Fig 4. Sequencing of 5’ RACE PCR products identified HIV-1 transcripts with intragenic start sites.

(A) Schematic of HIV-1 genome with putative promoters indicated as dotted red lines as predicated by Promoter 2.0 [33] and ElementNT V2 [34]. The green line represents the 5’ RACE gene-specific Nef primer. (B) 5’ RACE results analyzed using the NCBI Multiple Sequence Alignment Viewer. (Top) All 5’ RACE sequences aligned to NL4-3 sequence (N = 41 sequences). (Middle) 5’ RACE sequences which lacked 5’ UTR aligned to NL4-3 sequence (N = 19). (Bottom) 5’ RACE sequences which contained 5’ UTR aligned to NL4-3 sequence (N = 22). (C) Schematic alignment of 5’ RACE sequences lacking 5’ UTR sequence (gray bars, N = 19) with the NL4-3 env gene (blue bar). Green and black dotted lines indicate in-frame start codons (numbered using GenBank: AF324493.2). Yellow lines represent out of frame start codons. Orange lines (top) represent TATA box sequences. Red lines represent sequence mismatches between 5’ RACE reads and NL4-3 env. Orange lines (bottom) indicate that no nucleotide was assigned for that position during the sequencing reaction. Start codons and TATA box sequences were mapped and multiple sequence alignments were performed using Benchling Biology Software (https://benchling.com, 2021).