The 5´-leader of the HIV-1 genome regulates multiple functions during viral replication by mechanisms that have yet to be established. We developed an NMR 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 protein (NC), 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.

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

BackgroundRetroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism. Dimerization of human immunodeficiency virus Type-1 (HIV-1) genomes is initiated by “kissing” interactions between GC-rich palindromic loop residues of a conserved hairpin (DIS), and is indirectly promoted by long-range base pairing between residues overlapping the gag start codon (AUG) and an upstream Unique 5′ element (U5). The DIS and U5:AUG structures are phylogenetically conserved among divergent retroviruses, suggesting conserved functions. However, some studies suggest that the DIS of HIV-2 does not participate in dimerization, and that U5:AUG pairing inhibits, rather than promotes, genome dimerization. We prepared RNAs corresponding to native and mutant forms of the 5′ leaders of HIV-1 (NL4-3 strain), HIV-2 (ROD strain), and two divergent strains of simian immunodeficiency virus (SIV; cpz-TAN1 and -US strains), and probed for potential roles of the DIS and U5:AUG base pairing on intrinsic and NC-dependent dimerization by mutagenesis, gel electrophoresis, and NMR spectroscopy.ResultsDimeric forms of the native HIV-2 and SIV leaders were only detectable using running buffers that contained Mg2+, indicating that these dimers are more labile than that of the HIV-1 leader. Mutations designed to promote U5:AUG base pairing promoted dimerization of the HIV-2 and SIV RNAs, whereas mutations that prevented U5:AUG pairing inhibited dimerization. Chimeric HIV-2 and SIV leader RNAs containing the dimer-promoting loop of HIV-1 (DIS) exhibited HIV-1 leader-like dimerization properties, whereas an HIV-1NL4-3 mutant containing the SIVcpzTAN1 DIS loop behaved like the SIVcpzTAN1 leader. The cognate NC proteins exhibited varying abilities to promote dimerization of the retroviral leader RNAs, but none were able to convert labile dimers to non-labile dimers.ConclusionsThe finding that U5:AUG formation promotes dimerization of the full-length HIV-1, HIV-2, SIVcpzUS, and SIVcpzTAN1 5′ leaders suggests that these retroviruses utilize a common RNA structural switch mechanism to modulate function. Differences in native and NC-dependent dimerization propensity and lability are due to variations in the compositions of the DIS loop residues rather than other sequences within the leader RNAs. Although NC is a well-known RNA chaperone, its role in dimerization has the hallmarks of a classical riboswitch.

An estimated 35 million people live with Human Immunodeficiency Virus (HIV-1), with approximately 1.7 million associated deaths per year. HIV-1 is a retrovirus that infects the T cells of the host’s immune system. After infection, the virus reverse-transcribes its RNA genome into DNA and integrates the viral DNA into the host DNA, which is eventually transcribed into RNA by the host cell. The 5’ untranslated region (UTR) of the RNA, is the most conserved region of the genome. It exists in equilibrium between a monomeric form, important for translation, and a dimeric form, important for genome packaging. While gel electrophoresis is usually used to determine the equilibrium of monomer and dimer, we have developed a new technique that uses gel centrifugation. We are developing this technique as it may allow us to look at the monomer-dimer equilibria of different RNAs, such as HIV-2, which have weaker “kissing” dimer interactions. When gel electrophoresis is used on these dimers, the magnesium that stabilizes the dimer is drawn towards the opposite pole as the RNA, providing inaccurate data by dissociating the dimers and causing more monomer to be visualized. Magnesium containing gels are used to combat this, but it is difficult to determine how much magnesium is needed to obtain accurate results. Future work includes testing this method on retroviruses with a “kissing dimer” interaction and eventually determining a way to quantify the amount of monomer and dimer present.