A Nucleic Acid Switch Triggered by the HIV-1 Nucleocapsid Protein

DeCiantis, Christopher L.; Jensen, Danielle K.; Hudson, Bruce S.; Borer, Philip N.

doi:10.1021/bi700031j

Cited by 1 publication

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“…Implications for Drug Discovery. Beyond an interest in accurately defining K d and conditions for study of the 1:1 complex, the simple expedient of comparing affinities at 0.2 M NaCl or 18 mM MgCl 2 is sufficient to suppress the non-sequencespecific interactions to the point that anti-NC agents can be found to interrupt the specific interactions (43,52,53).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2010

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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...

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Section: Discussionmentioning

confidence: 99%