Monomer−Dimer Equilibrium Constants of RNA in the Dimer Initiation Site of Human Immunodeficiency Virus Type 1

Abstract

The genome of the human immunodeficiency virus (HIV) exists as a dimer of two identical RNA molecules hydrogen bonded to each other near their 5' ends. The dimer, known to be important for viral infectivity, is formed by two monomers interacting through a stem-loop structure called the dimer initiation site (DIS). An initially formed intermediate, the "kissing" dimer, is unstable and rearranges to the stable, duplex form. In this report we use nondenaturing polyacrylamide gel electrophoresis to measure the monomer-dimer equilibrium constant of three RNA sequences, 41-, 27-, and 19-mers, located in the DIS of the MAL isolate of HIV-1. Experiments in which the RNA was equilibrated at various temperatures before electrophoresis revealed that interconversion is rapid for all the sequences, so that they reach equilibrium in the loading well of the gel at 5 degrees C before they enter the gel proper. However, interconversion kinetics in the gel are slow, so autoradiographic spot intensities can be used to measure the amounts of monomer and dimer present when the sample entered the gel. After correction for the amount of RNA added with the radiolabel and dilution of samples in the loading well of the gel, dimerization equilibrium constants were calculated from spot intensities. The calculated values of the dimerization constant K at 5 degrees C were approximately 10(5), approximately 10(6), and approximately 10(8) M(-1) for the 41-, 27-, and 19-mers, respectively, in solutions of ionic strength, I, of about 100 mM. The decrease in K by three orders of magnitude between the 19-mer and 41-mer is due in part to the change in rotational entropy of rodlike molecules on dimerization and in part to the increased conformational entropy of the monomers. As expected, increased ionic strength increases the dimerization constant for all three RNAs. For the 41-mer, however, K has a maximum value at I approximately 140 mM. The origin of the decrease in K for higher I is unknown but it may be due to formation of species (perhaps higher order oligomers) that do not enter the gel. The 41-mer exists in two dimeric forms assigned to the kissing and duplex dimers. The ratio of kissing to duplex form at 5 degrees C is 0.48 +/- 0.22 at I = 113 mM and 0.91 +/- 0.35 at I = 183 mM. The observed decrease in K with RNA length suggests that the dimerization constant of the packaging region of HIV-1 is small, < approximately 10(5) M(-1), implying that the nucleocapsid protein is important in promoting dimerization in the capsid of the virus.

HIV-1 Nucleocapsid Protein Switches the Pathway of Transactivation Response Element RNA/DNA Annealing from Loop–Loop “Kissing” to “Zipper”

“…Interestingly, TAR annealing is ∼10-fold slower than mini-TAR annealing in the absence of protein chaperone 30. We hypothesize that this is due to the larger entropy loss upon intermediate complex formation in the case of the longer hairpins 44. Alternatively, slower annealing of longer hairpins may be due to a greater electrostatic repulsion.…”

Effect of Mg2+ and Na+ on the Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein: Implications for Reverse Transcription

“…The fact that this difference between mini and full-length TAR hairpins does not disappear in higher salt suggests that the difference is not due to the stronger electrostatic repulsion between the longer molecules. Shubsda et al also observed slower association of longer hairpins, and attributed this difference to the larger entropy loss upon complex formation 73.…”

Regulation of primate lentiviral RNA dimerization by structural entrapment

“…Tight dimers withstand dissociation during semi-native gel electrophoresis (Tris-borate EDTA (TBE) buffer at 26°C). By contrast, loose dimers are detected only by using native conditions, since they dissociate upon loading in semi-native TBE/26°C gels [33]. Loose dimers are thought to correspond to a loop-loop interaction between two SL1 motifs, whereas tight dimers are thought to represent a more extensive SL1–SL1 interaction due to the intra- to intermolecular conversion of the stems located below the SL1 loop ([2,4]).…”