral (green), non-polar neutral (purple), non-polar aliphatic (blue) and non-polar aromatic (pink). The used abbreviations correspond, by order of appearance, to the following species: Hosa – Human; Patr – Chimpanzee; Papa – Bonobo; Gogo – Gorilla; Poab – Orangutan; Mamu – Rhesus macaque; Sabo – Black-capped squirrel monkey; Caja – Marmoset; Mimu – Mouse lemur; Otga – Bushbaby; Bota – Cow; Ovar – Sheep; Susc – Pig; Tutr – Dolphin; Mylu – Little brown myotis; Ptva – Large flying fox; Ptal – Black flying fox; Loaf – Elephant; Mupu – Ferret; Aime – Giant panda; Calu – Dog; Feca – Cat; Eqca – Horse; Ocpr – American pika; Orcu – European rabbit; Ictr – Squirrel; Crgr – Chinese hamster; Mumu – Mouse; Rano – Rat; Capo – Guinea pig. </p

Structural representation and identification of positively-selected branches and codons in mammalian MDA5.

<p>(A) Based on the human protein structure, the key domains of MDA5 (<a href="http://www.uniprot.org/uniprot/q9byx4" target="_blank">http://www.uniprot.org/uniprot/Q9BYX4</a>) and the corresponding boundaries are schematically represented. Also, the human domain boundaries while in the mammalian MDA5 deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s005" target="_blank">Figure S5</a>) are shown in brackets. (B) Cladogram of 26 mammalian MDA5 genes collected from Ensembl and NCBI databases. Branch-site analyses were performed to identify specific branches episodic under positive selection. Branches with statistically significant likelihood ratio tests (LRTs) when performing PAML branch-site model A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t003" target="_blank">Table 3</a>) are colored in green; branches simultaneously identified by PAML branch-site model A and Hyphy branch-site REL method (Table 5) are colored in blue. (C) Positively-selected codons are exhibited in the table and numbered according to the mammalian MDA5 deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s005" target="_blank">Figure S5</a>). Symbol “?” represents an undetermined amino acid, while “-” symbolizes a deletion. Colors on the codon numbering row correspond to the MDA5 domain with the same color in the protein structural representation (A). The background colors on the identified sites match different amino acid properties: polar positive (yellow), polar negative (orange), polar neutral (green), non-polar neutral (purple), non-polar aliphatic (blue) and non-polar aromatic (pink). The used abbreviations correspond, by order of appearance, to the following species: Hosa – Human; Gogo – Gorilla; Patr – Chimpanzee; Papa – Bonobo; Poab – Orangutan; Nole – Gibbon; Mamu – Rhesus macaque; Sabo – Black-capped squirrel monkey; Caja – Marmoset; Otga – Bushbaby; Bota – Cow; Ovar – Sheep; Susc – Pig; Mupu – Ferret; Aime – Giant panda; Calu – Dog; Eqca – Horse; Mylu – Little brown myotis; Ptal – Black flying fox; Loaf – Elephant; Orcu – European rabbit; Crgr – Chinese hamster; Mumu – Mouse; Rano – Rat; Ictr – Squirrel; Capo – Guinea pig. </p

Maximum likelihood (ML) phylogenetic trees of mammalian MDA5 gene used for codon-based ML analysis.

<p>When testing mammalian MDA5 alignment for recombination, one significant breakpoint was detected at nucleotide position 903. (A) A phylogenetic tree was reconstructed for the first 903 nucleotides under the nucleotide substitution model TIM3+G. (B) A second ML tree was inferred for the remaining 2211 nucleotides and under the nucleotide substitution model TIM3+I+G. (C) A tree was also reconstructed for MDA5 total alignment without recombination testing and under the nucleotide substitution model GTR+G. Bootstrap values >50 are indicated on the branches. </p