Figure 5. 5′-L deletions lead to noninfectious particles lacking env incorporation.

(A) Deletions found in proviruses causing viremia were introduced in an NL4-3 expression plasmid by site-directed mutagenesis. Deletion start and end positions relative to HXB2 are indicated in parentheses. (B) Copies of HIV-1 RNA recovered at 72 hours after transfection of 293T cells, expressed as copies per mL (left) or normalized by p24 pg/mL (right). (C) Spinoculation of primary CD4⁺ T cells shows exponential increase in p24 levels only with WT NL4-3, in the absence of antiretrovirals (ARVs: TDF, FTC, DTG). (D) Reverse transcription was assessed by measuring late cDNA products by ddPCR targeting the U5-PBS junction. Primary CD4⁺ T cells were collected at 0, 6, and 12 hours after spinoculation with and without ARVs. U5-PBS copies detected in the presence of ARVs, which are the result of incomplete DNAseI digestion of plasmid carryover from transfection, were subtracted from copies detected in conditions without ARVs. (E) Virus produced upon 293T transfection was pelleted by ultracentrifugation, lysed, normalized by p24, and used for Western blots with primary antibodies specific to p24 and gp41. (F) Surface staining of HIV-1 env on 293T cells 24 hours after transfection. (G) Frequency of env-positive cells transfected with WT versus 5′-L deletions. Fold reduction relative to WT is indicated above each mutant. Results from 2 transfection experiments are shown. Each circle represents the average of 2 technical replicates. (H) Quantification of cell-associated spliced HIV-1 transcripts belonging to the 4 kb class or tat/rev mRNA normalized to RNA ng. (I) Percentage of spliced transcripts relative to total HIV-1 polyA RNA. Error bars indicate SEM (B, H and I) or SD (C and F). Statistical significance between conditions was determined by 1-way ANOVA. *P < 0.05; **P <0.01, ***P <0.001.