HIV-1 excludes non-guanosine nucleotides in major transcription start sites to ensure distinct unspliced RNA fates

HIV-1 expresses two unspliced RNA transcripts with distinct 5' untranslated region (UTR) structures-one optimized for packaging to serve as the viral genome and the other optimized to serve as an mRNA template for translation. This strategy requires precisely regulated production of two structurally distinct RNA isoforms, yet the mechanisms controlling this regulation remain poorly understood. In this work, we targeted the three guanosines in the HIV-1 genome that serve as start sites for the major transcripts and examined the impact of specific nucleotide substitutions on RNA expression, the resulting RNA structures, and viral replication. Our studies demonstrate that substituting the guanosines with pyrimidines severely perturbs transcription initiation from these sites. Adenosine substitutions are well tolerated as initiation sites for RNA polymerase II but eliminate the distinct post-transcriptional fates of the resulting transcripts through a structure-based mechanism. This work provides insights into the evolutionary pressures on HIV-1 to maintain three guanosines as major transcription start sites and the finely tuned regulation of HIV-1 RNA that ensures production of two structurally and functionally distinct unspliced RNAs. IMPORTANCE: To optimize replication fitness, HIV-1 expresses two major unspliced transcripts that are 99.9% identical, yet perform distinct functions. These transcripts are initiated at guanosines two nucleotides apart within the 5'-long terminal repeat (LTR). Through genetic and structural studies, we show that these guanosines serve two regulatory roles. At the DNA level, they control transcription initiation of the two RNA isoforms. At the RNA level, they control the interplay of two RNA hairpins at the start of the 5'-untranslated region, leading to divergent global RNA structures. This elegant mechanism enables functionally distinct transcripts to arise from nearly identical sequences and highlights how evolutionary pressures act at both the DNA and RNA levels to preserve guanosine identity at the major transcription start sites.