Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

Abstract

The nucleocapsid (NC) protein of human immunodeficiency virus HIV‐1 (NCp7) is responsible for packaging the viral RNA by recognizing a packaging site (PSI) on the viral RNA genome. NCp7 is a molecule of 55 amino acids containing two zinc fingers, with only the first one being highly conserved among retroviruses. The first zinc finger is flanked by two basic amino acid clusters. Here we demonstrate that chemically synthesized NCp7 specifically binds to viral RNA containing the PSI using competitive filter binding assays. Deletion of the PSI from the RNA abrogates this effect. The 35 N‐terminal amino acids of NCp7, comprising the first zinc finger, are sufficient for specific RNA binding. Chemically synthesized mutants of the first zinc finger demonstrate that the amino acid residues C‐C‐C/H‐C/H are required for specific RNA binding and zinc coordination. Amino acid residues F16 and T24, but not K20, E21 and G22, located within this zinc finger, are essential for specific RNA binding as well. The second zinc finger cannot replace the first one. Furthermore, mutations in the basic amino acid residues flanking the first zinc finger demonstrate that R3, 7, 10, 29 and 32 but not K11, 14, 33 and 34 are also essential for specific binding. Specific binding to viral RNA is also observed with recombinant NCp15 and Pr55Gag. The results demonstrate for the first time specific interaction of a retroviral NC protein with its PSI RNA in vitro.

Binding properties of human immunodeficiency virus type‐2 (HIV‐2) rna corresponding to the packaging signal to its nucleocapsid protein

“…ldentifieatlon of the specific binding region of the NCpS-derived sequence In several previous investigations into the interaction between HIV-1 RNA containing the packaging signal and HIV-1 NCp7, the specific binding regions in the amino acid sequence of NCp7 identified were not always identical (17,18). Our previous investigation using HIV-2…”

“…One of the candidate viral proteins in HIV-1 is the nucleocapsid (NC) protein NCp7, which is located within the Gag polyprotein NCp7-derived synthetic peptides demonstrated that the reactivity of the second ZFM flanked by basic amino acid clusters with the packaging signal on viral RNA was very weak and non-specif'fc and that just the first ZFM flanked by basic amino acid clusters was enough for specific RNA binding (1,11,17). On the other hand, a gel mobility shift assay was used recently to measure in vitro binding of the HIV-1 Gag proteins expressed as fusion proteins with glutathione Stransferase to viral RNA containing the packaging signal (18).…”

An Extended RNA Binding Site for the Yeast Branch Point-binding Protein and the Role of Its Zinc Knuckle Domains in RNA Binding

“…Zinc knuckles have been shown to be directly involved in RNA binding in other proteins. The best studied example is the HIV-1 nucleocapsid protein, which contains two zinc knuckles and binds single-stranded and stem-loop RNAs that are important for HIV genome recognition and encapsidation (23)(24)(25)(26)(27)(28)(29)(30). The human SR protein 9G8, also a splicing factor, binds the 3Ј splice site, and its zinc knuckle is important in RNA binding and in defining its specificity (31).…”

The gag proteins of human immunodeficiency virus type 1: mechanisms of virus assembly and possibilities for interference

“…During the following steps of HIV assembly two events have to occur: first, the viral mRNA genome must interact via its r which is located upstream of the start of the open reading frame coding for the gag protein and is present only in complete and unspliced messengers, with the RNA-binding domain in the nucleocapsid protein portion of the Pr55gag polyprotein [19]. This region shows similarities to nucleic acid binding zink-finger motifs [20][21][22][23]. Second, the individual protein molecules -Pr55gag, Prl60gag-pol, and gpl20/gp41 -interact to form clusters with high concentrations of the indicated proteins at the lipid bilayer [24,25].…”