Computational protocol: Structural Analysis of Monomeric RNA Dependent Polymerases: Evolutionary and Therapeutic Implications

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[…] The RdRp crystal structures employed in this study include: human rhinovirus polymerase (PDB:1XR7); poliovirus (PDB:3OL6); human enterovirus 71 (PDB:3N6L); foot-and-mouth disease virus (PDB:1U09); encephalomyocarditis virus (PDB:4NZ0); Norwalk virus (PDB:3BSO); rabbit hemorrhagic disease virus (PDB:1KHV); Sapporo virus (PDB:2CKW); Japanese encephalitis virus (PDB:4HDH); dengue virus (PDB:2J7U); bovine viral diarrhea virus (PDB:1S48); hepatitis C virus (PDB:1C2P); Qβ-phage (PDB:3MMP); infectious pancreatic necrosis virus (PDB:2YI9); infectious bursal disease virus (PDB:2PUS); φ6 phage (PDB:1HHS); reovirus (PDB:1MUK); simian rotavirus (PDB:2R7Q); human immunodeficiency virus type 1 (PDB:3DLK); moloney murine leukemia virus (PDB:4MH8); human immunodeficiency virus type 2 (PDB:1MU2); Tribolium castaneum telomerase (PDB:3KYL); bat influenza A virus (PDB:4WSB); human influenza B virus (PDB:4WRT); LaCrosse virus (PDB:5AMQ).Pairwise structural comparisons between the different RdRps and RTs mentioned above were performed with the Secondary Structure Matching (SSM) program [] included in the PDBe web server. In the case of reverse transcriptases, both the connection and the RNase H domains were deleted for the comparisons. All the crystals have a resolution of 3 Å or higher. The results of each set of comparisons allowed the construction of a matrix that included the number of residues in each of the structures, the Root-mean Square Deviation (RMSD), and the number of aligned residues.A geometric distance measure was then estimated for each of the comparisons using the Structural Alignment Score [], which is calculated according to the following formula: (RMSD x 100)/number of aligned residues.The program FITCH, included within the PHYLIP package, was used to transform the geometric distance into an evolutionary distance and FigTree (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize the resulting tree. [...] The search for homologs of the EBOV polymerase and the three-dimensional structure modeling of the EBOV RdRp domain were performed using the PHYRE web server version 2.0 []. One Zaire Ebola virus L protein sequence (Sierra Leona, Makona-G3686.1; AIE11922) from the current outbreak was downloaded from NCBI’s Viral Genome Resource (http://www.ncbi.nlm.nih.gov/genome/viruses/). The sequence was edited according to the information provided by the Conserved Domain Database [], leaving only the fragment of the sequence which corresponds to the entry “Mononegavirales RNA dependent RNA polymerase” (CDD 250248). This edited fragment of the protein was used as the PHYRE version 2.0 server query sequence.The three-dimensional model and its images were edited with Chimera 1.8 []. [...] Secondary structure-based multiple sequence alignments were built using the PROMALS3d web server []. For each EBOV species, one L protein sequence was randomly chosen with the exception of the Zaire EBOV, for which two sequences were chosen, one from the 1976 Yambuku-Mayinga outbreak and another one from the current outbreak. The accession codes for the sequences employed here are listed: Reston Ebola virus: NP_690587.1; Sudan Ebola virus: YP_138527; Tai Forest Ebola virus: YP_003815431; Bundibugyo Ebola virus: YP_003815440; Zaire Ebola virus 1976 Yambuku-Mayinga outbreak: NP_06625; Zaire Ebola virus 2014 sample ManoRiver-G3823: AIG96450.For the Mononegavirales multiple sequence alignment, the different L-protein “Mononegavirales RNA-dependent RNA polymerase” (CDD 250248) domains from the various genera belonging to the Mononegavirales families were chosen. A total of 52 sequences were included for the alignment. These are listed below, and include the Borna disease virus: NP_042024; Lloviu cuevavirus: YP_004928143; Marburg marburgvirus: YP_001531159.1; avian metapneumovirus: YP_443845; avian paramyxovirus 6: NP_150063; Beilong virus: YP_512254; bovine parainfluenza virus 3: NP_037646; bovine respiratory syncytial virus: NP_048058; canine distemper virus: NP_047207; dolphin morbillivirus:NP_945030; fer-de-lance virus:NP_899661; human metapneumovirus: YP_012613; human parainfluenza virus 1:NP_604442; human parainfluenza virus 2:NP_598406; human parainfluenza virus 3:NP_067153; human respiratory syncytial virus:NP_056866; Mapuera virus:YP_001249278; measles virus:NP_056924; mumps virus:NP_054714; Newcastle disease virus B1:NP_071471; parainfluenza virus 5:YP_138518; peste-des-petits-ruminants virus:YP_133828; pneumonia virus of mice J3666:YP_173335; porcine rubulavirus:YP_001331035; rinderpest virus: YP_087126; Sendai virus: NP_056879; simian virus 41:YP_138510; Australian bat lyssavirus:NP_478343; bovine ephemeral fever virus:NP_065409; European bat lyssavirus 1:YP_001285392; Hirame rhabdovirus: NP_919035; infectious hematopoietic necrosis virus:NP_042681; lettuce yellow mottle virus: YP_002308376; maize Iranian mosaic virus:YP_002308459; maize fine streak virus: YP_052849; Mokola virus:YP_142354; northern cereal mosaic virus:NP_597914; potato yellow dwarf virus:YP_004927971; rabies virus:NP_056797; rice yellow stunt virus:NP_620502; snakehead virus:NP_050585; sonchus yellow net virus:NP_042286; spring viraemia of carp virus:NP_116748; taro vein chlorosis virus:YP_224083; vesicular stomatitis Indiana virus:NP_041716; viral hemorrhagic septicemia virus Fil3:NP_049550. […]

Pipeline specifications

Software tools PHYLIP, FigTree, Phyre, PROMALS3D
Databases CDD PDBe
Applications Phylogenetics, Amino acid sequence alignment
Diseases Hemorrhagic Fever, Ebola
Chemicals Aspartic Acid, Lamivudine