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Session 120
Poster Abstracts Interactions among RTI Resistance Mutations Wednesday, 1:30 - 3:30 pm Hall A |
Background: L100I arises during efavirenz
(EFV) therapy and adds to the resistance of K103N. In early studies,
K103N+L100I was rarely present in patients failing
EFV. In ACTG 368, when present, K103N+L100I was
frequently associated with L74V, despite the lack of concomitant nucleoside (NRTI)
therapy. We postulated that L74V was selected for in ACTG 368 because it
compensated for the fitness defect of K103N+L100I. We performed growth
competition experiments in H9 cells in the absence of EFV to test this hypothesis.
Methods: Wild type and mutant NL4-3 virus were produced
by transfection of 293 cells with site-directed
mutant molecular clones. Inputs of each virus pair based on p24 concentration
were used to infect H9 cells, absent EFV. The RT region amplified from proviral DNA was sequenced at days 4, 7, 11, and 14. The proportion
of each strain was determined by measuring relative peak heights. The fitness
coefficient (s) was calculated from
the slope of the line best fitting the equation ln[(qt/q0)/(pt/p0)]=st (t = time after co-infection; qt and q0 = proportions of mutant at times t and 0; pt
and p0 = proportions of
the reference strain at times t and
0.) Infections were performed in triplicate and verified in ≥ 2 separate
experiments.
Results: K103N+L100I had significantly reduced fitness
relative to K103N. L74V+K103N+L100I had a relative fitness indistinguishable
from K103N. The relative fitness of the L74V triple mutant was significantly
higher than the K103N+L100I double mutant. The improved fitness of the L74V
triple mutant was confirmed in direct competitions with K103N+L00I.
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Variant (vs reference strain) |
% variant @ each time |
relative Fitness |
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D4 |
d14 |
Δ·sd) |
1+s (SD) |
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K103N+L100I (vs K103N) |
77% |
45% |
-32%
(4.5) |
0.80
(0.03) |
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l74V+K103N+L100I (vs K103N) |
53% |
54% |
+1%
(7.7) |
1.01
(0.05)** |
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L74V+K103N+L100I (vs K103N+L100I) |
23% |
57% |
+33%
(4.8) |
1.12 (0.03) |
*% variant @ D14
minus % @ D4
**p<0.001,
compared to K103N+L100I,t-test
Conclusions: L74V compensates for the fitness defect
conferred by K103N+L100I. This may explain why L74V was selected for in
isolates with K103N+L100I, despite the lack of NRTI therapy. These data also
suggest that drug resistance mutations may be selected for on the basis of
their effects on fitness rather than resistance. These results are consistent
with the growing body of evidence that NRTI- and NNRTI-resistance mutations
interact, and suggest that a better understanding of these interactions may
lead to more rational design of NNRTI+NRTI regimens.
Keywords: viral fitness; drug resistance; L100I
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