Distinct nucleic acid interaction properties of HIV-1 nucleocapsid protein precursor NCp15 explain reduced viral infectivity.
Bottom Line: To understand the strict requirement for NCp15 processing, we compared the chaperone function of the three forms of NC.Dynamic light scattering studies reveal that NCp15 forms much smaller aggregates relative to those formed by NCp7 and NCp9.Neutralizing the acidic residues in p6 improves the annealing and aggregation activity of NCp15 to the level of NCp9 and increases the protein-NA aggregate size.
Affiliation: Department of Chemistry and Biochemistry, Center for Retrovirus Research and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.Show MeSH
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Mentions: The NA chaperone function of retroviral NC proteins depends on two major properties—the capability of the protein to destabilize stable duplex structures and to aggregate NAs (14,16,17,24). Fast NA interaction kinetics also plays a role in chaperone function (84). To further characterize the chaperone activity of NC, Förster resonance energy transfer-based cTAR DNA hairpin destabilization assays were adopted to evaluate HIV-1 NC's duplex destabilization activity (29,50,78). When NC interacts with cTAR, up to four populations of DNA can be detected [a ‘dark state’ (α0) and three more open states (α1, α2, α3)]. The ‘dark state’ is completely closed and becomes less populated with increasing binding of NC, while the open states become more populated as a result of NC's preferential binding to ssDNA. Figure 4 shows a plot of α0 (Figure 4A) and α3, which is the most open state (Figure 4B), as a function of NC concentration. Based on these data, we conclude that the three forms of NC show comparable duplex destabilization activity. High concentrations of NCp9 induced aggregation of the DNA and thus the destabilization activity of NCp9 cannot be studied at the highest protein concentrations. (Supplementary Figure S5 summarizes the population shifts observed at the highest NC concentration used for all three forms of NC.)
Affiliation: Department of Chemistry and Biochemistry, Center for Retrovirus Research and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.