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Replicative Capacity Effect of Mutations that Are Associated with Efavirenz Hypersusceptibility
Nancy Shulman*1, J Mellors2, S Clark2, R Bosch3, and N Parkin4
1Stanford Univ Sch of Med, Palo Alto, CA, US; 2Univ of Pittsburgh Sch of Med, PA, US; 3Statistical and Data Analysis Ctr, Harvard Sch of Publ Hlth, Boston, MA, US; and 4Monogram Biosci, South San Francisco, CA, US
Background: Hypersusceptibility to efavirenz (EFV) has
been associated with an improved response to EFV-containing regimens. Prior cART
and regression analysis of a large genotypic and phenotypic dataset identified T215Y,
H208Y, and V118I as the mutations most associated with EFV hypersusceptibility.
The mechanisms by which these mutations confer hypersusceptibility are not
clear. In some cases, hypersusceptibility to protease inhibitors (PI) is
associated with reduced replication capacity (RC). Site-specific mutagenesis
studies have confirmed the importance of the T215Y and H208Y in EFV hypersusceptibility,
but the impact of these mutations on RC has not been explored.
Methods: Mutations were introduced into the RT gene
of pNL43 by polymerase chain reaction (PCR)-based mutagenesis. Mutants made and
analyzed were T215Y, H208Y, T215Y+H208Y, T215Y+H208Y+V118I, and H208Y+V118I. Phenotypes
(IC50 fold change, FC) and RC of the mutant clones were analyzed
with PhenoSense HIV. The T215Y+V118I and V118I alone mutant data are pending.
Results: All mutants except T215Y alone were hypersusceptibility
to EFV and nevirapine (NVP) (FC <0.4, see the table). The H208Y mutation
reduced RC markedly when present alone but less so in combination with T215Y. Consistent
with the RC data, of 781 isolates with H208Y in the Stanford database, 100%
have 215Y/F/C/D/S either alone (96%) or as a mixture (4%). Of the 215 mutants
found with H208Y, 75% were T215Y.
|
RT Mutations
|
ZDV FC
|
DLV FC
|
EFV FC
|
NVP FC
|
RC
|
|
T215Y
|
5.0
|
0.31
|
0.46
|
0.55
|
100%
|
|
T215Y + H208Y
|
2.3
|
0.13
|
0.23
|
0.29
|
70%
|
|
T215Y + H208Y + V118I
|
1.2
|
0.06
|
0.12
|
0.17
|
56%
|
|
H208Y
|
0.39
|
0.10
|
0.12
|
0.21
|
9%
|
|
H208Y + V118I
|
0.27
|
0.07
|
0.08
|
0.13
|
12%
|
Conclusions: T215Y and H208Y confer hypersusceptibility
to EFV, confirming previous results. H208Y alone is an unfit mutant, but in
combination with T215Y, it is reasonably fit. H208Y always occurs in the
presence of mutations at 215 in vivo.
Low RC did not explain the NNRTI hypersusceptibility seen with this combination
of mutations.
|
