Proof of concept
Successful preparation of HIV-1 RRE DNA templates

Successful acquisition of HIV-1 RRE RNA for in vitro transcription is firstly to obtain high quality HIV-1 RRE template after optimized PCR process and primers by constructed and prepared plasmid. After reacting the designed primers and enzyme selection in PCR, we finally succeeded in obtaining a high-quality HIV-1 RRE DNA template.

Figure 1 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 .

HIV-1 RRE RNA obtained by in vitro transcription

After the successful preparation of HIV-1 RRE DNA template, HIV-1 RRE RNA was obtained by in vitro transcription, and the high quality and stability of HIV-1 RRE RNA was obtained by isolation and purification, thus realizing the stable in vitro transcription of RNA process. The results showed that the stable DNA template provided us with a high-quality template for the preparation of transcribed HIV-1 RRE RNA. After in vitro transcription, isolation and purification, we obtained the transcribed HIV-1 RRE RNA with a single tight band and symmetrical peaks, which can provide the necessary conditions for EMSA experiments of interaction with viral proteins.

Fig. 2 a) Diagram of molecular sieves for HIV-1 RRE RNA isolation and purification, with inset showing gel electrophoresis of 8% TBM of fractions collected from HIV-1 RRE RNA

Fig. 2 b) HIV-1 RRE RNA sequences
HIV-1 Rev protein overexpression, isolation and purification

The results of overexpression, isolation, purification and characterization of HIV-1 Rev protein showed that a single target protein, HIV-1 Rev protein, was obtained after purification, and the purification results showed that the target protein had a single band, which basically meets the requirements for further studies on the interaction between Rev and HIV-1 RRE RNA, as well as the basis for exploring the interaction site, and it can provide a stable sample for interaction with HIV-1 RRE RNA.

Fig. 3 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

Interaction between HIV-1 RRE RNA and HIV-1 Rev protein

Successfully transcribed and prepared HIV-1 RRE RNA and HIV-1 Rev proteins were subjected to gel migration electrophoresis experiments via a gradient series of RNA-to-protein molar ratios as a means of verifying the interactions between the two, and the results showed that the bands of the complexes of HIV-1 RRE RNA and HIV-1 Rev were clearly present, and with the increasing RNA-to-Rev molar ratios, different low and high Rev-RRE multimers could be form different low, medium and high level Rev-RRE multimers until the monomeric RNA is completely consumed to form stable Rev-RRE complexes. The results of full-length HIV-1 RRE RNA and full-length HIV-1 Rev interaction are consistent with the facts reported in the literature, which suggests that the interaction between full-length RNA and full-length Rev proteins is not affected [1], and this also provides a valid basis for further studies on the sites of HIV-1 RNA and viral protein interaction.

Fig. 4. 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.