Figure 5
Structural analysis of viral DNA via Southern blot. HIV-1IIIB-infected C8166 cells were co-cultured with SJP-L-5 (50, 5, and 0.5 μM) or NVP (1000, 100, and 10 nM) for 48 h. Viral DNA was extracted using Hirt′s method and denatured by boiling in a water bath. The DNA samples were digested with Pst I, subjected to electrophoresis in 0.7% agarose gel with 0.5×TBE and hybridized using DIG-labelled probes. (a) Southern blot with double-stranded DNA detection. The upstream half-genome levels of the plus-strand DNA were not reduced with SJP-L-5 but were reduced with NVP. (b) Southern blot with downstream plus-strand DNA-specific detection. The downstream half-genome levels of the plus-strand DNA were accumulated with SJP-L-5, possibly due to a delay in full-length viral DNA maturation, displaying a different mechanism from that observed with NVP. RAL (1 µM) was used to prevent integration of viral DNA into host chromosomes in all groups. Full-length blots are presented in Supplementary Figure S1. (c) Gray analysis of down-stream plus-strand DNA probe hybridization. Half genomic plus-strand DNA of SJP-L-5 accumulated in a dose-responsive manner, but was maintained at a base level of NVP at different concentrations. Furthermore, the total amounts of viral DNA were accumulated via SJP-L-5 in treatment with 50 and 5 μM. The genomic or 1/2LTR plus-strand DNAs were not affected by SJP-L-5 in a series of concentrations. The total amounts of viral DNA and genomic or 1/2LTR plus-strand DNAs decreased by NVP in a dose-responsive manner.
