ated genome from nuclease degradation. In the HIV-1 RNA genome, a G-4 structure has been proposed to promote dimerization of the two viral genome copies. We present here the first comprehensive analysis of putative G-4 forming sequences in 9723957 the HIV-1 nef coding region. We showed that three contiguous putative G-4 regions are present and that at least two are extremely conserved among most circulating HIV-1 strains. We provide evidence of their G-4 folding and stabilization in the presence of cations and G-4 binding compounds by spectroscopic and electrophoretic techniques. Finally, we demonstrate that G-4 ligands impaired Nef expression and significantly suppressed Nef-dependent enhancement of HIV-1 infectivity. Materials and Methods All oligonucleotides were purchased from Sigma-Aldrich. TMPyP4 and PIPER were purchased from Calbiochem,. BRACO-19 was obtained from ENDOTHERM GmbH,. G-4 analysis of the HIV-1 genome The HIV-1 Nef coding region was analyzed by QGRS Mapper for prediction of G-4 forming sequences in both coding and non-coding strands. The putative G-4s were identified by the motif GxNy1GxNy2GxNy3Gx, where x was the number of guanine tetrads and yn the length of loops connecting the G tetrads. The following restrictions have been applied: i) the number of tetrads had to be 2; ii) maximum length of QGRS was set to 30 bases; iii) at most one of the loops was allowed to be of zero length; iv) loop size from 0 to 15. The found QGRS were ranked based on the G-score, which is the likelihood to form a stable G-4, according to the following principles: a) shorter loops are more common than longer loops; b) G-4s tend to have loops roughly equal in size; c) the greater the number of G tetrads, the more stable the G-4. All oligonucleotides were diluted from stock to the final concentration in lithium cacodylate buffer and, where appropriate, KCl 100 mM. All samples were annealed by heating at 95 C for 5 min, gradually cooled to room temperature and measured after 24 h. Compounds at 16 M final concentration were added after DNA annealing. CD spectra were recorded on a Jasco-810 spectropolarimeter equipped with a Peltier temperature controller using a quartz cell of 5-mm optical path length and an instrument scanning speed of 100 nm/min with a KU55933 chemical information response time of 4s over a wavelength range of 230-600 nm. The reported spectrum of each sample represents the average of 2 scans at 20C and is baseline-corrected for signal contributions due to the buffer. Observed ellipticities were converted to mean residue ellipticity = deg 10877822 cm2 dmol-1. For the determination of Tm, spectra were recorded over a temperature range of 20-95 C, with temperature increase of 5 C/min. Tm values were calculated as the first derivative of the melting profiles. For UV thermal unfolding, DNA oligonucleotides were diluted to final concentration of 4 M or 40 M. UV spectra were recorded on Lamba25 UV/Vis spectrometer equipped with a Peltier temperature controller using a quartz cell of 10-mm optical path length and measuring absorbance at 295 nm. UV spectra were recorded over a temperature range of 20-95C, with temperature increase of 1C/min. The autozero function was applied on the corresponding buffer sample at 20C. underlined nucleotides are those complementary to the templates. Primers were 5-end-labeled with ATP using T4 polynucleotide kinase for 30 min at 37C and purified with Illustra MicroSpin G-25 Column. DNA templates were diluted from stock to the final concentration i