Figure 2

Mutations present in the TW10 epitope of plasma viral variants dramatically impact viral fitness. (A) Schematic for the development of NL43nGFP based viruses to test the impact of plasma TW10 variants on viral fitness. Gag from ES8-1a virus which contains the TSTLAEQVAW TW10 mutant was mutated using site-directed mutagenesis to produce gag with the six other variants present in the plasma, as well as wild type TW10 and T242N on this ES8-1a backbone. Site-directed mutagenesis was also utilized to insert T242N into the NL43 gag, and gag from ES8-17 was amplified. Each of these gag mutants were then inserted into NL43eGFP backbone. All viruses derived from ES8-1a, the viral variant representing plasma viral variants, also had the I147M mutation, as indicated. (B) Fitness of viral variants was measured on day 6 post infection. Data shown are infection of two uninfected donors infected with two separate virus preparations in duplicate. Percent infection was normalized to transfection efficiency, and to the percent infection of WT NL43 which was on average 42%. (C) Replication of viral variants ES8-17 and ES8-1a. ES8-17 is wild type at the TW10 epitope and was cultured from resting CD4⁺ T cells from ES8. ES8-1a has mutation at TW10 and was cultured from activated CD4⁺ T cells. Results are the average of triplicate samples of p24 supernatant of activated CD4⁺ T cells infected with each virus. P-values shown above each time point compare the p24 of ES8-17 and ES8-1a using a one-tailed Student's T-test.