Nucleocapsid (NC) protein possesses nucleotide-annealing activities, which are used in various processes in retroviral life cycle. As conserved characters, the NC proteins have one or two zinc fingers of CX 2 CX 4 HX 4 C motif surrounded by basic amino acid sequences. Requirement of the zinc fingers for the annealing activities of NC protein remains controversial. In this study, we focused the requirement in the process of maturation of dimeric viral RNA. Discrimination between immature and mature dimers of synthetic RNA corresponding to the dimerization initiation site of human immunodeficiency virus type 1 (HIV-1) genomic RNA was performed based on their Mg 2؉ -dependent stability in gel electrophoreses and on their distinct signal pattern from NMR analysis of imino protons. Chaperoning activity of the HIV-1 NC protein, NCp7, and its fragments for maturation of dimeric RNA was investigated using these experimental systems. We found that the two basic regions flanking the N-terminal zinc finger of NCp7, which are connected by two glycine residues instead of the zinc finger, were sufficient, although about 10 times the amounts of peptide were needed in comparison with intact NCp7. Further, it was found that the amount of basic residues rather than the amino acid sequence itself is important for the activity. The zinc fingers may involve the binding affinity and/or such a possible specific binding of NCp7 to dimerization initiation site dimer that leads to the maturation reaction.
Nucleocapsid (NC)1 protein is a component in retroviral particles and takes various functional roles in retrovirus life cycle, involving encapsidation of the genomic RNA, maturation of the dimeric RNA, annealing of tRNA onto primer binding site (PBS) of the viral RNA, protection of reverse transcriptase (RT) pausing, and strand transfer during reverse transcription (for review, see Refs. 1 and 2 and references therein). These functions are associated with the specific binding and the annealing activities to viral RNA. The NC proteins of all retroviruses except those of spumaviruses have one or two zinc fingers of CX 2 CX 4 HX 4 C motif surrounded by basic amino acid sequences. Three-dimensional structures of NC proteins in several retroviruses are known (3-10). The crystal structure of complex of the NC protein and packaging signal of the genomic RNA in the human immunodeficiency virus type 1 (HIV-1) demonstrated two zinc finger knuckles bind to G-rich loop of the RNA in a stem-loop structure and N-terminal basic sequence forms a 3 10 -helix and binds to major groove of stem (5). Alternations and deletions of the zinc fingers impaired the specific RNA binding activity (11-13) and packaging of the viral RNA (11,14), and extinguished viral infectivity (11, 14), but did not much affect nonspecific RNA binding activity (13,15) and the annealing activities such as annealing of complementary DNA and RNA (16), tRNA-PBS annealing (16)(17)(18)(19) strand transfer,(20), and reduction of RT pausing (21). On the other hand, mutations and deletions ...