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. 2023 Mar 15;133(6):e165245. doi: 10.1172/JCI165245

Figure 4. 5′-L-defective proviruses are inducible and their genomic RNA is packaged and can use alternative splicing donors.

Figure 4

(A) CD4+ T cells from P1 and P2 were cultured for 48 hours in the presence of dolutegravir (DTG) and anti-CD3/CD28 beads. Cells and supernatants were collected at 0, 24, and 48 hours. Right panel shows the location of primers and probes used to quantify viral RNA. Shaded areas indicate 5′-L deletion used to measure provirus-specific transcription. (B) Mean levels of read-through, total, and provirus-specific RNA detected in cells and supernatant upon T cell activation. Gray circles represent digital PCR replicate reactions. Error bars indicate SEM. (C) Singly and multiply spliced transcripts amplified at limiting dilution from cells at 48 hours. Arrows indicate primer locations. Ticks indicate the locations of splicing donors and acceptors in the HIV-1 genome. Major and alternative splicing donor sites are labeled in black. Mapped splicing junctions are underlined and in bold; 22 nt deletion is represented by dashed lines. Numbers in parentheses indicate the number of sequences recovered for each type of spliced variant.