Background:  We recently observed that K101E+G190S reduces the replication fitness of NL4-3. We identified a clinical RT sequence, D10, which contains M41L+T215Y and K101E+G190S, in addition to several polymorphisms. We evaluated the replication fitness and efavirenz (EFV) susceptiblity of the D10 clone. In addition to M41L+T215Y, we also evaluated the effect of L74V on fitness and EFV susceptibility of K101E+G190S. 

Methods:  We measured fitness using a growth competition assay in PM1 cells. The proportion of test and reference strains at days 3 to 6 was measured with direct sequencing. Relative fitness was quantified using the production rate ratio (PRR). Effects of EFV on viral growth were measured using a modification of the ACTG/DoD method in PM1 cells. IC₅₀ was estimated using the median-effect equation. In order to further evaluate the genetic basis of the properties of the D10 clone, mutations were introduced into NL4-3, using polymerase chain reaction (PCR) -mediated site-directed mutagenesis, and verified by double-stranded sequencing of RT. M230L, which was reported to confer EFV-dependent stimulation, was included as a reference.

Results:  See the table.

Conclusions:  K101E reduces fitness and confers EFV-dependent stimulation of growth when combined with G190S. Both fitness and EFV stimulation are augmented in the clinical RT sequence, D10, which contains M41L+T215Y, in addition to several RT polymorphisms. In NL4-3, both M41L+T215Y and L74V reduce EFV-dependent stimulation and improve fitness. The genetic basis for replication fitness and EFV-dependent stimulation in the D10 clone is complex, and influenced by both RT polymorphisms and nucleoside reverse transcriptase inhibitor resistance mutations.