Dimer Initiation Site Hairpin Research Articles

Genome sequence analysis suggests coevolution of the DIS, SD, and Psi hairpins in HIV-1 genomes

HIV-1 RNA dimerization is a critical step in viral life cycle. It is a prerequisite for genome packaging and plays an important role in reverse transcription and recombination. Dimerization is promoted by the DIS (dimerization initiation site) hairpin located in the 5′ leader of HIV-1 genome. Despite the high genetic diversity in HIV-1 group M, only five apical loops (AAGCGCGCA, AAGUGCGCA, AAGUGCACA, AGGUGCACA and AGUGCAC) are commonly found in DIS hairpins. We refer to the parent DISes with these apical loops as DISLai, DISTrans, DISF, DISMal, and DISC, respectively. Based on identity or similarity of DIS hairpins to parent DISes, we distributed HIV-1 M genomes into five dimerization groups. Comparison of the primary and secondary structures of DIS, SD and Psi hairpins in about 3000 HIV-1 M genomes showed that the mutation frequencies at particular nucleotide positions of these hairpins differ among the dimerization groups, and DISF may be an origin of other parent DISes. We found that DIS, SD and Psi hairpins have hundreds of variants, only some of them occurring rather frequently. The lower part of DIS hairpin with G x AGG internal loop is highly conserved in both HIV-1 and SIV genomes. We supposed that the G-quadruplex, located 56 nts downstream of the Gag start codon, may participate in switching of HIV-1 leader RNA from BMH (branched multiple hairpins) to LDI (long distance interaction) conformation.

A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site

Dimerization of the genomic RNA is an important step of the HIV-1 replication cycle. The Dimerization Initiation Site (DIS) promotes dimerization of the viral genome by forming a loop–loop complex between two DIS hairpins. Crystal structures of the DIS loop–loop complex revealed an unexpected and strong similitude with the bacterial 16S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. As a consequence of these structural and sequence similarities, the HIV-1 DIS also binds some aminoglycosides, not only in vitro, but also ex vivo, in lymphoid cells and in viral particles. Crystal structures of the DIS loop–loop in complex with several aminoglycoside antibiotics provide a detailed-view of the DIS/drug interaction and reveal some hints about possible modifications to increase the drug affinity and/or specificity.

Polymorphism of Bulged-out Residues in HIV-1 RNA DIS Kissing Complex and Structure Comparison with Solution Studies

All retroviruses encapsidate their genome as a dimer of homologous single-stranded RNAs. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) is located in the 5'-untranslated region of the viral genome and consists of a hairpin with a 6 nt self-complementary loop sequence. Genomic RNA dimerization, a crucial step for virion infectivity, is promoted by the formation of a loop-loop complex (or kissing complex) between two DIS hairpins. Crystal structures for the subtypes A, B and F of the HIV-1 DIS kissing complex have now been solved at 2.3 A, 1.9 A and 1.6 A, respectively. They revealed a polymorphism of bulged-out residues showing clearly that their conformation is not a mere consequence of crystal packing. They also provide more insights into ion binding, hydration, and RNA conformation and flexibility. In particular, we observed the binding of spermine to the loop-loop helix, which displaced a magnesium cation important for subtype A DIS dimerization. The excellent agreement between X-ray structures and the results of chemical probing and interference data on larger viral RNA fragments shows that the crystal structures are relevant for the DIS kissing complex present in solution and in viral particles. Accordingly, these structures will be helpful for designing new drugs derived from aminoglycoside antibiotics and targeted against the RNA dimerization step of the viral life-cycle.

The HIV-1 Leader RNA Conformational Switch Regulates RNA Dimerization but Does Not Regulate mRNA Translation

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a long distance interaction (LDI) and a branched multiple-hairpin (BMH) structure. The BMH structure exposes the dimer initiation site (DIS) hairpin, whereas this motif is occluded in the LDI structure. Consequently, these structures differ in their capacity to form RNA dimers in vitro. The BMH structure is dimerization-competent, due to DIS hairpin formation, but also presents the splice donor (SD) and RNA packaging (Psi) hairpins. In the LDI structure, an extended RNA packaging (Psi(E)) hairpin is folded, which includes the splice donor site and gag coding sequences. The gag initiation codon is engaged in a long distance base pairing interaction with sequences in the upstream U5 region in the BMH structure, thus forming the evolutionarily conserved U5-AUG duplex. Therefore, the LDI-BMH equilibrium may affect not only the process of RNA dimer formation but also translation initiation. In this study, we designed mutations in the 3'-terminal region of the leader RNA that alter the equilibrium of the LDI-BMH structures. The mutant leader RNAs are affected in RNA dimer formation, but not in their translation efficiency. These results indicate that the LDI-BMH status does not regulate HIV-1 RNA translation, despite the differential presentation of the gag initiation codon in both leader RNA structures.

A riboswitch regulates RNA dimerization and packaging in human immunodeficiency virus type 1 virions.

The genome of retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA-the Psi and the dimerization initiation site (DIS) hairpins. The HIV-1 leader can adopt two alternative conformations that differ in the presentation of the DIS hairpin and consequently in their ability to dimerize in vitro. The branched multiple-hairpin (BMH) structure folds the poly(A) and DIS hairpins, but these domains are base paired in a long distance interaction (LDI) in the most stable LDI conformation. This LDI-BMH riboswitch regulates RNA dimerization in vitro. It was recently shown that the Psi hairpin structure is also presented differently in the LDI and BMH structures. Several detailed in vivo studies have indicated that sequences throughout the leader RNA contribute to RNA packaging, but how these diverse mutations affect the packaging mechanism is not known. We reasoned that these effects may be due to a change in the LDI-BMH equilibrium, and we therefore reanalyzed the structural effects of a large set of leader RNA mutations that were presented in three previous studies (J. L. Clever, D. Mirandar, Jr., and T. G. Parslow, J. Virol. 76:12381-12387, 2002; C. Helga-Maria, M. L. Hammarskjold, and D. Rekosh, J. Virol. 73:4127-4135, 1999; R. S. Russell, J. Hu, V. Beriault, A. J. Mouland, M. Laughrea, L. Kleiman, M. A. Wainberg, and C. Liang, J. Virol. 77:84-96, 2003). This analysis revealed a strict correlation between the status of the LDI-BMH equilibrium and RNA packaging. Furthermore, a correlation is apparent between RNA dimerization and RNA packaging, and these processes may be coordinated by the same LDI-BMH riboswitch mechanism.

RNA LEGO: magnesium-dependent assembly of RNA building blocks through loop-loop interactions.

We describe the construction of nano-molecular assemblies using RNA building blocks the human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) RNA, that forms stable base pairing through a magnesium-dependent loop-loop interaction ("kissing"). RNA building blocks containing two DIS or DIS-like hairpins connected by a two nucleotide linker self-assembled to form specific structures as observed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Furthermore, observation of "real time" formation of the molecular assemblies by circular dichroism (CD) spectroscopy was attempted.

The Dimer Initiation Site Hairpin Mediates Dimerization of the Human Immunodeficiency Virus, Type 2 RNA Genome

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.

Role of the DIS hairpin in replication of human immunodeficiency virus type 1.

The virion-associated genome of human immunodeficiency virus type 1 consists of a noncovalently linked dimer of two identical, unspliced RNA molecules. A hairpin structure within the untranslated leader transcript is postulated to play a role in RNA dimerization through base pairing of the autocomplementary loop sequences. This hairpin motif with the palindromic loop sequence is referred to as the dimer initiation site (DIS), and the type of interaction is termed loop-loop kissing. Detailed phylogenetic analysis of the DIS motifs in different human and simian immunodeficiency viruses revealed conservation of the hairpin structure with a 6-mer palindrome in the loop, despite considerable sequence divergence. This finding supports the loop-loop kissing mechanism. To test this possibility, proviral genomes with mutations in the DIS palindrome were constructed. The appearance of infectious virus upon transfection into SupT1 T cells was delayed for the DIS mutants compared with that obtained by transfection of the wild-type provirus (pLAI), confirming that this RNA motif plays an important role in virus replication. Surprisingly, the RNA genome extracted from mutant virions was found to be fully dimeric and to have a normal thermal stability. These results indicate that the DIS motif is not essential for human immunodeficiency virus type 1 RNA dimerization and suggest that DIS base pairing does not contribute to the stability of the mature RNA dimer. Instead, we measured a reduction in the amount of viral RNA encapsidated in the mutant virions, suggesting a role of the DIS motif in RNA packaging. This result correlates with the idea that the processes of RNA dimerization and packaging are intrinsically linked, and we propose that DIS pairing is a prerequisite for RNA packaging.