Construction of A Preliminary Three-Dimensional Structure Simian betaretrovirus Serotype-2 (SRV-2) Reverse Transcriptase Isolated from Indonesian Cynomolgus Monkey

Abstract

Simian betaretrovirus serotype-2 (SRV-2) is an important pathogenic agent in Asian macaques. It is a potential confounding variable in biomedical research. SRV-2 also provides a valuable viral model compared to other retroviruses which can be used for understanding many aspects of retroviral-host interactions and immunosuppression, infection mechanism, retroviral structure, antiretroviral and vaccine development. In this study, we isolated the gene encoding reverse transcriptase enzyme (RT) of SRV-2 that infected Indonesian cynomolgus monkey (Mf ET1006) and predicted the three dimensional structure model using the iterative threading assembly refinement (I-TASSER) computational programme. This SRV-2 RT Mf ET1006 consisted of 547 amino acids at nucleotide position 3284-4925 of whole genome SRV-2. The polymerase active site located in the finger/palm subdomain characterised by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165 and Asp166. We estimated that this SRV-2 RT Mf ET1006 structure has the accuracy of template modelling score (TM-score 0.90 ± 0.06) and root mean square deviation (RMSD) 4.7 ± 3.1Å, indicating that this model can be trusted and the accuracy can be seen from the appearance of protein folding in tertiary structure. The superpositionings between SRV-2 RT Mf ET1006 and Human Immunodeficiency Virus-1 (HIV-1) RT were performed to predict the structural in details and to optimise the best fits for illustrations. This SRV-2 RT Mf ET1006 structure model has the highest homology to HIV-1 RT (2B6A.pdb) with estimated accuracy at TM-score 0.911, RMSD 1.85 Å, and coverage of 0.953. This preliminary study of SRV-2 RT Mf ET1006 structure modelling is intriguing and provide some information to explore the molecular characteristic and biochemical mechanism of this enzyme.

Keywords: 3D Structure Model; Reverse Transcriptase; SRV-2 Indonesian Isolates.

Figure 1

PCR amplification to SRV-2 RT gene isolated from Indonesian cynomolgus monkeys (SRV-2 RT Mf ET1006) resulting 1641 bp amplicon. (M) 100 bp DNA ladder (Invitrogen), (1) SRV-2 RT Mf ET1006, (2) SRV-2/A549 positive control (obtained from National Primate Research Centre, University of Washington, USA).

Figure 2

Phylogenetic tree of SRV-2 RT Mf ET1006 amino acids sequences relatedness to others retroviruses constructed using the neighbor-joining method. The bootstrap percentages of 500 replicate are shown at the branch nodes. The evolutionary distances are estimated by branch lengths of the tree indicated and drawn to scale, which represents the number of base substitutions per site.

Figure 3

Amino acid sequences alignment and secondary structural motifs prediction of the SRV-2 RT Mf ET1006 and HIV-1 RT. Amino acid sequence of SRV-2 RT Mf ET1006 from the position 1–547 and HIV-1 RT from 18–553 were aligned based on sequences and structural motifs using the programs ClustalW, STRAP2 and I-TASSER. The motifs were indicated as α-helixes (H), β-sheets (S), and coils (C). The amino acids active site was indicated in red and highlighted red, the DNA binding sequences were indicated in green, dNTPs binding site were indicated in blue, NNRTI binding site were indicated in brown, RNA/DNA hybrid (RNase H) were indicated in purple and highlighted purple. Meanwhile, the YXDD sequences were over-scored with a red line. The boundaries of the Finger/Palm, Thumb, Connection and RNase H were shown with a vertical line.

Figure 4

(A) Three-dimensional structure model of the full-length SRV-2 RT Mf ET1006. The models were generated using I-TASSER method and visualised using Pymol program. The estimated accuracy of model is 0.90 ± 0.06 (TM-score), 4.7 ± 3.1Å (RMSD) and C-score = 1.33. The fingers/palm, thumb, connection and RNaseH subdomains are coloured in dark red, purple, dark blue and green, respectively. Light blue spheres represent the residues of the polymerase active site and dNTPs binding site. Yellow spheres represent the YXDD motif in the finger/palm. Meanwhile, the brown spheres indicated the DED motif of RNaseH active site. (B) Superpositioning model between SRV-2 RT Mf ET1006. with HIV-1 RT (2B6A.pdb), estimated accuracy is 0.911 (TM-score), 1.85 Å (RMSD), and 0.953 (coverage).

Figure 5

Ribbon diagram of the SRV-2 RT Mf ET1006 bound to a stick model of an 18–19-base RNA/DNA duplex oligonucleotide substrate. The various domains of the enzyme were colour-coded as presented in Fig. 4. The active site amino acids of the DNA polymerase and RNase H are shown in yellow spheres.

Figure 6

Interaction of amino acids that play roles in polymerase site active of SRV-2 RT Mf ET1006 with terminal portion of the RNA/DNA duplex. DNA was indicated with red and blue stick, while RNA in red, white and blue stick, signed with −1, −2 and −3 as duplex RNA/ DNA; overhang RNA was indicated by +1 as round edges stick. (A) SRV-2 RT Mf ET1006 site active is represented in green surface with conserved amino acids Asp165, Gln131, Gly132 and Arg58 (shown in green stick); (B) SRV-2 RT Mf ET1006 amino acids site active represented in green stick.