One guanosine determines transcript fate: Transcripts of the HIV-1 RNA genome can be either spliced and translated into viral proteins or packaged into new virions as a progeny genome. The path taken depends on whether the transcript contains one guanosine at the 5′ terminus (1G) rather than two or three (2G or 3G). Brown et al. used nuclear magnetic resonance spectroscopy to show that 1G transcripts adopt a dimeric structure that sequesters a terminal cap required for translation and splicing but exposes sites that bind to the HIV-1 Gag protein, which recruits the genome during viral assembly. Conversely, 2G or 3G transcripts have the cap accessible, but Gag-binding sites are sequestered. Therefore, a single guanosine acts as a conformational switch to determine the fate of HIV-1 transcripts. Science , this issue p. 413

The genomes of several plant viruses contain RNA structures at their 3′ ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5′ cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5′ cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3′ CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5′ cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE–eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5′ cap for eIF4E binding. Such 5′ cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5′ capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5′ polyA element, resulting in formation of an extended 5′ polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5′ polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5′ polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5′ cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5′ cap exposure were both poorly packaged and more efficiently translated than those that favored 5′ cap sequestration. We propose that structural plasticity of 5′ polyA and other conserved RNA elements place the 5′ leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.

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