Combinations of mutations in the connection domain of human immunodeficiency virus type 1 reverse transcriptase: assessing the impact on nucleoside and nonnucleoside reverse transcriptase inhibitor resistance

Abstract

Recent reports have described the effect of mutations in the connection and RNase H domains of reverse transcriptase (RT) on nucleoside and nonnucleoside reverse transcriptase inhibitor (NRTI and NNRTI, respectively) resistance in the presence of thymidine analog resistance mutations (TAMs) and NNRTI mutations (J. H. Brehm, D. Koontz, J. D. Meteer, V. Pathak, N. Sluis-Cremer, and J. W. Mellors, J. Virol. 81:7852-7859, 2007; K. A. Delviks-Frankenberry, G. N. Nikolenko, R. Barr, and V. K. Pathak, J. Virol. 81:6837-6845, 2007; G. N. Nikolenko, K. A. Delviks-Frankenberry, S. Palmer, F. Maldarelli, M. J. Fivash, Jr., J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 104:317-322, 2007; G. N. Nikolenko, S. Palmer, F. Maldarelli, J. W. Mellors, J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 102:2093-2098, 2005; and S. H. Yap, C. W. Sheen, J. Fahey, M. Zanin, D. Tyssen, V. D. Lima, B. Wynhoven, M. Kuiper, N. Sluis-Cremer, P. R. Harrigan, and G. Tachedjian, PLoS Med. 4:e335, 2007). In the present study, novel mutations in the connection domain of RT (T369I/V), first identified in patient-derived viruses, were characterized, and their effects on NNRTI and NNRTI susceptibility were determined. Furthermore, the effect of N348I on NRTI and NNRTI resistance was confirmed. HIV-1 with either N348I or T369I/V demonstrated reduced susceptibility to nevirapine (NVP), efavirenz (EFV), delaviridine (DLV), and zidovudine (ZDV) compared to wild-type HIV-1. However, HIV-1 with T369I and N348I demonstrated 10- to 60-fold resistance to these same drugs. In clinical samples, these two connection domain RT mutations were predominantly observed in viruses containing TAMs and NNRTI mutations and did not alter the susceptible-resistant classifications of these samples. Introduction of T369I, N348I, or T369I/N348I also reduced replication capacity (RC). These observations suggest that it may be of scientific interest to test these mutations against new NNRTI candidates.

FIG. 1.

Schematic representation of RH/IN resistance test vectors. RH/IN RTVs were assembled by cloning pol sequences encoding the C terminus of RT (codons 317 to 426), RNase H (RH), and IN from individual patient viruses into a HIV-1 (NL4-3) vector containing a luciferase expression cassette inserted into the env region.

FIG. 2.

NNRTI and ZDV susceptibility of RH/IN RTVs derived from 154 patient viruses. The distribution of EC₅₀ FCs of 154 RTVs containing patient-derived RH/IN sequences is shown for EFV, NVP, DLV, and ZDV. One, 2, 6, and 13 of the 154 samples demonstrated reduced susceptibility to DLV, EFV, NVP, and ZDV, respectively. The horizontal dotted lines represent the biological cutoff for susceptibility to each drug (22).

FIG. 3.

Replication capacity of RH/IN RTVs containing site-directed mutations in the C terminus of RT. The data are shown as mean values obtained from the results of three independent experiments, and the error bars represent the standard errors of measurement (SEM). Replication capacity is expressed as a percentage of that of the wild-type reference virus (defined as 100%).