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Distinct nucleic acid interaction properties of HIV-1 nucleocapsid protein precursor NCp15 explain reduced viral infectivity.

Wang W, Naiyer N, Mitra M, Li J, Williams MC, Rouzina I, Gorelick RJ, Wu Z, Musier-Forsyth K - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Center for Retrovirus Research and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.

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Annealing of 10-nM human tRNALys3 to 25-nM HIV-1 shortPBS as a function of time in the presence of different forms of HIV-1 NC. (A) Comparison of the kinetics of annealing in the presence of the indicated concentration of NCp7, NCp9 and NCp15. (B) Comparison of the annealing kinetics in the presence of 600-nM NCp9, WT NCp15 and NCp15 acidic residue variants. (C) Effect on annealing of negatively charged peptides HIV-1 p6 and polyglutamic acid or neutral peptide CA helix 11.
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Figure 3: Annealing of 10-nM human tRNALys3 to 25-nM HIV-1 shortPBS as a function of time in the presence of different forms of HIV-1 NC. (A) Comparison of the kinetics of annealing in the presence of the indicated concentration of NCp7, NCp9 and NCp15. (B) Comparison of the annealing kinetics in the presence of 600-nM NCp9, WT NCp15 and NCp15 acidic residue variants. (C) Effect on annealing of negatively charged peptides HIV-1 p6 and polyglutamic acid or neutral peptide CA helix 11.

Mentions: We next compared the NA annealing activities of NCp7, NCp9 and NCp15 (29,71,77). In these assays, 32P-labeled in vitro transcribed human tRNALys3 (76 nt) was incubated with shortPBS (105 nt) in the presence of different forms of HIV-1 NC (Figure 1). Time-course annealing assays at 600-nM protein concentration showed that NCp15 can anneal human tRNALys3 to the shortPBS at a similar rate as the mature NCp7 and the reactions reach similar extents of annealed product (Figure 3A and Table 2; gel examples are shown in Supplementary Figure S2). Surprisingly, HIV-1 NCp9 is the best annealing agent among the three forms of HIV-1 NC. However, increasing the protein concentration of NCp7 and NCp15 to 2 μM resulted in increased rates and extents of annealing that approached the level of NCp9 (Figure 3A and Table 2). Immunoblots of samples taken from WT HIV-1 failed to detect NCp15 in rapid harvest (30 min) or 46-h virus (Supplementary Figure S3). In contrast, whereas 46-h virus contained only NCp7, rapid harvest virus particles contained a mixture of NCp7 and what appears to be NCp9 as the migration distance is similar to that of purified recombinant NCp9 (Supplementary Figure S3). Based on these findings in rapid harvest virus, preliminary annealing assays with mixtures of NC protein were also carried out. Adding NCp7 or NCp15 (600 nM or 2 μM) to reactions containing 600-nM NCp9 does not negatively affect annealing (Supplementary Figure S4).


Distinct nucleic acid interaction properties of HIV-1 nucleocapsid protein precursor NCp15 explain reduced viral infectivity.

Wang W, Naiyer N, Mitra M, Li J, Williams MC, Rouzina I, Gorelick RJ, Wu Z, Musier-Forsyth K - Nucleic Acids Res. (2014)

Annealing of 10-nM human tRNALys3 to 25-nM HIV-1 shortPBS as a function of time in the presence of different forms of HIV-1 NC. (A) Comparison of the kinetics of annealing in the presence of the indicated concentration of NCp7, NCp9 and NCp15. (B) Comparison of the annealing kinetics in the presence of 600-nM NCp9, WT NCp15 and NCp15 acidic residue variants. (C) Effect on annealing of negatively charged peptides HIV-1 p6 and polyglutamic acid or neutral peptide CA helix 11.
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Related In: Results  -  Collection

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Figure 3: Annealing of 10-nM human tRNALys3 to 25-nM HIV-1 shortPBS as a function of time in the presence of different forms of HIV-1 NC. (A) Comparison of the kinetics of annealing in the presence of the indicated concentration of NCp7, NCp9 and NCp15. (B) Comparison of the annealing kinetics in the presence of 600-nM NCp9, WT NCp15 and NCp15 acidic residue variants. (C) Effect on annealing of negatively charged peptides HIV-1 p6 and polyglutamic acid or neutral peptide CA helix 11.
Mentions: We next compared the NA annealing activities of NCp7, NCp9 and NCp15 (29,71,77). In these assays, 32P-labeled in vitro transcribed human tRNALys3 (76 nt) was incubated with shortPBS (105 nt) in the presence of different forms of HIV-1 NC (Figure 1). Time-course annealing assays at 600-nM protein concentration showed that NCp15 can anneal human tRNALys3 to the shortPBS at a similar rate as the mature NCp7 and the reactions reach similar extents of annealed product (Figure 3A and Table 2; gel examples are shown in Supplementary Figure S2). Surprisingly, HIV-1 NCp9 is the best annealing agent among the three forms of HIV-1 NC. However, increasing the protein concentration of NCp7 and NCp15 to 2 μM resulted in increased rates and extents of annealing that approached the level of NCp9 (Figure 3A and Table 2). Immunoblots of samples taken from WT HIV-1 failed to detect NCp15 in rapid harvest (30 min) or 46-h virus (Supplementary Figure S3). In contrast, whereas 46-h virus contained only NCp7, rapid harvest virus particles contained a mixture of NCp7 and what appears to be NCp9 as the migration distance is similar to that of purified recombinant NCp9 (Supplementary Figure S3). Based on these findings in rapid harvest virus, preliminary annealing assays with mixtures of NC protein were also carried out. Adding NCp7 or NCp15 (600 nM or 2 μM) to reactions containing 600-nM NCp9 does not negatively affect annealing (Supplementary Figure S4).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus