Structural studies of HTLV-1 reverse transcriptase for antiviral drug design
Human T-cell leukaemia virus type 1 (HTLV-1) is a major problem in highly endemic regions and is the causative agent for adult T-cell leukaemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. To date, no cure is available for either of these diseases, yet prevention of HTLV-1 infection has proven to be a viable option. The HTLV-1 reverse transcriptase (RT) enzyme provides an interesting drug target due to its vital function in the viral replication. In this thesis, we aim to obtain the structure of HTLV-1 RT via single-particle cryogenic electron microscopy (cryo-EM) and gain insight into the mechanisms it employs to perform its DNA polymerase function. In the first part, an extensive analysis was conducted on phylogeny, gene ontology, and homology, where we found HTLV-1 RT to have great similarity to both human immunodeficiency virus type 1 RT and human endogenous retrovirus K RT. Following this, a physiologically relevant model of HTLV-1 RT in complex with a nucleic acid dimer was constructed and used in an antiviral compound docking study. In the second part, multiple plasmids were designed to express HTLV-1 RT in various forms and were transformed into multiple Escherichia coli (E. coli) strains for recombinant protein expression. After induction, HTLV-1 RT was isolated using nickel-histidine affinity chromatography. Here, the recombinant protein yield remained very low, which prompted us to perform an in-depth analysis of the HTLV-1 RT encoding mRNA. Via RT-qPCR, significant variation in mRNA expression levels between constructs was observed, but the presence of HTLV-1 RT encoding mRNA was confirmed. In silico RNA folding of the HTLV-1 RT mRNA suggested the occurrence of a highly stable GC-rich hairpin loop at the 5’ end of the mRNA. In conclusion, we substantiated the link between HIV-1 RT and HTLV-1 RT necessary to validate the effort in
screening anti-HIV-1 RT compounds against HTLV-1 RT. Furthermore, by docking these compounds into a physiologically relevant in silico model of HTLV-1 RT, we showed that some of those would favourably bind to HTLV-1 RT’s polymerase active site with sub-nanomolar affinity. Additionally, we successfully expressed and isolated HTLV-1 RT in low quantities. This low yield is most likely explained by the presence of a highly stable GC-rich hairpin loop at the 5’ end of the mRNA, reducing the ribosomes' binding capacity for successful translation. Although we succeeded in expressing HTLV-1 RT, higher quantities are required to perform structural studies. In the future, once sufficient protein is obtained, a high-resolution model of HTLV-1 RT can be constructed, which will allow for high-throughput drug screening and the development of HTLV-1 RT-specific drugs, for which there is a great need.
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