Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution‐state structure of the nucleocapsid protein from HIV‐1

Abstract

All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys‐1His array (CCHC = C‐X2‐C‐X4‐H‐X4‐C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc‐binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc‐edge extended X‐ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus‐bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840–847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys‐1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV‐1 NC protein and have determined its three‐dimensional solution‐state structure. The CCHC arrays of HIV‐1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV‐1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid‐binding results provide a basis for the rational design of new NC‐targeted antiviral agents and vaccines for the control of AIDS.

Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity

“…much attention has focused on the Zn(I1) domains as providing at least a subset of important protein-RNA interactions (Delahunty et al, 1992;Summers et al, 1992).…”

“…The molecular details of RNA binding of any kind by NCs and the role that Zn(1I) plays remain poorly defined. Recent solution structural studies of the Zn(I1)-complexed form of NC from HIV-1 virions (a 55-amino acid protein) and related synthetic peptides reveal that this molecule is remarkably lacking in stable secondary structure outside of the two structurally well-defined Zn(I1)-coordination 23 1 domains (Omichinski et al, 1991;Summers et al, 1992). The zinc-finger structure itself is probably highly conserved in the retroviral NC family (Berg, 1986).…”

“…Because NC shows so little regular structure, a well-defined RNA binding groove or surface is difficult to identify. Recent studies have focussed on the zinc-fingers themselves as primary RNA binding determinants (Delahunty et al, 1992;Summers et al, 1992), but other biochemical and molecular genetic studies (Fu et al, 1988;Cornille et al, 1990;Secnik et al, 1990; suggest the highly positively charged N-terminus and/or interfinger regions may be involved. Indeed, these regions are perhaps ideally suited to potentially adopt one or more defined conformations in the presence of a wide range of RNA structural contexts, exactly the type of binding one would expect to find in sequence nonspecific genome condensation (Churchill & Travers, 1991).…”

RNA-binding activities of barley stripe mosaic virus b fusion proteins

“…From these results, Prats et al (1991) postulated that interactions of the finger motif with zinc influence the conformation requirements of the nucleocapsid protein for specific packaging of the retroviral RNA genome. Additional studies that are relevant to our findings with BSMV suggest that the human immunodeficiency virus (HIV) nucleocapsid protein, which contains two zinc finger-like motifs flanked by basic amino acid residues, has RNA annealing activities that are independent of the activity of the two zinc fingers (Berkowicz et al, 1993;Berkowicz & Goff, 1994;De Rocquigny et al, 1992;Summers et al, 1992). Nonspecific RNA binding is thought to occur via electrostatic and hydrophobic interactions involving basic amino acids with some similarity to those composing the BSMV basic motif, whereas the finger-like regions appear to govern basespecific RNA sequence interactions.…”

DNA binding properties of the zinc‐bound and zinc‐free HIV nucleocapsid protein: supercoiled DNA unwinding and DNA‐protein cleavable complex formation

“…They are also required for the subsequent functions of NC within the mature virions, such as the dimerization of the genomic viral RNA [16] and the binding of the primer tRNA to the primer binding sequence in this genomic RNA [17]. Nucleocapsid proteins from several retroviruses bind singlestranded DNA and RNA [18 20] and are capable of unwinding folded RNA structures by binding selectively to single-stranded RNA [21]. Moreover, the NC of HIV-1 and HTLV-I have recently been shown to be zinc finger proteins capable of binding two Zn 2+ cations for each NC molecule [21].…”