Development of serotype-2 simian betaretrovirus reverse transcriptase recombinant enzyme isolated from indonesian cynomolgus monkey

Abstract

Reverse transcriptase (RT) is a multifunctional enzyme that catalyzes the formation of double-stranded DNA from single-stranded RNA viral genomes. Reverse transcription is an essential and critical step in life cycle of all retroviruses replication. RT has become an important enzyme in molecular biology, genetics and medicine for the synthesis of complementary DNA (cDNA) from messenger RNA (mRNA). The application of this enzyme combining with PCR amplification technique (RT-PCR) has expanded our knowledge of numerous cellular regulatory mechanisms and has become a gold standard to facilitate the coding region cloning of any gene of interest. The development of biotechnology has been relying on the availability of this efficient RT. In biomedical fields, RT of human immunodeficiency virus (HIV), the virus causing acquired immunodeficiency syndrome (AIDS) has been the most studied enzyme due to the prime target for the development of antiretroviral drug therapy of HIV/AIDS. Simian betaretrovirus serotype-2 (SRV-2) is the causative agent of simian acquired immunodeficiency syndrome (SAIDS) in Asian macaques with varying severity. While SRV is an important pathogen in Asian macaque and causes a potential confounding variable in biomedical research, SRV also provides a valuable viral model to compare with other retrovirals for a better understanding of aspects of retroviral–host interactions, infection mechanism, retroviral structure, antiretroviral and vaccine development. As is characteristic of all retroviruses, SRV-2 can transcribe its own RNA genome into a double-stranded DNA by the action of reverse transcriptase enzyme. The presence of gene encoding RT enzyme in SRV-2 genome potentially be studied and utilized further as RT enzyme model for developing the anti RT drug of others retrovirus. On the other hands, the gene encoding SRV-2 RT can be isolated and cloned to produce RT recombinant enzyme and applied in molecular biology techniques. In this study, we isolated the gene encoding RT enzyme of SRV-2 that infected Indonesian cynomolgus monkey and predicted the three dimensional (3D) structure model using I-TASSER (Iterative Threading ASSEmbly Refinement) computational program and visualized with PyMol program. Gene expression was constructed in Escherichia coli expression system with Gateway technology methods using pENTR/SD/TOPO entry vector and pDEST17 destination vector. Purification was performed using Ni2+-NTA affinity chromatography. Expression was analyzed using SDS-PAGE and Western blot techniques; meanwhile enzyme activity was measured using RT colorimetric assay and RT-PCR technique. We isolated the gene encoding RT enzyme of SRV-2 Indonesian isolate at nucleotide position 3284-4925 consisted of 547 amino acids. The I-TASSER program has predicted the three dimensional structure model of RT SRV-2 enzyme that consisted of palm/finger, thumb, connection and RNase H domain. The polymerase active site located in the finger/palm subdomain characterized by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165, and Asp166. This SRV-2 RT structure model has the highest homology to HIV-1 RT (2B6A.pdb). Meanwhile, the structure of SRV-2 RNase H closely related to its ortologs such as Escherichia coli RNase H (1GOA) and HIV-1 RNase H (3QIO, 3HYF). The catalytic active site of SRV-2 RNase H contains three amino acid carboxylateresidues as Asp436, Glu465 and Asp486. Analysis of expression using SDS PAGE and Western blot techniques showed a specific band of 64.9 kDa, indicating SRV-2 RT recombinant enzyme. Purification of SRV-2 RT recombinant enzyme produced 312 μg mL-1 protein with 7.1 Unit (U) μL-1 enzyme activities. Application of this recombinant enzyme in reverse transcription-PCR (RT-PCR) of -globin and -actin genes produced DNA fragments of 206 base pairs (bp) and 350 bp, indicating amplification of - globin and -actin genes, respectively. Therefore, the expressed SRV-2 RT enzyme was proven to be functional, although the activity was low.