Viruses is unclear. Our studies also showed that the envelope protein
Viruses is unclear. Our studies also showed that the envelope protein of SRV2 was very stable suggesting a high degree of adaptation of SRV-2 to its host. In all likelihood, simian retroviruses have evolved very slowly in geographically distinct macaque species, and only the physical contact with otherPage 12 of(page number not for citation purposes)Virology Journal 2006, 3:http://www.virologyj.com/content/3/1/To search for SRV-2 variants in the same animal, a 439 bp fragment of the env PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26866270 gene was PCR-amplified from genomic DNA of two macaques (T82422, F91249) using Platinum Taq polymerase and the primers: SRV2-env 1a (5′-GAGTGCTGGCTATGCTTACCATC-3′) and SRV2-env b (5′-CAGTTGAGACGGCAGTGGTT-3′). The fragments were subcloned into the pCR-Blunt vector (Invitrogen Life Technologies, Carlsbad CA) using standard procedures. Eight clones were obtained from each animal for sequence analysis. Sequencing was performed on an ABI model 310 automated sequencer with Prism Big Dye terminator cycle sequencing ready reaction kit with Amplitaq DNA polymerase FS (Applied Biosystems). DNA sequences were analysed using Sequencher 4.1.4 (GeneCodes).Sequence analysis Multiple nucleotide and encoded amino acid alignments were done with Vector NTI 9.0.0 (InforMax) based on the ClustalW algorithm (EMBL, Heidelberg, Germany) or with the ClustalW program [55] directly. The location of the signal peptide was determined by SignalP 3.0 [56], SigCleave [57], and iPSORT [58]. The location of transmembrane domains was determined with MEMSAT2 [59] and Tmpred [60]. Putative glycosylation sites were determined with NetNGLyc 1.0 [61]. Three-dimensional structure analysis The program 3D-PSSM [62] was used to identify proteins with structural similarities to the SRV2 env prototype (D2/ CEL/OR). The structures for the six highest scoring proteins were obtained from the Molecular Modeling Database (MMDB)[63] which contains experimentally determined three-dimensional biomolecular structures obtained from the Protein Data Bank. Matching structures were aligned with SRV2 env using NCBI’s 3D-structure viewer Cn3D v4.1 [64]. Using NCBI’s vector alignment search tool (VAST)[65], we determined further protein structure neighbors by direct comparison of LixisenatideMedChemExpress Lixisenatide 3-dimensional protein structures stored in MMDB. This allowed us to further evaluate possible structural and functional domains of the SRV2 env protein. Phylogenetic analysis Multiple sequence alignments were performed with ClustalW [55]. Phylogenetic analysis of amino acid sequences was done with protein maximum-likelihood (ProML) method from the PHYLIP package version 3.62 of phylogenetic analysis programs (.)[66] (University of Washington, Seattle). Bootstrap analysis was performed using the programs Seqboot and Consense from the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25681438 PHYLIP package. The treefiles were displayed with TreeView [67]. Identical tree topologies were obtained by using neighborjoining analysis of the protein distance matrices and by aparsimony method as implemented in the PHYLIP package. All of the major branch points were strongly supported by boot-strap analysis and statistical evaluations within ProML.Competing interestsThe author(s) declare that they have no competing interests.Authors’ contributionsJP-S carried out the sequence analysis, the three-dimensional structure analysis, the phylogenetic analysis, as well as the cloning studies, participated in the PCR and cloning efforts, and drafted the manuscript. TM participated in the PCR and cloning efforts. AG.
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