Fig. 3
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 “Methods” section. Representative gels can be found in Additional file 1: 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