Heterogeneous transcriptional start site usage by HIV-1 produces 5'-capped RNAs beginning with one, two, or three 5'-guanosines ((Cap)1G, (Cap)2G, or (Cap)3G, respectively) that are either selected for packaging as genomes ((Cap)1G) or retained in cells as translatable messenger RNAs (mRNAs) ((Cap)2G and (Cap)3G). To understand how 5'-guanosine number influences fate, we probed the structures of capped HIV-1 leader RNAs by deuterium-edited nuclear magnetic resonance. The (Cap)1G transcript adopts a dimeric multihairpin structure that sequesters the cap, inhibits interactions with eukaryotic translation initiation factor 4E, and resists decapping. The (Cap)2G and (Cap)3G transcripts adopt an alternate structure with an elongated central helix, exposed splice donor residues, and an accessible cap. Extensive remodeling, achieved at the energetic cost of a G-C base pair, explains how a single 5'-guanosine modifies the function of a similar to 9-kilobase HIV-1 transcript.