Figure 1

Experimental strategy for investigating HIV-1 and HIV-2 mutagenesis by Illumina DNA sequencing. Vector virus stocks were produced by co-transfecting 293T cells with HIV-1 or HIV-2 Env-deficient vectors and HIV-1 or HIV-2 CXCR4-tropic Env expression constructs. Virus stocks were concentrated, DNase I-treated to reduce plasmid carryover, and titered in U373-MAGI cells. To prepare samples for Illumina sequencing, 1 × 10⁶ U373-MAGI cells were infected at an MOI of 1.0, generating approximately 1 × 10⁶ proviruses per experimental replicate. This assay represents a single round of viral replication, as producer cells and target cells cannot be re-infected, due to a lack of receptor or Env expression, respectively. Polymerase chain reaction (PCR) of five amplicons (Gag, Vif, HSA, EGFP-1, and EGFP-2) was performed from the proviral DNA. Amplicons from the HIV-1 and HIV-2 proviral DNAs were either identical (HSA, EGFP-1 and 2) or homologous (Gag and Vif) in sequence. The EGFP-1 and EGFP-2 amplicons represent non-overlapping segments of the egfp gene. Sequencing libraries were prepared from the amplicons, pooled in an equimolar fashion to normalize coverage, and subjected to 2 ×150 bp sequencing on the Illumina MiSeq, generating approximately 4.7 million read pairs after data processing.