Conformational and Nucleic Acid Binding Studies on the Synthetic Nucleocapsid Protein of HIV-1

Surovoy, Andrej; Dannull, Jens; Moelling, Karin; Jung, Günther

doi:10.1006/jmbi.1993.1011

Cited by 62 publications

(72 citation statements)

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“…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%

“…In the case of HIV-1, the mature NC product, NCp7 (12), is a small basic protein (72 amino acids) containing two copies of a conserved zinc finger-like domain with the sequence Cys-X 2 -Cys-X 4 -His-X 4 -Cys, designated as the CCHC motif (13). NCp7 has been synthesized by a stepwise solid phase method (14,15), and the synthetic protein shown to possess all the in vitro biological activities of the native protein extracted from virions (14). NMR studies have shown that, whereas the N-and C-terminal parts of the protein are flexible, the finger domains are folded around the divalent ion (16,17).…”

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confidence: 99%

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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

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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.

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Section: Discussionmentioning

confidence: 99%

mentioning

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

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“…Numerous studies looking at the importance of the NC zinc finger motifs have generally concluded that this domain is required for the proper selection and packaging of genomic RNA but that the motif is dispensable for such activities as RNA dimerization, annealing of the tRNA to genomic RNA, strand renaturation, and nonspecific RNA binding (25,51,52,(63)(64)(65). On the other hand, several studies have provided evidence that the zinc finger domain is critical for viral infectivity beyond the requirement for proper RNA packaging.…”

Section: Discussionmentioning

confidence: 99%

“…NC is a small, basic protein that possesses either one or two zinc finger motifs in conventional retroviruses. NC binds nucleic acids with some apparent cooperativity, shows a higher binding affinity for RNA over DNA with a preference for single strands, and promotes renaturation between complementary nucleic acid chains (25)(26)(27)(28)(29)(30)(31). These properties are reminiscent of those associated with SSBs (32)(33)(34), thus leading to the proposal that NC may serve to facilitate reverse transcription.…”

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confidence: 99%

Characterization of RNA Strand Displacement Synthesis by Moloney Murine Leukemia Virus Reverse Transcriptase

Kelleher

Champoux

1998

Journal of Biological Chemistry

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The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H ؊ mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 5-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H ؊ RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain.Retroviral replication requires the single-stranded RNA genome of the virus to be converted into double-stranded DNA through a complex series of reactions termed reverse transcription. This process appears to be catalyzed solely by the viral reverse transcriptase (RT) 1 that possesses the following two distinct enzymatic activities: a polymerase activity that synthesizes DNA using either RNA or DNA templates, and an RNase H activity that cleaves RNA in hybrid duplex with DNA (1, 2).The current model of reverse transcription proposes that the RNase H activity of RT is critical for several steps including degradation of the 5Ј end of the RNA genome following minusstrong-stop DNA synthesis to facilitate the first jump, specific cleavage at the polypurine tract to create the plus strand primer, and removal of the plus and minus strand primers (3). Additionally, it is presumed that the RNase H activity is required to degrade the RNA genome following minus strand synthesis to free the minus strand DNA for use as a template during plus strand synthesis (reviewed in Ref. 3). In vitro studies, however, suggest that RNA fragments that are too large to spontaneously dissociate from the minus strand remain following cleavage of the genome (4 -11). Furthermore, evidence that plus strand synthesis in several retroviral systems is discontinuous demonstrates that stably annealed RNA fragments persist in vivo (3,(12)(13)(14). This raises the interesting possibility that reverse transcription requires a mechanism for the displacement of genomic RNA fragments during plus strand synthesis.Most replicative polymerases require accessory proteins such as helicases and single-strand binding proteins (SSBs) ...

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“…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.

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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)....

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Conformational and Nucleic Acid Binding Studies on the Synthetic Nucleocapsid Protein of HIV-1

Cited by 62 publications

References 0 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

Characterization of RNA Strand Displacement Synthesis by Moloney Murine Leukemia Virus Reverse Transcriptase

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

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