Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein

Kanevsky, Igor; Chaminade, Françoise; Chen, Yingying; Godet, Julien; René, Brigitte; Darlix, Jean‐Luc; Mély, Yves; Mauffret, Olivier; Fossé, Philippe

doi:10.1093/nar/gkr526

Cited by 20 publications

(29 citation statements)

References 69 publications

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“…As previously reported (29), KMnO 4 is expected to interact with the nucleobases of unpaired thymine residues that interact with the N-terminal zinc finger of NC. Indeed, a close examination of the structures of the NC⅐DNA complexes (53,54) shows that the 5,6-double bond of the nucleobase thymine in these complexes is accessible to KMnO 4 .…”

Section: Resultssupporting

confidence: 62%

“…Our results show that the cpoly(A) and the cTAR hairpins are formed in the full-length ssDNA. Previous studies showed that short DNAs corresponding to the cTAR sequence (55 or 59 nucleotides) adopt two alternative hairpin conformations in the absence of NC: the closed form and the partially open Y form (20,27,29). Interestingly, our probing data indicate that the cTAR sequence in ssDNA adopts the partially open Y form but not the closed form (conformer 1 in Fig.…”

Section: Discussionmentioning

confidence: 51%

“…Several studies performed with the cTAR sequence (55 to 59 nucleotides) support a dynamic structure of the cTAR hairpin, involving equilibrium between both the closed conformation and the partially open "Y" conformation (20,(25)(26)(27)(28). Recently, we showed that NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the Y conformation exhibiting at least 12 unpaired nucleotides in its lower part (29). We also showed that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively (29).…”

Section: Invmentioning

confidence: 87%

“…Recently, we showed that NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the Y conformation exhibiting at least 12 unpaired nucleotides in its lower part (29). We also showed that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively (29). To date, the interactions between NC and the full-length ssDNA have not been studied.…”

Section: Invmentioning

confidence: 99%

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Structural Insights into the HIV-1 Minus-strand Strong-stop DNA

Chen¹,

Maskri²,

Chaminade³

et al. 2016

Journal of Biological Chemistry

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An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3-end of the genomic RNA with the complementary r region at the 3-end of minusstrand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex).Two strand transfers are required for reverse transcription of the human immunodeficiency virus type 1 (HIV-1) 5 genome(1). These strand transfers are enhanced by the HIV-1 nucleocapsid protein (NC) (2-6). During the first strand transfer, which involves reverse transcriptase (RT) with its RNase H activity to promote degradation of the 5Ј-copied genomic RNA (donor RNA), the 3Ј-end of the minus-strand strong-stop DNA (ssDNA) is transferred to the 3Ј-end of the genomic RNA (acceptor RNA). This transfer can occur in an intra-(i.e. transfer of ssDNA to the original RNA template) or intermolecular (i.e. transfer of ssDNA to the other genomic RNA copy in the virion) manner (1). The first requirement for synthesis of ssDNA is the annealing of the human primer tRNA 3 Lys onto the primer binding site of the genomic RNA (Fig. 1A). The model of reverse transcription presumes that the first strand transfer occurs after synthesis of the full-length ssDNA. More precisely, the transfer is mediated by base pairing of the R region at the 3Ј-end of the genomic RNA with the complementary r region at the 3Ј-end of ssDNA. The effect of shortening the length of complementarity has been studied using murine leukemia virus-based vectors capable of only a single round of replication (7). This study showed that removing more than 80% of the length of 3Ј R, from 69 to 12 nucleotides, did not significantly affect the efficiency of the first strand transfer. These results cannot be extrapolated directly to HIV-1, because the R regions of murine leukemia virus and HIV-1 are different. The effect of shortening the length of 3Ј R has also been studied in HIV-1 (8). This study showed that production of viral particles was barely affected by a...

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Section: Resultssupporting

confidence: 62%

Section: Discussionmentioning

confidence: 51%

Section: Invmentioning

confidence: 87%

Section: Invmentioning

confidence: 99%

See 2 more Smart Citations

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA

Chen¹,

Maskri²,

Chaminade³

et al. 2016

Journal of Biological Chemistry

Self Cite

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show abstract

“…In addition, NC binding to TAR RNA and its role in destabilizing the hairpin during the minus-strand transfer step of reverse transcription is well established (Kanevsky et al 2005(Kanevsky et al , 2011Vo et al 2009b;Levin et al 2010;Heng et al 2012). In an HIV-1 variant that does not depend on Tat-TAR interaction for transcription activation, TAR is not required for gRNA packaging (Das et al 2007), but TAR destabilization leads to aberrant RNA dimerization and packaging (Das et al 2012).…”

Section: Discussionmentioning

confidence: 99%

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Webb

Jones

Parent

et al. 2013

RNA

144

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Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z eff ) and nonelectrostatic (i.e., specific) component of binding, K d(1M) . Gag binds to Psi RNA with a dramatically reduced K d(1M) and lower Z eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z eff . These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

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2022

Structures and Functions of Retroviral RNAs

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Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein

Cited by 20 publications

References 69 publications

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

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