a) By analyzing the electrophoresis results on the agarose gel Figure 4, we can see that each electrophoretic band is at the location of our target gene. Therefore our team came up with the result that each of our wet team members were able to complete the template experiment and get a stable HIV-1 RRE DNA template.

Figure 4 HIV-1 RRE DNA template agarose electropherogram. The gel above is a 3% gel showing a DNA template near the target position of a gel .

b) The stable DNA template provided a high-quality template for our preparation of transcribed HIV-1 RRE RNA. After in vitro transcription, isolation and purification, the purified peak graph in Figure 5 shows a single symmetrical peak of the transcribed RNA, and the inlay gel electrophoresis indicates that the purified HIV-1 RRE RNA has a single, tight and high quality band, which is essential for EMSA experiments of interaction with viral proteins.

Fig. 5 a) Diagram of molecular sieves for HIV-1 RRE RNA isolation and purification, inset shows 8% TBM plot of gel electrophoresis of fractions collected from HIV-1 RRE RNA; b) HIV-1 RRE RNA sequence

c) HIV-1 Rev protein overexpression, isolation, purification and characterization. HIV-1 Rev protein was cultured at 37°C until the OD600 was at 0.6-0.8, induced with 1 mM IPTG, and overexpressed overnight at 16°C for 12-16 h. Bacteria were collected. Rev was purified on nickel resin by 5M urea denaturation and re-denaturation for nucleic acid removal and gradient imidazole elution of heteroproteins, followed by overnight dialysis and Superdex 75 molecular sieves for purification and target buffer replacement. As can be seen on the 10% SDS-PAGE electropherogram embedded in Figure 6, the purified Rev has a single band, which is basically sufficient for further study of the interaction of Rev with HIV-1 RRE RNA, as well as the basis for exploring the interaction site.

Fig. 6 After dialysising at 4 degree for overnight in the (NH4)2SO4 buffer, HIV-1 Rev is further changed into the store buffer through Superdex 75 column. Inset shows the result of 10% SDS-PAGE gel of fractions.

d)The results of the experimental validation of HIV-1 RRE RNA and HIV-1Rev protein binding to each other are shown in Figure 7, which indicates that bands of HIV-1 RRE RNA complexed with HIV-1 Rev are clearly present and can form low, medium, and high levels of Rev-RRE multimers as the RNA-to-Rev molar ratio continues to increase until the monomeric RNAs are completely consumed to form a stable Rev -RRE complexes. The full-length HIV-1 RRE RNA and full-length HIV-1 Rev interaction results are consistent with the facts reported in the literature, which suggests that the full-length RNA and full-length Rev protein interactions are not affected[1],and this also provides a valid basis for further investigation of the HIV-1 RNA and viral protein sites of action.

Fig. 7. Plot of EMSA electrophoresis results of HIV-1 RRE RNA binding to HIV-1 Rev each other.


[1] Chringma Sherpa, et al. The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication. Nucleic Acids Research, 2015, 43(9) , 4676–4686.