Structural signals that direct HIV packaging: During the viral replication cycle of HIV, unspliced dimeric RNA genomes are efficiently packaged into new virions at the host cell membrane. Packaging is directed by a region at the start of the genome, the 5′ leader. The architecture of the 5′ leader remains controversial. Keane et al. developed nuclear magnetic resonance methods to determine the structure of a 155-nucleotide-long region of the 5′ leader that can direct viral packaging. The structure shows how the 5′ leader binds to the HIV protein that directs packaging, how unspliced dimeric genomes are selected for packaging, and how translation is suppressed when the genome dimerizes. Science , this issue p. 917

The 5′-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5′ region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.

SUMMARY: The genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from a combination of point mutations and genetic recombination, and rates of both processes are unusually high. This review focuses on the mechanisms and outcomes of HIV-1 genetic recombination and on the parameters that make recombination so remarkably frequent. Experimental work has demonstrated that the process that leads to recombination—a copy choice mechanism involving the migration of reverse transcriptase between viral RNA templates—occurs several times on average during every round of HIV-1 DNA synthesis. Key biological factors that lead to high recombination rates for all retroviruses are the recombination-prone nature of their reverse transcription machinery and their pseudodiploid RNA genomes. However, HIV-1 genes recombine even more frequently than do those of many other retroviruses. This reflects the way in which HIV-1 selects genomic RNAs for coencapsidation as well as cell-to-cell transmission properties that lead to unusually frequent associations between distinct viral genotypes. HIV-1 faces strong and changeable selective conditions during replication within patients. The mode of HIV-1 persistence as integrated proviruses and strong selection for defective proviruses in vivo provide conditions for archiving alleles, which can be resuscitated years after initial provirus establishment. Recombination can facilitate drug resistance and may allow superinfecting HIV-1 strains to evade preexisting immune responses, thus adding to challenges in vaccine development. These properties converge to provide HIV-1 with the means, motive, and opportunity to recombine its genetic material at an unprecedented high rate and to allow genetic recombination to serve as one of the highest barriers to HIV-1 eradication.

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 (^Cap1G) 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 (^Cap2G or ^Cap3G) 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 ^Cap1G, ^Cap2G, and ^Cap3G 5′-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the ^Cap1G 5′-leader RNA adopts a dimeric structure, the ^Cap2G and ^Cap3G 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 dimer-promoting 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. Significance Current dogma states that the integrated HIV type 1 provirus encodes a single RNA transcript that serves as both mRNA for generating viral proteins and as genomic RNA that is packaged and used for reverse transcription. We now show that multiple transcripts with different functions are generated in infected cells, a consequence of heterogeneous transcriptional start site usage. Transcripts that begin with a single capped guanosine are specifically selected for packaging, whereas those that begin with two or three capped guanosines are enriched on polysomes and used for translation. In vitro studies with recombinant 5′-leader transcripts reveal a mechanism by which the incorporation of a single 5′ guanosine dramatically alters the structure, function, and fate of the viral RNA.

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