PMC

DLS measurements for HIV-1 and SIV NC proteins in the presence of SIV Psi RNA. The size distributions of NA aggregates formed in the presence of the indicated NC or a no NC control are shown. Each curve represents the average of at least three independent experiments

Sequence and mfold-predicted secondary structure of TAR and Psi RNA constructs used in this study. a HIV-1 TARpolyA. b HIV-1 Psi. c SIVmac TAR. d SIVmac Psi. In all cases, numbering refers to the nt position in gRNA. The box in (a) indicates HIV-1 TAR RNA. The boxes in (b, d) indicate the ∆DIS mutation, where DIS loop residues are replaced with a GNRA-type tetraloop (GAGA) to eliminate dimerization

Plot of the parameters determined from measuring the interaction between HIV-1 NC, SIV NC, and HIV-1 Gag and the indicated HIV-1 and SIV RNAs as a function of salt concentration. The dark gray circles indicate the fitted a K _d(1M) (M = molarity) and b Z _eff parameters from each individual salt-titration experiment, while each light gray bar is the average of at least three independent trials

Sequence and structural features of HIV-1 and SIV NC proteins. a Schematic diagrams of NC proteins: HIV-1 NL4-3 NC and SIVmne NC. Basic residues are colored blue, acidic residues are colored red, the CCHC residues that coordinate the Zn²⁺ ions in the ZFs are colored gray, and the aromatic residue in each finger is underlined. The numbering is based on the sequence of the mature NC protein in each case. b Sequence alignment of HIV-1 and SIV NC proteins. Coloring and underlining are the same as in (a). The boxes indicate the sequence comprising each ZF

a Force-extension curves for dsDNA stretch (solid lines) and return (dashed lines) with no protein and in the presence of 30 nM SIV NC or HIV-1 NC. b, c Dependence of the measured transition slope (b) and hysteresis area ratio (c) on protein concentration (see Additional file ) for HIV-1 NC and SIV NC. The lines in (b) are fits to a simple binding isotherm (Additional file : Eq. 6), revealing equilibrium dissociation constants K _d = 5.5 ± 0.4 nM for SIV NC and K _d = 4.2 ± 0.4 nM for HIV-1 NC. Error bars are standard errors for three or more measurements

a Method for calculating the compaction force (F_c) induced by protein-DNA interactions. Inset shows stretch (solid lines) and return (dashed lines) curves for dsDNA in the absence of protein and in the presence of near saturated (30 nM) HIV-1 NC protein. F_c is calculated in the low force-extension regime denoted within the gray box in the inset and magnified in the main figure. The DNA only extension curve is fit to the WLC model (Additional file : Eq. 1). The force difference (F_c) between the return curve in the presence of high protein concentration and the DNA only stretching curve is averaged over measured extensions <0.31 nm/bp to obtain F_c. b F_c for SIV NC and HIV-1 NC for concentrations of 30 nM and 60 nM. Error bars are standard errors for three or more measurements

Kinetics of SIV minus-strand transfer in the presence of SIV and HIV-1 NC proteins. Reactions were incubated with the indicated concentrations of SIV or HIV-1 NC for 60 min at 37 °C and were analyzed as described in “” section. a Representative gels showing DNA species present in reactions with 1.25 µM SIV or HIV-1 NC. The transfer product (T) and (−) SSDNA are indicated to the left of the gel image and these were the only two bands that appeared on the gel. Note that self-priming products [, , , ] were not formed under the conditions used for these assays. Lane c shows the migration of (−) SSDNA in the absence of other reactants. b, c The % strand transfer product formed was plotted against time of incubation for reactions with SIV NC (b) or HIV-1 NC (c). Error bars represent the SD from three or more independent experiments

Kinetics of minus-strand annealing with SIV and HIV-1 substrates in the presence of SIV and HIV-1 NC proteins. a Reconstituted system used to assay minus-strand annealing and transfer. The diagram shows the acceptor RNA with a portion of U3 and the R sequence at the 3′ end of the viral genome annealed to (−) SSDNA with the complementary r sequence and a portion of u5, complementary to the U5 sequence. For the SIV substrates, the nt lengths of u5, R/r, and U3 sequences are as follows: u5, 20 nt; R/r, 176 nt; and U3, 52 nt. For the HIV-1 substrates, the lengths are: u5, 34 nt; R/r, 94 nt; and U3, 54 nt. The asterisk indicates that the (−) SSDNA is labeled at its 5′ end with ³²P. Annealing of the complementary R regions is indicated by vertical lines. The U3 sequence serves as the template for RT-catalyzed extension of annealed (−) SSDNA. The final DNA transfer product is 248 nt (SIV) or 182 nt (HIV-1). The diagram is not drawn to scale. b-1, b-2, c-1, c-2 Reactions were incubated with SIV (b-1, b-2) or HIV-1 substrates (c-1, c-2) and different concentrations of SIV NC or HIV-1 NC for 30 min at 37 °C and analyzed as described in “” section. Representative gels can be found in Additional file : Fig. S1. The percent (%) annealed product was plotted against time of incubation. Error bars represent the standard deviation (SD) from three or more independent experiments