Dimerization of retroviral genomic RNAs: structural and functional implications

Paillart, Jean-Christophe; Marquet, Roland; Skripkin, Eugene; Ehresmann, Chantal; Ehresmann, Bernard

doi:10.1016/s0300-9084(96)80010-1

Cited by 138 publications

(144 citation statements)

References 128 publications

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“…1 The use of fluorescent nucleotide base analogs as structural probes, however, has been limited owing to the practical size limit for chemical synthesis of RNA (≤80 nt). To address this issue, here we have described the use of the T4 RNA ligase to couple chemically synthesized 2-AP labeled sequence fragments with unlabeled RNA sequences to produce large RNA oligonucleotides site-specifically labeled with the nucleotide base analog.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of HIV-1 Ψ-site RNA sequences with site specific incorporation of the fluorescent base analog 2-aminopurine

Zhao

Marino

2007

Tetrahedron

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Fluorescent nucleotide base analogs can serve as sensitive probes of the local structure and chemical environment of the base within a nucleic acid sequence. A significant strength of these base analogs is their similarity in molecular constitution and chemical properties to natural bases. While chemical synthesis has afforded the ability to generate oligonucleotides in good yield with sequence-specific incorporation of fluorescent base analogs, this method is limited in practice to the synthesis of relatively small RNAs of less than ~ 80 nucleotides. Since most RNAs of biological interest are greater than 80 nucleotides in length, methods for synthesizing these larger RNAs in good yield, while maintaining the ability to site-specifically incorporate base analogs that allow for fluorescence measurements, could be of broad interest. Here we describe an approach for synthesis of large RNA molecules (>100 nt) that uses T4 RNA ligase to segmentally join a sequence fragment of an RNA, chemically synthesized with a fluorescent base analog, with the remaining unmodified portion of the RNA oligonucleotide, synthesized through in vitro transcription with T7 polymerase. This method is demonstrated through synthesis of packaging sequences (Ψ-site) derived from HIV-1 genomic RNA leader sequence (~ 120 nt) with the fluorescent base analog, 2-aminopurine (2-AP), selectively incorporated into the dimerization initiation site (DIS) stem-loop sequence. Using 2-AP fluorescence, RNA conformational changes associated with the formation of non-covalent DIS mediated Ψ-site dimers have been analyzed.

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Section: Resultsmentioning

confidence: 99%

Synthesis of HIV-1 Ψ-site RNA sequences with site specific incorporation of the fluorescent base analog 2-aminopurine

Zhao

Marino

2007

Tetrahedron

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“…The structure and the location of this element are homologous to those of the HIV-1 SL1, which is the element that mediates dimerization of HIV-1 RNA fragments in vitro (5)(6)(7)13,14,33,34). However the HIV-2 SL1 sequence does not mediate the formation of loose dimers in vitro, either as an isolated structure (9) or as a structural element in the 1-561 RNA (17).…”

Section: Discussionmentioning

confidence: 99%

“…In general, loose dimers can be observed on a native gel in Tris-borate magnesium (TBM) buffer at 4°C but are absent when assayed either on a semi-denaturing gel in Tris-borate EDTA (TBE) buffer or on a TBM gel at 24°C, presumably because the dimers dissociate under these electrophoresis conditions (12). Loose dimers of HIV-1 RNA are thought to be associated via an intermolecular kissing loop interaction (10,11,(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

et al. 2003

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An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1 a 5′ leader region site referred to as stem-loop1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5′-end of the primerbinding site (PBS) and a stem loop homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization/electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5′ and/or 3′ ends and by making compensatory base substitutions we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1- † This research was supported by the National Institutes of Health grant number AI45388 to J.S.L.

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“…Different structures and sequences within ψ may be required for distinct dimer formation, gag binding and RNA packaging steps. Guanine quartets (33) and kissing loops (34)(35)(36)(37)(38)(39) have been proposed to be important for some of these steps. We considered whether the nucleocapsid-binding RNAs described here could form such structures.…”

Section: Minimal Nucleocapsid Ligand Sequencesmentioning

confidence: 99%

In vitro selection of RNAs that bind to the human immunodeficiency virus type-1 gag polyprotein

Lochrie

Waugh²,

Pratt³

et al. 1997

Nucleic Acids Research

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Dimerization of retroviral genomic RNAs: structural and functional implications

Cited by 138 publications

References 128 publications

Synthesis of HIV-1 Ψ-site RNA sequences with site specific incorporation of the fluorescent base analog 2-aminopurine

Synthesis of HIV-1 Ψ-site RNA sequences with site specific incorporation of the fluorescent base analog 2-aminopurine

Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

In vitro selection of RNAs that bind to the human immunodeficiency virus type-1 gag polyprotein

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