Figure 2

Phenotypic impact of mutations Q91R and I175M on susceptibility to RAL (A) and on viral titers in vitro (B). (A) 3 × 10⁴ MT-4 cells were infected with 3 × 10⁶ TCID₅₀ (M.O.I. of 100) of ROD or ROD-Q91R + I175M variant in the presence of serial RAL dilutions ranging from 3.763 × 10^-5 nM to 20 nM. Infection was quantified by measuring MTT in culture supernatants after 3 days. Infections were performed in quadruplicate wells. 4 independent experiments were performed. The percentage of protection (PP = (O.D. measured - O.D. infected cells without RAL)/O.D. uninfected cells - O.D. infected cells without RAL) × 100). RAL IC₅₀ corresponds to the RAL concentration that protects 50% of the culture from virus-induced cytopathic effect, i.e. inhibition of infection. Percent protection is reported as a function of log₁₀ RAL concentration (nM). The mean of 4 independent experiments, each performed in quadruplicate wells, is presented. (B) Two million MT-4 cells were infected with 2 × 10⁸ TCID₅₀ of HIV-2 ROD for at least two hours, then washed and cultured in the presence of RAL. New infections were performed twice a week, and virus titers were determined once a week by RT-PCR. Suboptimal concentrations were used during the first passages, then raised gradually by 3-fold. The titers are only shown for the two last drug increases, when the variant carrying Q91R and I175M mutations was selected.