Abstract
Background
Human T-cell leukemia virus-1 (HTLV-1) was the first identified human retrovirus and was shown to be associated with diseases such as adult T-cell leukemia lymphoma and tropical spastic paraparesis/HTLV-1 associated myelopathy. Retroviral proteases (PRs) are essential for viral replication by processing viral Gag and Gag-(Pro)-Pol polyproteins during maturation. Full-length HTLV-1 PR is 125 residues long; whether the C-terminal region is required for catalytic activity is still controversial. In this study, we characterized the effect of C-terminal amino acids of HTLV-1 PR for PR activity and examined the binding of compounds identified by in silico screening. One compound showed inhibition against the virus in infected cells.
Methods
Truncated (116-, 121- and 122-residue) forms of HTLV-1 PR were prepared and proteins from expression of the genes were purified. In silico screening was performed by docking small molecules into the active site of HTLV-1 PR. The kinetic constants k cat , K m , k cat /K m and inhibition constants K i for inhibitors identified by the computational screening were determined. Western blot and ELISA analyses were used to determine the effect of the most potent PR inhibitors on HTLV-1 protein processing in infected cells.
Results
The constructs showed similar catalytic efficiency constants (k cat /K m ); thus HTLV-1 PR C-terminal amino acids are not essential for full activity. Computational screening revealed new PR inhibitors and some were shown to be inhibitory in enzyme assays. In HTLV-1-infected cells, one of the small molecules inhibited HTLV-1 gag cleavage and decreased the amount of HTLV-1 p19 produced in the cells.
Conclusions
We have identified an HTLV-1 PR inhibitor that is biologically functional. Inhibitor screening will continue to develop possible drugs for therapy of HTLV-1 infection.