Fig. 5

RNA chromatin immunoprecipitation (ChIP) assay to determine the interaction of ZIKV protein with viral RNA. a The diagram of ZIKV genome and the area covered by the primers listed in Table 2: 5′ side, from 1 to 1051; 3′ side, from 10,031 to 10,776. b RNA ChIP assay examined by regular RT-PCR. The Vero cells were infected with ZIKV MR766 at an MOI of 0.5 for 24 h. Then the cells were fixed with 4% formaldehyde. The cells were then sonicated to shear the RNA to 250–500 nt. The samples were then divided into 3 part: 1) input, 2) normal mouse IgG-precipitation, and 3) anti-ZIKV antibody precipitation. In both 2) and 3), the RNA-protein complex was pulled down using an anti-ZIKV mouse serum generated from a ZIKV-infected mouse or the normal mouse IgG (as negative control) with the beads. After washing for 3 cycles, the protein was digested with proteinase K, and the DNA was removed by DNase I (RNase free). The input RNA and the ChIPed RNA samples were then precipitated and the samples were used for RT-PCR. The PCR products were applied to run an agarose gel and visualized. c RNA ChIP assay examined by real-time RT-PCR. The same as B, but used a real-time RT-PCR. The yields of PCR from ChIPed RNA or from input RNA were normalized to IgG. The ratio of PCR products from ChIPed RNA were then compared to the input shown as % Input