FIG 11.
Viral replicative fitness of V1 substitutions, V4 substitutions, and V4 deletions relative to SIVmac239. Rhesus 221 and 444 immortalized T-lymphocyte cell lines were infected with a pooled mixture of SIVmac239 and Env single-point mutant virus stocks (A and B) or with a pooled mixture of SIVmac239 and Env V4 loop deletion mutant virus stocks (C) at a low MOI (≤0.01). Viral RNA extracted from mixed-infection supernatants was used as the template in RT-PCR using target region-specific primers. Amplicon products were barcoded to produce libraries that were deep sequenced by Illumina MiSeq. Reads were directly assembled to the SIVmac239 reference sequence. The frequency of reads with the introduced point mutation relative to the total number of reads was plotted over time for V1 loop mutants (A), V4 loop mutants (B), and V4 loop deletion mutants (C) in Rh 221 cells and Rh 444 cells. The results are reported as the means ± standard deviations (SD) from four replicates performed in parallel. Linear regression analysis was performed to determine the linear slope of the frequency of the mutant virus over time (reported as the relative replication capacity of the mutant virus), and the R2 value of each line and the P value of an F-test, testing whether the slope of the line is significantly different from zero, was determined.