Effects of the Nature and Concentration of Salt on the Interaction of the HIV-1 Nucleocapsid Protein with SL3 RNA

Abstract

The mature nucleocapsid protein of HIV-1, NCp7, and the NC-domains in gag-precursors are attractive targets for anti-AIDS drug discovery. The stability of the 1:1 complex of NCp7 with a 20mer mimic of stem-loop 3 RNA (SL3, also called psi-RNA, in the packaging domain of genomic RNA) is strongly affected by changes in ionic strength. NC-domains recognize and specifically package genomic HIV-1 RNA, while electrostatic attractions and high concentrations of protein and RNA drive NCp7 to completely coat the RNA in the mature virion. The specific interactions from NCp7-binding to loop bases of SL3 produce 1:1 complexes in solutions that have [NaCl] at or above 0.2 M, while the electrostatic interactions can dominate at and below 0.15 M NaCl, leading to complexes that have mainly 1:2 RNA:protein. Persistent, non-equilibrium mixtures of 1:1 and protein-excess complexes can exist at these lower salt concentrations, where the distribution of complexes depends on the order of addition of RNA and protein. Adding salt causes rapid rearrangement of metastable multi-protein complexes to 1:1. The stability of complexes is also affected by the nature of the added salt, with 0.018 M MgCl 2 and 0.200 M added NaCl producing the same K d (21 ± 2 nM); acetate ion stabilizes the 1:1 complex by more than a factor of two compared to the same concentration of chloride ion. Maintaining a salt concentration of 0.2 M NaCl or 18 mM MgCl 2 is sufficient for experiments to distinguish drug candidates that disrupt the specific SL3-NCp7 interactions in the 1:1 complex. Keywords RNA; HIV-1; nucleocapsid protein; fluorescenceThe nucleocapsid protein of HIV-1 is an attractive anti-AIDS drug target. In addition to its role in packaging the RNA (1-4), it has chaperoning functions (5-6), helps refold the RNA dimer (7-9), and anneals the primer tRNA onto genomic RNA for reverse transcription (10-11). It also interacts with viral proteins including reverse transcriptase (12-13), and the accessory protein, Vpr, to play a role in stable integration of the proviral DNA in the chromosomes of infected cells (14). Drugs targeted at NC have the potential to interfere with critical functions at many stages of the viral infection cycle (6,9,11,(15)(16)(17)(18). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 May 4. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIn retroviruses similar to HIV-1 about 2,000 polyprotein precursors (gag and gag-pol) assemble at the inner membrane of the forming virion (19)(20)(21). Each of these proteins contains a nucleocapsid domain that is required for packaging genomic RNA into new virus particles. The 55 kD gag precursor polyprotein is later processed by the viral protease to structural proteins, including the mature NCp7 (20). NC-domains within gag precursors bind to the RNA with several RNA-NC interactions responsible for full discrimination of genomic from nongenomic RNA (22-27). Both sequence-specific and non-sequence-specific contacts contribut...

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

“…This finding is in agreement with previous studies showing ∼100-fold affinity range for the K d(1M) values for NC binding to 6-nt ssDNA oligos (Vuilleumier et al 1999). NC/TARPolyA and NC/Psi RNA interactions were both characterized by a Z eff of ∼3-4, in agreement with previous studies (Vuilleumier et al 1999;Vo et al 2009a;Athavale et al 2010). Furthermore, the nonelectrostatic binding component of Gag, K d(1M) was even more selective for Psi RNA relative to TARPolyA (∼1000-fold) than NC (Table 1).…”

“…3A) and NC (Fig. 3B) (Rouzina and Bloomfield 1997;Vo et al 2006;Athavale et al 2010), using this analysis K d may be predicted for a particular protein-RNA pair under different buffer conditions. K d(1M) reflects the binding under conditions in which all electrostatic interactions are screened out by salt, and the only contribution to binding comes from specific contacts such as aromatic residue stacking with unpaired nucleic acid bases or specific hydrogen bonds.…”

“…Within ψ, SL1 contains the dimerization initiation site (DIS), a palindromic sequence (GCGCGC in the NL4-3 isolate) responsible for dimerization of the two copies of packaged gRNA (Skripkin et al 1994; for review, see Moore and Hu 2009;Lu et al 2011b). SL2 contains the splice donor site, critical for splicing of the viral RNA for translation of viral accessory proteins, and SL3 is a sequence shown to contribute to gRNA packaging (Clever and Parslow 1997;McBride and Panganiban 1997;Russell et al 2003) and high-affinity NC binding (De Guzman et al 1998;Athavale et al 2010). In HIV-1, genome packaging and dimerization are hypothesized to be coordinated processes, as virions contain only dimeric gRNA (Moore and Hu 2009;Nikolaitchik et al 2013).…”

“…1) that are critical for RNA-binding specificity, nucleic acid chaperone activity Darlix et al 2011), and gRNA packaging (Gorelick et al 1999b;Kafaie et al 2008). Investigations into the RNAbinding specificity of Gag have previously studied the binding of the NC protein to RNA and DNA (Dannull et al 1994;Fisher et al 1998Fisher et al , 2006Vuilleumier et al 1999;Avilov et al 2009;Athavale et al 2010), and more recently the binding of Gag to short oligonucleotides has also been investigated (Stephen et al 2007;Wu et al 2010;Jones et al 2011). In this study, we investigate the binding of Gag and NC to longer ∼100-nt RNAs derived from the gRNA 5 ′ UTR.…”

Flexible Nature and Specific Functions of the HIV-1 Nucleocapsid Protein

“…In-line with the basic character of NCp7 and the strong ionic strength dependence of its binding to numerous oligonucleotides, [82][83][84] NCp7 is able to bind, through an unspecific low-affinity electrostatic mode, to nearly any NA sequence of 5-8 nt in length. This binding mode is certainly the predominant one in the virus, where due to the small size of the viral core, the concentrations of NCp7 and the relevant NAs are in the millimolar range.…”