Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

Abstract

The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3-R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5′ capped guanosine ( Cap 1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines ( Cap 2G or Cap 3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer-dimer equilibrium properties of Cap 1G, Cap 2G, and Cap 3G 5′-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the Cap 1G 5′-leader RNA adopts a dimeric structure, the Cap 2G and Cap 3G 5′-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimerpromoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5′ guanosine.HIV-1 | 5′-leader | transcription | RNA | structure

NMR Studies of the Structure and Function of the HIV-1 5′-Leader

“…The lack of base pair covariation has led some to question the validity of the tandem three-way junction structure [40], especially in view of strong evidence that residues of SD adopt a hairpin structure [8,40,41]. Although it is unfortunate that sequence conservation was too high for a conclusive phylogenetic assessment, it is certainly possible (likely, we believe) that the three-way junction and hairpin structures are both formed in infected cells, possibly a consequence of heterogeneous transcription start site usage (see below) [42]. …”

“…An additional layer of regulation may come from the sequence of the RNA itself, determined by the transcription start site (TSS) used during genome synthesis [42,62]. Shortly after initiation of RNA synthesis, the HIV-1 genome is co-transcriptionally capped by a 5′-5′ triphosphate linked 7-methylguanosine moiety (7MeG) [62,63,64,65,66].…”

“…Recent reports suggest that HIV-1 utilizes three different transcription start sites (1G, 2G, and 3G), creating an array of genomes with varying 5′ end identity, each of which is co-transcriptionally capped ( Cap 1G, Cap 2G, Cap 3G). Interestingly, the Cap 1G genome is predominately packaged into virions (70 to ~100%) [42,62], whereas Cap 2G and Cap 3G RNAs are enriched on polysomes [42]. The distinct function of these transcripts appears to correlate with a distinct structural rearrangement.…”

“…The distinct function of these transcripts appears to correlate with a distinct structural rearrangement. In vitro transcribed 5′-leader (5′-L) RNAs beginning with either 1G, 2Gs, or 3Gs displayed strikingly different dimerization profiles, with 1G and 2G 5′-L RNA favoring the dimer and 3G 5′-L RNA favoring the monomer [42]. The authors went on to examine the effect that capping played on these in vitro transcribed leader RNAs.…”

“…Mutagenesis studies show that destabilizing the predicted base of the PolyA stem promoted the monomeric RNA conformation while stabilizing the base of the hairpin promoted dimerization. These studies suggest a new paradigm for RNA fate, in which function is not encoded by an intrinsic monomer–dimer equilibrium, but rather by the transcriptionally encoded sequence of the capped 5′-terminus [42]. …”