The strictly conserved GGG-tracts in the 5' and 3' long terminal repeat of HIV-1 are critical to control multiple steps of HIV-1 replication to prevent acquisition of unwanted mutations in the region

Abstract

Three consecutive deoxyguanosine residues (the GGG-tract) in the U3/R junction of the 5 long terminal repeat (LTR) are strictly conserved among all HIV-1 subtypes. Each deoxyguanosine within the tract has been reported to function as transcription initiation site for HIV-1 RNA. Furthermore, RNAs whose transcription initiates from the third deoxyguanosine in the tract (1G form RNAs) are predominant in virus particles and serve as the primary templates for reverse-transcription. In this study, we generated mutant HIV-1s by replacing the tracts in both the 5 and 3 LTR with other nucleotides to elucidate their functional significance. We identified several proviral sequences containing unexpected mutations near the 5 tract after infection with the mutant, but not the wild-type virus. Five-prime rapid amplification of cDNA end (5 RACE) analyses of RNAs purified from mutant virus particles revealed multiple RNA variants with 5 terminal sequences differing from the plasmid used for producing the particles. Some of the unexpected proviral sequences likely arise directly from these variants during reverse-transcription. We also found that replacing all three nucleotides in the 3 tract with deoxyadenosines decreased the proportion of the 1G form RNAs in particles to 32.6%. Nevertheless, up to 88% of provirus was likely generated with the 1G form RNAs, although they were no longer absolutely predominant in particles. Our results demonstrate that the GGG-tracts in the 5 and 3 LTR are conserved to maintain the integrity of reverse-transcription for LTR sequence generation by controlling multiple steps of HIV-1 replication, including transcription, RNA packaging and reverse-transcription. ImportanceHIV-1 is highly mutable, yet certain conserved sequences remain consistent across most strains. These sequences are thought to be maintained because mutations in these regions typically impair viral fitness, leading to the elimination of such variants through natural selections. This study suggests that HIV-1 has a unique mechanism to autonomously prevent acquisition of mutations in certain regions. Specifically, the GGG-tracts in the 5 and 3 LTR ensure accurate transcription of HIV-1 RNAs, selective packaging of the 1G form RNAs, and preferential use of these 1G forms as a template for reverse-transcription. GGG preservation minimizes unwanted mutations in this region, allowing progeny virus to inherit intact LTR sequences. Interestingly, the preferential usage of 1G form RNAs as template for reverse-transcription is not due to their predominance in virus particles. This suggests that genome packaging independently contributes to reverse-transcription as preparatory stage by concentrating the most suitable RNAs for reverse-transcription in particles.