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The roles of five conserved lentiviral RNA structures in HIV-1 replication.

ABSTRACT: The HIV-1 RNA genome contains complex structures with many structural elements playing regulatory roles during viral replication. A recent study has identified multiple RNA structures with unknown functions that are conserved among HIV-1 and two simian immunodeficiency viruses. To explore the roles of these conserved RNA structures, we introduced synonymous mutations into the HIV-1 genome to disrupt each structure. These mutants exhibited similar particle production, viral infectivity, and replication kinetics relative to the parent NL4-3 virus. However, when replicating in direct competition with the wild-type NL4-3 virus, mutations of RNA structures at inter-protein domain junctions can cause fitness defects. These findings reveal the ability of HIV-1 to tolerate changes in its sequences, even in apparently highly conserved structures, which permits high genetic diversity in HIV-1 population. Our results also suggest that some conserved RNA structures may function to fine-tune viral replication.

SUBMITTER: Liu Y

PROVIDER: S-EPMC5766380 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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The roles of five conserved lentiviral RNA structures in HIV-1 replication.

Liu Yang Y Chen Jianbo J Nikolaitchik Olga A OA Desimmie Belete A BA Busan Steven S Pathak Vinay K VK Weeks Kevin M KM Hu Wei-Shau WS

Virology 20171109

The HIV-1 RNA genome contains complex structures with many structural elements playing regulatory roles during viral replication. A recent study has identified multiple RNA structures with unknown functions that are conserved among HIV-1 and two simian immunodeficiency viruses. To explore the roles of these conserved RNA structures, we introduced synonymous mutations into the HIV-1 genome to disrupt each structure. These mutants exhibited similar particle production, viral infectivity, and repli ...[more]

PMID: 29128752

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Project description:Replication and pathogenesis of the human immunodeficiency virus (HIV) is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001) SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further application of this technology will make possible newly informative analysis of any RNA in a cellular transcriptome.

Project description:Retroviruses 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.Dimeric forms of the native HIV-2 and SIV leaders were only detectable using running buffers that contained Mg(2+), 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.The 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.

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The roles of five conserved lentiviral RNA structures in HIV-1 replication.

Publications

The roles of five conserved lentiviral RNA structures in HIV-1 replication.

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