Selective packaging of HIV-1 RNA genome is guided by the stability of 5′ untranslated region polyA stem

Abstract

To generate infectious virus, HIV-1 must package two copies of its full-length RNA into particles. HIV-1 transcription initiates from multiple, neighboring sites, generating RNA species that only differ by a few nucleotides at the 5′ end, including those with one (1G) or three (3G) 5′ guanosines. Strikingly, 1G RNA is preferentially packaged into virions over 3G RNA. We investigated how HIV-1 distinguishes between these nearly identical RNAs using in-gel chemical probing combined with recently developed computational tools for determining RNA conformational ensembles, as well as cell-based assays to quantify the efficiency of RNA packaging into viral particles. We found that 1G and 3G RNAs fold into distinct structural ensembles. The 1G RNA, but not the 3G RNA, primarily adopts conformations with an intact polyA stem, exposed dimerization initiation site, and multiple, unpaired guanosines known to mediate Gag binding. Furthermore, we identified mutants that exhibited altered genome selectivity and packaged 3G RNA efficiently. In these mutants, both 1G and 3G RNAs fold into similar conformational ensembles, such that they can no longer be distinguished. Our findings demonstrate that polyA stem stability guides RNA-packaging selectivity. These studies also uncover the mechanism by which HIV-1 selects its genome for packaging: 1G RNA is preferentially packaged because it exposes structural elements that promote RNA dimerization and Gag binding.

Retroviral RNA Processing

“…How and where is this selection between packaging and translation made? Alternative RNA structures in the 5′ UTR have been proposed to distinguish genomic from translated RNAs [ 26 , 27 , 28 , 29 ].…”

“…However, Gag and Pol proteins are still translated from unspliced RNA and Env from singly spliced mRNA. Over 50 different HIV-1 mRNAs have been described, all derived from a single full-length primary transcript (with 2 or 3 Gs at the 5′ end [ 28 , 29 , 30 ]). These fall into three size classes: full-length (10 kb), singly spliced (4 kb), and completely (or multiply) spliced (1 kb).…”

Anomalous HIV-1 RNA, How Cap-Methylation Segregates Viral Transcripts by Form and Function

“…As discussed above, cell-based experiments have documented HIV TMG-cap engages NCBP3/CBP80 heterodimer for translation in a Rev/RRE-dependent manner [ 20 ]. Taken together, in-cellulo data, in-solution analyses and in silico modeling converge on the hypothesis that TSS heterogeneity modulates HIV translation control, as well as the short- and long-range interactions in the HIV 5′ UTR that regulate genome dimerization and packaging [ 68 , 175 , 176 ]. Future studies are warranted to document the TSS of the TMG-capped RNA.…”

“…Analysis of the HIV-1 TSS initially identified guanosine-guanosine as the primary sequence at the 5′ end of HIV-1 in lymphocytes [ 65 , 66 , 67 ]. Recently, an adenosine at the HIV TSS was detected infrequently [ 68 ]. Thus, PCIF1 improbably contributes to HIV evasion of innate immune suppression directly [ 69 ].…”

Phosphomimetic S207D Lysyl–tRNA Synthetase Binds HIV-1 5′UTR in an Open Conformation and Increases RNA Dynamics

“…The catalytically inactive dimeric pS207–LysRS/tRNA complex is then recruited to sites of viral assembly via interactions with HIV-1 Gag [ 5 , 48 ]. The 5′UTR can adopt various monomeric and dimeric conformations, including a “kissing loop” dimer [ 49 , 50 , 51 , 52 , 53 , 54 ] ( Figure 6 , Step 2). The anticodon-like TLE of an HIV-1 gRNA dimer then competes for binding to pS207–LysRS resulting in an increase in dynamics of the gRNA in this region and release of the tRNA primer proximal to the PBS ( Figure 6 , Step 3).…”