Prior studies have shown that the K65R mutation in HIV-1 RT decreases susceptibility to various NRTIs, including ddC, PMEA, PMPA, d-
DXG, (+/-)dOTC, d-
d4FC, and d4T (2
). Our study was the first to systemically analyze the structural components of NRTIs that influence activity against HIV-165R
by the use of the same susceptibility assays. We found that all d
and acyclic NRTIs tested had significantly reduced activity against HIV-165R
except those containing a 3′-azido moiety (AZT and AZA). In addition, the structural features of NRTIs that influence activity were identified.
The pseudosugar structure had the greatest impact on activity against HIV-165R
. Comparing cytidine analogs that differed only by pseudosugar structure, the greatest loss of activity was seen with (+)-3TC (77-fold), whereas the least was seen with ddC (5.0-fold). The rank order of greatest to least loss of activity against HIV-165R
or (+) cytidine pseudosugars is as follows: 3TC
FTC > dOTC > DOC > d4C > ddC. The fold resistance for 3TC was significantly (P
≤ 0.05) different from DOC, d4C and ddC results, with a trend towards significance for dOTC (P
= 0.06). The fold resistance results for dOTC and DOC were also significantly (P
< 0.05) different from ddC results. A similar pattern was seen for different pseudosugars with other base components, although the differences in severalfold resistance were not significant. For example, DXG showed greater loss of activity than ddG (6.0-fold versus 4.0-fold), as did d4T (3.8-fold) compared with ddT (2.2-fold). A conclusion cannot be made for DXT (data not shown), because this compound was poorly active in both P4/R5 cells and MT-2 cells.
The role of HIV-165R
in resistance to d4T has not been well defined. In our study, HIV-165R
did exhibit significantly reduced susceptibility to d4T in P4/R5 cells (3.8-fold; P
< 0.001) and in MT-2 cells (2.1-fold; P
< 0.05). The recent report by J. G. Garcia-Lerma indicates that 65R can be selected by D4T in vitro (5
). In addition, 65R has been reported to be selected in two clinical trials of d4T-containing regimens that excluded tenofovir. In the Gilead 903 study, two patients (0.7%) who failed d4T/3TC/efavirenz therapy had virus with a 65R mutation (4
), and in a Danish study, five out of eight patients who failed d4T/ddI/ABC therapy had virus with a 65R mutation (14
). Collectively, these data support the view that the 65R mutation reduces susceptibility to D4T and that this reduction in susceptibility can be clinically relevant.
As noted above, (+)- or (−)-3TC and (+)- or (−)-FTC showed the greatest loss of activity against HIV-165R
. In addition, a greater than 10-fold loss of activity was also observed for most of the dioxolane compounds and the dioxathiolane dOTC. This suggests that the 65R mutation in RT preferentially interferes with incorporation of analogs containing pseudosugars with 3′ oxygen or sulfur components. The 3′ oxygen or sulfur, which is larger and more negatively charged than the normal 3′ carbon, may distort the positioning of the analog triphosphates to a greater extent than for other pseudosugars. Further, the 3′ hydroxyl of the incoming dNTP makes a stabilizing intramolecular hydrogen bond with one oxygen of the β-phosphate (15
). The lack of the 3′ hydroxyl group and the negatively charged oxygen or sulfur at the 3′ position is likely to further distort the configuration of the analog triphosphate. As a consequence, natural dNTPs are likely to be much more efficiently incorporated by HIV65R
RT than 3′ oxygen- or sulfur-substitution analogs, leading to high-level resistance to these analogs.
Our results show that the base component has a moderate effect on NRTI activity against HIV-165R
. Comparing compounds that were structurally identical except for base component shows that the rank order of greatest to least loss of activity is C > G > A > T. For example, the rank order of activity loss for the dideoxy compounds is ddC > ddG > ddI > ddT. However, only the differences in activity of ddC versus ddI or ddT are statistically significant (P
= 0.02). Although not all the possible variations of base component for other pseudosugar structures are active against HIV-1, a similar pattern is apparent for the active analogs tested. Specifically, the loss of activity was greater for d-
DOC than for d-
= 0.02), for FLG than for FLT (P
= 0.04), and for d4C than for d4T (P
= 0.13). Biochemical studies have showed a similar trend with HIV65R
RT, showing greater discrimination against ddCTP than against ddTTP (15
). The influence of base component on activity against HIV-165R
is unexplained but may be mediated through differences in binding affinity or position of the analog in the substrate binding site.
Stereochemical differences (l- versus d-enantiomer) did not influence NRTI activity against HIV-165R. Six sets of compounds that differed only by stereochemistry were tested: (+)- and (−)-3TC, (+)- and (−)-FTC, l- and d-DOFC, l- and d-dOTC, l- and d-DOC, and l- and d-d4FC. The loss of activity was not significantly (P > 0.5) different for any enantiomeric pair. One hypothesis to explain this is that altering the enantiomer from d to l does not further affect the position of the 5′ triphosphates of the analog, which are linked to the pseudosugar by flexible single bonds.
AZT and AZA showed no loss of activity against HIV-165R
, whereas AZG and AZC showed a trend toward a significant loss of activity (2.0- and 2.5-fold decrease, respectively). Thus, the base component has a role in the activity of the 3′-azido-containing compounds, with C and G analogs showing a greater loss of activity than A or T analogs, as was observed with other pseudosugar structures. The relative preservation of activity of 3′-azido analogs may be explained by interaction of the 3′-azido with the β- and γ-phosphates of the analog triphosphate, restoring the correct configuration of the analog for incorporation. RT with a 65R mutation has reduced excision capability compared to the wild type, allowing AZT to remain incorporated (11
; U. Parikh, D. Koontz, N. Sluis-Cremer, J. Hammond, L. Bacheler, R. Schinazi, and J. Mellors, Abstr. 11th Conf. Retrovir. Opportunistic Infect., abstr. 54, 2004). The decreased excision capacity of 65R may help explain the preservation of activity of compounds with 3′-azido groups against HIV-165R
. The use of AZT in combination with other NRTIs is likely to prevent the emergence of HIV-165R
, because this substitution does not confer a selective advantage for the virus in the presence of AZT. Recent clinical data support this hypothesis (9
; R. Elion, C. Cohen, E. DeJesus, R. Redfield, J. Gathe, R. Hsu, L. Yau, L. Ross, B. Ha, R. Lanier, T. Scott, and the COL40263 Study Team, Abst. 11th Conf. Retrovir. Opportunistic Infect., abstr. 53, 2004).
In summary, the NRTI structures that retain activity against HIV-165R are those having a thymine or adenine base and a 3′-azido component. NRTIs with the 3′ carbon replaced by a sulfur or oxygen have the least activity in either the d or l conformation. The data presented provide evidence that 65R is a multi-NRTI-resistance mutation and support the use of AZT in drug combinations to treat patients with HIV-165R and to prevent its emergence.