Nucleic Acid Binding Properties of the Simian Immunodeficiency Virus Nucleocapsid Protein NCp8

Abstract

The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G ϳ I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence-specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes.

Rous Sarcoma Virus Gag Protein-Oligonucleotide Interaction Suggests a Critical Role for Protein Dimer Formation in Assembly

“…From pairwise comparisons of the K a of the protein with three GT oligonucleotides, the binding site size of ⌬MBD⌬PR-Zn was estimated to be ca. 8 nt (Table 3), a finding consistent with the results previously reported for retroviral NC proteins (32,56,57,67). If we assume that the binding site sizes of ⌬MBD⌬PR and ⌬MBD⌬PR-Zn are 12 and 8 nt, respectively, the intrinsic binding constants for the two forms of the protein would be similar, approximately 0.7 ϫ 10 5 to 1.7 ϫ 10 5 M Ϫ1 , a value that also is consistent with the binding constant previously reported for various retroviral NC proteins at 100 mM NaCl (32,67).…”

“…The conclusion that this ratio reflects the binding site size of the protein is indirect, being based on the assumption that in the particles all protein molecules contact nucleic acid in the same way and cover the entire nucleic acid used in assembly. However, all published calculations of the binding site size of retroviral NC proteins are in the range of 5 to 8 nt, smaller than the value we estimate here for ⌬MBD⌬PR (20,32,56,57,67). We envisioned two possible explanations for this discrepancy.…”

“…Fluorescence-quenching measurements for MLV NC and RSV NC originally indicated that that protein covers approximately 6 to 8 nt (32). Later, HIV-1 and SIV NC were shown to have similar binding site sizes (56,57,67). In the presence of EDTA, the binding site size of HIV-1 NC was reported to increase from 6 to 9 nt, paralleling our observation with ⌬MBD⌬PR (56).…”

Distinct Roles for Nucleic Acid in In Vitro Assembly of Purified Mason-Pfizer Monkey Virus CANC Proteins

“…Calculations based on experimental data show that the RSV NC binding site for nucleic acids spans eight nucleotides and therefore a 16-mer should be sufficient for bridging together two NC molecules (53,54). HIV-1 and simian immunodeficiency virus NC were shown to have binding site sizes similar to those of RSV NC (73,74,80). For HIV-1 it has been proposed that binding of nucleic acid to NC concentrates the Gag polyproteins and induces CA-CA interactions that lead subsequently to the assembly of immature capsids.…”

dUTPase and Nucleocapsid Polypeptides of the Mason-Pfizer Monkey Virus Form a Fusion Protein in the Virion with Homotrimeric Organization and Low Catalytic Efficiency

“…37 and 38). Other studies of DNA-NC protein interaction reported high preference of NC for binding to TG dinucleotide repeats (39,40). Therefore the octanucleotide (TG) 4 was also synthesized and used in parallel experiments.…”