Phosphorescence and Optically Detected Magnetic Resonance Investigation of the Binding of the Nucleocapsid Protein of the Human Immunodeficiency Virus Type 1 and Related Peptides to RNA

Lam, Wai-Chung; Maki, August H.; Casas‐Finet, José R.; Erickson, John W.; Kane, Bradley; Sowder, Raymond C.; Henderson, Louis E.

doi:10.1021/bi00201a017

Cited by 29 publications

(42 citation statements)

References 24 publications

(30 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The isolated different domains of the (12-53)NCp7 are unable to anneal tRNA 3 Lys to RNA-(77-257). Fixed concentrations of [ 33 P]tRNA 3 Lys and RNA-(77-257) (5.10 Ϫ12 mol of each denaturated RNA/ assay) were incubated in a buffer with 25 mM Tris-HCl (pH 7.5), 0.1 mM MgCl 2 , and 80 mM NaCl, for 30 min at 37°C, in the presence of 20, 80, and 150 molar equivalents of (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) NCp7 (lanes 2-4, respectively), (34 -51)NCp7 (lanes 5-7, respectively), and (13-56)NCp7dd (lanes 8 -10, respectively). Lane 1 shows the free tRNA 3 Lys .…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, several studies have underlined the importance of Trp 37 in HIV-1 (19,26,27), its replacement by a nonaromatic amino acid leading to a loss of viral infectivity. When, Trp 37 was replaced by a hydrophobic amino acid such as leucine in Leu 37 (12-53)NCp7, no ternary complex was observed, illustrating the crucial importance of Trp for the NCp7 hybridase activity.…”

Section: Ncp7 Zinc Fingers Are Required For Trna Annealing 4821mentioning

confidence: 99%

See 1 more Smart Citation

The Annealing of tRNA3Lys to Human Immunodeficiency Virus Type 1 Primer Binding Site Is Critically Dependent on the NCp7 Zinc Fingers Structure

Remy¹,

Rocquigny²,

Petitjean³

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

The nucleocapsid protein NCp7 of the human immunodeficiency virus type 1 contains two zinc fingers of the CX 2 CX 4 HX 4 C type, flanked by several basic residues, and plays a major role in viral infectivity. Thus, NCp7 was shown to promote annealing of the tRNA 3 Lys to the primer binding site, a key step in reverse transcription. However, previous in vitro experiments were unable to clarify the role of the zinc fingers in this process, due to nucleic acid aggregation induced by the basic N-and C-terminal domains of NCp7. We show here that deletion of these sequences in (12-53)NCp7 strongly reduces the formation of aggregates and allows a direct visualization of the binary or ternary complexes between NCp7 and nucleic acids by gel electrophoresis. (12-53)NCp7 is able to induce hybridization of the 33 P tRNA 3 Lys and the human immunodeficiency virus type 1 viral RNA-(77-257), which contains the primer binding site. Modification of the proximal zinc finger conformation in Cys 23 (12-53)NCp7 led to a large reduction in this hybridization process, while replacement of Trp 37 by Leu in the distal zinc fingers resulted in a complete absence of annealing activity. These data account for the in vivo loss of viral infectivity following these mutations and emphasize the critical role of the structure of the zinc finger domain of NCp7. This could facilitate a rational approach to new antiviral agents directed toward NCp7.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Ncp7 Zinc Fingers Are Required For Trna Annealing 4821mentioning

confidence: 99%

The Annealing of tRNA3Lys to Human Immunodeficiency Virus Type 1 Primer Binding Site Is Critically Dependent on the NCp7 Zinc Fingers Structure

Remy¹,

Rocquigny²,

Petitjean³

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The peptide VKCFNCGKEG-HIARNCRA and similar peptides have been extensively studied by numerous investigators as model zinc fingers (2,(11)(12)(13)(14)(15)(16)(17). This peptide has been shown to adopt zinc finger-like structure and bind double-stranded deoxyribonucleotides in solution (18).…”

Section: Binding Of Cobalt Complexes To a Model Zinc Finger Peptidementioning

confidence: 99%

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Louie

Meade

1998

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1 H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.Zinc finger domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated about a zinc ion. Detailed structural information supports the existence of multiple classes of zinc fingers (1-3), and these include domains in which the zinc ion is coordinated by Cys 2 His 2 (e.g., TFIIIA), Cys 3 His (e.g., retroviral nucleocapsid proteins), or Cys 4 (e.g., glucocorticoid receptor). The zinc finger motif is crucial for specific DNA recognition by zinc finger transcription factors.In this work, we describe a family of cobalt(III)-Schiff-base complexes [Co(III)-sb] that are effective zinc finger inhibitors. Related complexes were shown to have potent antiinflammatory properties (4). The general structure of this class of complexes is shown in Fig. 1. We investigated the effect of a series of Co(III) complexes, cobalt(III) complex of bis(acetylacetone)ethylenediimine [acacen], reacted with zinc finger proteins including a synthetic peptide representing the first zinc finger region from HIV-1 nucleocapsid protein (NCp7) and Sp1, a human zinc finger transcription factor (5). CD and 1 H NMR studies were performed to determine perturbations to zinc finger structure caused by reaction with the Co(III)-sb complexes. Gel-shift and filter-binding assays revealed that the Co(III)-sb effectively inhibited DNA binding by Sp1 and demonstrated that the Co(III)-sb could be specifically targeted by covalent attachment to an Sp1 recognition oligonucleotide. MATERIALS AND METHODSSynthesis of Cobalt Complexes. The syntheses of cobalt(III)-Sb complexes were performed as described (6). Synthesis of Oligonucleotides.Oligonucleotides were prepared on an Applied Biosystems 394 synthesizer, with addition of the phosporamidite of 2Ј-amino-2Ј-deoxyuridine as the terminal base for the sense strand and deprotected overnight at 55°C. DMT-2Ј-N-trifluoroacetyl-protected phosphoramidite was synthesized as reported (7). Oligonucleotides were purified over C18 SepPak columns (Waters)....

show abstract

“…For instance, binding of an intercalating drug induces a specific modification of the 260-nm band in the RNA transactivation response element RNA CD spectrum (28). Phosphorescence and optically detected magnetic resonance studies reported modifications of tRNA base stacking induced by NCp7 binding (29). Moreover, RNA unwinding should induce more significant changes in CD spectra.…”

Section: /Hismentioning

confidence: 99%

No tRNA3Lys Unwinding in a Complex with HIV NCp7

Grégoire¹,

Gautheret²,

Loret³

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

Phosphorescence and Optically Detected Magnetic Resonance Investigation of the Binding of the Nucleocapsid Protein of the Human Immunodeficiency Virus Type 1 and Related Peptides to RNA

Cited by 29 publications

References 24 publications

The Annealing of tRNA3Lys to Human Immunodeficiency Virus Type 1 Primer Binding Site Is Critically Dependent on the NCp7 Zinc Fingers Structure

The Annealing of tRNA3Lys to Human Immunodeficiency Virus Type 1 Primer Binding Site Is Critically Dependent on the NCp7 Zinc Fingers Structure

A cobalt complex that selectively disrupts the structure and function of zinc fingers

No tRNA3Lys Unwinding in a Complex with HIV NCp7

Contact Info

Product

Resources

About