Formation of Stable and Functional HIV-1 Nucleoprotein Complexesin Vitro

Tanchou, Valérie; Gabus, Caroline; Rogemond, Véronique; Darlix, Jean Luc

doi:10.1006/jmbi.1995.0520

Cited by 103 publications

(119 citation statements)

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“…This could further be facilitated by the high local concentrations of NC and RT in the complexes. Tanchou et al 40 have shown that strand transfer occurs within HIV-1 nucleoprotein complexes in vitro. Further, time-course experiments using donor and acceptor RNAs showed that the long DNA products resulted from internal strand transfer events ( Figure 4), and other experiments showed that a change in processivity was not a factor ( Figure 8).…”

Section: Discussionmentioning

confidence: 99%

“…40,42,43 The complexes were large and could be pelleted by slow speed centrifugation and were found to be competent for DNA synthesis. 40 To test for the formation of aggregates in the reactions that produce long DNAs, the assay was performed using the 1.9 kb RNA as template in the presence or absence of NC. After 1 h the material was centrifuged for 1 min at 12,000g in a microfuge.…”

Section: Determination Of the Rate Of Strand Transfermentioning

confidence: 99%

“…[40][41][42][43] The complexes are functional as isolated complexes can synthesize DNA in the presence of dNTPs, use tRNA to prime viral RNA synthesis, and catalyze minus strand strong-stop strand transfer. 40 As part of a longstanding effort in our laboratory to produce an in vitro system that more closely mimics cellular replication, we describe here conditions that produced DNA products up to 4 kb from genomic RNA of HIV at relatively high efficiency. These reactions included high concentrations of RT and enough NC to completely coat all the nucleic acids in the reaction.…”

Section: Introductionmentioning

confidence: 99%

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In vitro Synthesis of Long DNA Products in Reactions with HIV-RT and Nucleocapsid Protein

Anthony¹,

DeStefano²

2007

Journal of Molecular Biology

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In vitro reaction conditions using HIV reverse transcriptase (RT) and nucleocapsid protein (NC) that allowed efficient synthesis of single-stranded DNA products over a thousand nucleotides in length from genomic HIV RNA were characterized. Consistent with previous reports, the reactions required high concentrations of NC and RT. Long products were produced as a result of frequent strand transfer between RNA templates, averaging at least one transfer per 300 nucleotides synthesized. No change in RT processivity was observed in the reactions in the presence versus absence of NC. Synthesis of long products required formation of a high molecular mass aggregate between NC and nucleic acids. The aggregate formed rapidly and pelleted with low speed centrifugation. The aggregate was accessible to RT as pre-formed aggregates synthesized long products when RT was added. NC finger mutants lacking either finger one or two or with the finger positions switched were all effective in promoting long products. This suggests that the aggregation/condensation but not helix-destabilizing activity of NC was required. We propose that these high molecular mass aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC to close proximity, thereby mimicking the role of the capsid environment within the host cell.

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Section: Discussionmentioning

confidence: 99%

Section: Determination Of the Rate Of Strand Transfermentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In vitro Synthesis of Long DNA Products in Reactions with HIV-RT and Nucleocapsid Protein

Anthony¹,

DeStefano²

2007

Journal of Molecular Biology

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“…In addition to its involvement in dimerization and packaging of genomic RNA and in virus morphogenesis (2), several studies suggested that NCp7 may function as a key element of the reverse transcriptionally active ribonucleoprotein complex. Thus, in vitro, NCp7 was shown (i) to promote annealing of the tRNA 3 Lys to the primer binding site of HIV-1 genome (16,17), (ii) to accelerate minusstrand DNA transfers during proviral DNA synthesis (18 -21), and (iii) to enhance processivity and RNase H activity of the reverse transcriptase (23)(24)(25)(26). Most of these functions are related to the chaperone activity of NCp7 (34), which shows a high affinity for nucleic acids, in particular for single-stranded RNA.…”

Section: Discussionmentioning

confidence: 99%

“…NCp7 was shown to reduce nonspecific reverse transcription (18,21,22) and to enhance the efficiency and processivity of RT (19,(23)(24)(25)(26), suggesting that it could activate reverse transcription through direct interaction with the enzyme (18, 20, 22-24, 26, 27). Accordingly, NCp7 was shown to be capable of re-establishing strand transfer efficiency and RNase H activity of a defective RT mutant (14).…”

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confidence: 99%

Evidence of Interactions between the Nucleocapsid Protein NCp7 and the Reverse Transcriptase of HIV-1

Druillennec

Caneparo

Rocquigny³

et al. 1999

Journal of Biological Chemistry

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The human immunodeficiency virus (HIV-1) nucleocapsid protein NCp7 containing two CX 2 CX 4 HX 4 C-type zinc fingers was proposed to be involved in reverse transcriptase (RT)-catalyzed proviral DNA synthesis through promotion of tRNA 3 Lys annealing to the RNA primer binding site, improvement of DNA strand transfers, and enhancement of RT processivity. The NCp7 structural characteristics are crucial because mutations altering the finger domain conformation led to noninfectious viruses characterized by defects in provirus integration. These findings prompted us to study a putative RT/NCp7 protein-protein interaction. Binding assays using far Western analysis or RT immobilized on beads clearly showed the formation of a complex between NCp7 and RT. The affinity of NCp7 for p66/p51RT was 0.60 M with a 1:1 stoechiometry. This interaction was confirmed by chemical cross-linking and co-immunoprecipitation of the two proteins in a viral environment. Competition experiments using different NCp7 mutants showed that alteration of the finger structure disrupted RT recognition, giving insights into the loss of infectivity of corresponding HIV-1 mutants. Together with structural data on RT, these results suggest that the role of NCp7 could be to enhance RT processivity through stabilization of a p51-induced active form of the p66 subunit and open the way for designing new antiviral agents.

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Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein

et al. 1997

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Formation of Stable and Functional HIV-1 Nucleoprotein Complexesin Vitro

Cited by 103 publications

References 0 publications

In vitro Synthesis of Long DNA Products in Reactions with HIV-RT and Nucleocapsid Protein

In vitro Synthesis of Long DNA Products in Reactions with HIV-RT and Nucleocapsid Protein

Evidence of Interactions between the Nucleocapsid Protein NCp7 and the Reverse Transcriptase of HIV-1

Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein

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