Total chemical synthesis of human T-cell leukemia virus type 1 protease via native chemical ligation

Human T-cell leukemia virus type 1 (HTLV-1) – the first human retrovirus discovered by Gallo and colleagues in the late 1970s,1 is clinically associated with adult T-cell leukemia, tropical spastic paraparesis or HTLV-1 associated myelopathy, and several other chronic diseases.2–5 It is estimated that 20–30 million of the world population is infected with HTLV-1, and 5–10% of these infected individuals will develop HTLV-1 associated diseases.6,7 No effective treatment is currently available for HTLV-1 infection, making the virus a dangerous emerging pathogen as classified by CDC. Virally encoded proteases (PR) play an essential role in the life cycle of all retroviruses as viral assembly, maturation and replication necessitate proteolytic cleavage of Gag and Gag-Pro-Pol polyproteins into structural (matrix, capsid, and nucleocapsid) as well as functional (reverse transcriptase and integrase) proteins.8 This, along with the successful development of HIV-1 PR inhibitors for the treatment of AIDS, validates HTLV-1 PR as an attractive viral target for therapeutic intervention in HTLV-1 infection. HTLV-1 PR of 125 amino acid residues and HIV-1 PR of 99 amino acid residues are members of the aspartic acid protease family, and function as a structurally conserved homodimer.9 Aside from their significantly different substrate specificity and inhibition profile due to relatively low sequence identity,10 the two viral proteases differ in the C-terminal region. HTLV-1 PR C-terminally is elongated by 10 amino acid residues (P116EAKGPPVIL125) as compared with HIV-1 PR – an attribute shared only by leukemia virus proteases.11 Hayakawa et al. first reported in 1992 that while the very last five residues (P121PVIL125) were functionally dispensable, residues 116–120 (P116EAKG120) were required for the auto-processing activity of HTLV-1 PR.12 However, sequence alignment, structure modeling, and functional characterizations of truncation mutants of HTLV-1 PR suggest that the extra C-terminal residues exist as a flexible tail distal to the active site of the enzyme, thus playing limited structural or functional roles.10,12,13 This tenet recently received support from structural studies by Wlodawer and colleagues of a truncated HTLV-1 PR (1–116), which retained 60% of the catalytic activity of its full-length counterpart.14 Nevertheless, the controversy still remains as evidenced by a latest report in 2008 that deletion of residues 116–125 or 117–125 in HTLV-1 PR rendered the protease completely inactive.15 The authors attributed the lack of activity of HTLV-1 PR (1–115) or HTLV-1 PR (1–116) to its inability to dimerize. These conflicting findings triggered us to carry out comparative functional studies of a full-length protease and its C-terminally truncated form HTLV-1 PR (1–116), both of which were chemically synthesized using the native chemical ligation (NCL) technique pioneered by Kent and colleagues.16 Stepwise chemical syntheses of active bovine leukemia virus PR (1–126) and (1–116) were reported in 1992 and 1993,17,18 followed by the synthesis of HTLV-1 PR (1–125) first in 1997,19 and then in 2002.20 However, no comparative activity data were generated for those synthetic enzymes to allow the delineation of the functional effect of C-terminal truncation. Using three different peptide substrates, we demonstrated in this report that truncation of the extra C-terminal residues of HTLV-1 PR reduced the catalytic efficiency of the viral protease by tenfold and lowered its turnover number by twofold.