Role of the Trans-activation Response Element in Dimerization of HIV-1 RNA

Andersen, Ellen Juul; Contera, Sonia; Knudsen, Bjarne; Damgaard, Christian Kroun; Besenbacher, Flemming; Kjems, Jørgen

doi:10.1074/jbc.m314326200

Cited by 74 publications

(108 citation statements)

References 31 publications

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“…No difference was observed between cells and virions in the TAR loop, despite the fact that NC induces TAR-TAR dimerization in vitro (15). Similarly, the reactivity of the poly(A) loop was unchanged, although this hairpin has been proposed to participate in the RNA dimerization process (16).…”

Section: The Genomic Rna Undergoes a Few Limited Structural Rearrangementioning

confidence: 89%

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First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions

Paillart

Dettenhofer

et al. 2004

Journal of Biological Chemistry

127

143

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With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5 -untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5 -untranslated region in infected cells points to the existence of the various stemloop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA 3 Lys annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.

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Section: The Genomic Rna Undergoes a Few Limited Structural Rearrangementioning

confidence: 89%

“…Beside these intramolecular interactions, symmetric intermolecular interactions take place in vitro at the DIS (12)(13)(14) and could also occur at TAR (15) and poly(A) hairpin (16). In addition, tRNA 3 Lys , the reverse transcription primer, is selectively encapsidated into virions and is annealed to the PBS during or after budding (17,18).…”

mentioning

confidence: 99%

First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions

Paillart

Dettenhofer

et al. 2004

Journal of Biological Chemistry

127

143

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“…through nonspecific electrostatic attachment onto the positively charged spermine-coated surfaces (Fig. 3A) through a recently developed procedure (23). The oligomers showed a characteristic bagel-like appearance, with the globular CRDs forming a circular ring.…”

Section: Afm Imaging Of Mbl On Spermine-coated Surfacesmentioning

confidence: 99%

“…Modified mica surfaces were prepared by the immersion of freshly cleaved mica surfaces in a solution of 0.05 mg/ml spermine (S4264; SigmaAldrich) for 10 min, followed by a rinse in deionized water and drying under a steam of N 2 , which creates stable and flat surfaces (23). Subsequently, 2 l of a solution with 7 g/ml unfractionated MBL in a buffer (16).…”

Section: Surface Binding Of Mbl To Modified Mica Surfacesmentioning

confidence: 99%

Conformational Changes in Mannan-Binding Lectin Bound to Ligand Surfaces

Dong

Oliveira³

et al. 2007

The Journal of Immunology

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The binding of soluble proteins to target surfaces is vital in triggering the immune response. However, structural insight into such processes is still lacking. Mannan-binding lectin (MBL) is a classic example of a pattern recognition molecule with important roles in innate immunity against microbial infections. By small angle x-ray scattering analysis we show that the large MBL complex in solution is folded into a ramified structure with a striking rotational symmetry and a structure permissive of elongation by unbending. Nevertheless, the structure in solution is found to be very stable. However, when the MBL molecule interacts with surface-immobilized ligands, the stable MBL structure is broken into a stretched state with separation of the ligand-binding domains as shown by high resolution atomic force microscopy. These studies provide a snapshot of the single molecule mechanics of MBL and the first direct evidence that the transition from the soluble state to surface-bound protein involves large conformational changes in the quaternary structure, thus highlighting the role of surface topography in immune recognition.

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“…The R region of HIV-1 has been proposed to be involved in RNA dimerization with intermolecular base-pairing of nucleotides of the trans-activation response (TAR) element (Chang and Tinoco 1994;Andersen et al 2004). The results reported here raise the possibility of a similar role of the psls in the R region of RD114 RNA.…”

Section: Discussionmentioning

confidence: 99%

A unique, thermostable dimer linkage structure of RD114 retroviral RNA

Kharytonchyk

Pedersen²

2010

RNA

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Retroviruses package their genome as RNA dimers linked together primarily by base-pairing between palindromic stem-loop (psl) sequences at the 59 end of genomic RNA. Retroviral RNA dimers usually melt in the range of 55°C-70°C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87°C. We here report that the high thermal stability of RD114 RNA dimers generated from in vitro synthesized RNA is an effect of multiple dimerization sites located in the 59 region from the R region to sequences downstream from the splice donor (SD) site. By antisense oligonucleotide probing we were able to map at least five dimerization sites. Computational prediction revealed a possibility to form stems with autocomplementary loops for all of the mapped dimerization sites. Three of them were located upstream of the SD site. Mutant analysis supported a role of all five loop sequences in the formation and thermal stability of RNA dimers. Four of the five psls were also predicted in the RNA of two baboon endogenous retroviruses proposed to be ancestors of RD114. RNA fragments of the 59 R region or prolonged further downstream could be efficiently dimerized in vitro. However, this was not the case for the 39 R region linked to upstream U3 sequences, suggesting a specific mechanism of negative regulation of dimerization at the 39 end of the genome, possibly explained by a long double-stranded RNA region at the U3-R border. Altogether, these data point to determinants of the high thermostability of the dimer linkage structure of the RD114 genome and reveal differences from other retroviruses.

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Role of the Trans-activation Response Element in Dimerization of HIV-1 RNA

Cited by 74 publications

References 31 publications

First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions

First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions

Conformational Changes in Mannan-Binding Lectin Bound to Ligand Surfaces

A unique, thermostable dimer linkage structure of RD114 retroviral RNA

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