Abstract
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.
Highlights
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
Rong et al [30] reported on functional difference of RNA complexes annealed by NC protein and its mutants. They showed that NC protein devoid of the zinc fingers could anneal tRNA onto the viral RNA, but the resultant primer-template complex was not elongated by reverse transcriptase (RT) to the full-length negative-strand of strongstop DNA, unlike the primer-template complex yielded by wildtype NC protein
The immature dimer and NCp7-converted mature dimer of DIS39 can be distinguished by their different Mg2ϩ-dependent stabilities when they are subjected to gel electrophoreses with and without Mg2ϩ
Summary
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. As reported in the previous study [40], DIS39 mostly forms the kissing-loop dimer by itself at 37 °C (Fig. 2, lane 1) and is converted into the extended-duplex dimer when incubated with an equimolar NCp7 at 37 °C (Fig. 2, lane 2).
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