Impact of novel human immunodeficiency virus type 1 reverse transcriptase mutations P119S and T165A on 4'-ethynylthymidine analog resistance profile

Abstract

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a derivative of stavudine (d4T), has potent activity against human immunodeficiency virus and is much less inhibitory to mitochondrial DNA synthesis and cell growth than its progenitor, d4T. 4'-Ed4T triphosphate was a better reverse transcriptase (RT) inhibitor than d4T triphosphate, due to the additional binding of the 4'-ethynyl group at a presumed hydrophobic pocket in the RT active site. Previous in vitro selection for 4'-Ed4T-resistant viral strains revealed M184V and P119S/T165A/M184V mutations on days 26 and 81, respectively; M184V and P119S/T165A/M184V conferred 3- and 130-fold resistance to 4'-Ed4T, respectively. We investigated the relative contributions of these mutations, engineered into the strain NL4-3 background, to drug resistance, RT activity, and viral growth. Viral variants with single RT mutations (P119S or T165A) did not show resistance to 4'-Ed4T; however, M184V and P119S/T165A/M184V conferred three- and fivefold resistance, respectively, compared with that of the wild-type virus. The P119S/M184V and T165A/M184V variants showed about fourfold resistance to 4'-Ed4T. The differences in the growth kinetics of the variants were not more than threefold. The purified RT of mutants with the P119S/M184V and T165A/M184V mutations were inhibited by 4'-Ed4TTP with 8- to 13-fold less efficiency than wild-type RT. M184V may be the primary resistance-associated mutation of 4'-Ed4T, and P119S and T165A are secondary mutations. On the basis of our findings and the results of structural modeling, a virus with a high degree of resistance to 4'-Ed4T (e.g., more than 50-fold resistance) will be difficult to develop. The previously observed 130-fold resistance of the virus with P119S/T165A/M184V to 4'-Ed4T may be partly due to mutations both in the RT sequence and outside the RT sequence.

FIG. 1.

Chemical structures of d4T and 4′-Ed4T.

FIG. 2.

Relationship of dTTP to the side chains of P119, T165, and M184 in the HIV-1 RT ternary complex. (A) Overall structure; (B) close-up view of the dNTP binding site. The catalytic P66 subunit, the noncatalytic P51 subunit, the template, and the primer strands of the DNA duplex are shown in gold, yellow, cyan, and green, respectively. The distance of C-4′ of dTTP, which is the attachment site of 4′-Ed4T, to the carbon of M184 of P66, which we previously described as possibly having a role in interactions with 4′-Ed4T, is 6.7 Å (50).