Our project provides a concrete research program for basic research on retroviral non-coding long-strand RNA and its viral proteins. HIV-1 RRE RNA aims to efficiently transcribe RNA with high quality and interact with viral Rev proteins to determine whether non-coding long-strand RNA is stably transcribed in vitro, to provide a reliable reference condition for, for example, RNA vaccine development and to identify targeting sites through interaction with viral proteins to provide a favorable molecular mechanism for target drug development. Interact with viral proteins to identify targeting sites and provide favorable molecular mechanisms for targeted drug development. The following describes how we designed the HIV-1 RRE RNA and the viral Rev protein (see Figure 1).

With the rapid development of life sciences, more and more evidence shows that the development of a series of major diseases (hepatitis C, AIDS, encephalitis, myelitis, tumors, etc.) is related to the imbalance of non-coding RNA regulation. Meanwhile, RNAs longer than 200nt are called long chain non-coding RNAs (long chain RNAs), which have mRNA-like structures, are spliced, and participate in the regulation of various processes in the body, and play an important role in genome defense, RNA generation, modification, splicing, gene expression regulation and other processes[1]. However, retroviral replication and translocation of endogenous reverse transcription factors take advantage of a unique post-transcriptional regulatory mechanism of partially spliced/unspliced mRNA export into the cytoplasm, such that these viral mRNAs, as genomic RNAs, will be packaged into zygotic viral particles, which will then ultimately be exported to the nucleus through a high degree of coordination of structural interactions of the RNA genomes with a wide range of specialized viral proteins and host proteins[2]. HIV-1 RRE RNA, as a long-stranded noncoding RNA, is not yet sufficiently well documented to study the mechanism of action of full-length RRE RNA together with full-length viral Rev proteins, suggesting that additional studies are needed.

HIV RNA Selection criteria:

We chose the genome of HIV-1 as the subject of our HIV study. HIV-1, as the most common and highly infectious subtype, accounts for more than 95% of global infections and is the main infector of AIDS in our country. Meanwhile, HIV has the ability to mutate over time like other viruses, and its HIV-1 isolate ARV-2/SF2 strain belongs to the major epidemic subgroup, which is mainly responsible for the global HIV epidemic. Therefore, we specifically chose the genome of the HIV-1 isolate ARV-2/SF2 strain because the long-term lack of an effective vaccine and the unsatisfactory results of integrated nuclease therapy to contain the disease is a very worrisome scenario, and can also lead to a high prevalence of a variety of other hematologic and malignant complications with long-term systemic effects[3]. Studies of HIV-1 RRE RNA and viral Rev proteins will indicate possibilities for HIV-1 RNA vaccine development and targeted drug development.

Prepare RNA/Protein:

In order to prepare the RNA and protein for this project, we selected the HIV-1 RNA genome and constructed HIV-1 RRE RNA and HIV-1 Rev plasmid through synthetic biology to provide the basis for the subsequent development of this project. The HIV-1 RRE RNA we constructed was 354 nt, and the HIV-1 Rev consisted of 116 amino acids. HIV-1 RRE RNA was transcribed in vitro by optimized RNA polymerase to obtain high-quality HIV-1 RRE RNA, and HIV-1 Rev was overexpressed in E. coli and isolated and purified to obtain high-quality HIV-1 Rev monomers. Therefore, in this project, we can obtain high quality RNA and protein by designing and constructing HIV-1 RRE RNA and Rev protein. Although it is not yet suitable for direct use in RNA vaccine and drug target development, it can provide reliable reference conditions and molecular mechanisms for the preparation of stable RNA vaccines and possible target drug development, thus contributing to the end of HIV disease.

Our program is conducted primarily in a room temperature research facility, where transcriptional preparation of HIV-1 RNA and over-expression of Rev proteins is accomplished in an in vitro reaction, without particularly harsh experimental and equipment conditions. To use our HIV-1 samples, the researcher first prepares normal ice bath cassettes to eliminate prolonged periods of time (more than 24h) when the samples are exposed to contamination from other environments. The next step is to complete the project normally under certain conditions according to the project requirements.

In the future, we plan to validate RNA through single-molecule fluorescence technology, which can enable early and highly sensitive detection for intervention-blocking therapy. We will also work further on the vaccine development of HIV-1 RNA, as well as study the site of action through the interaction between HIV-1 RRE RNA and HIV-1 Rev protein, which will provide the possibility and mechanism of action for realizing the development of highly effective target drugs.

Ultimately, our team hopes to utilize its fundamental research on HIV-1 RRE RNA and HIV-1 Rev to provide an effective reference for HIV vaccine development and target drug screening.Each step in the process of HIV-1 RRE RNA from construction, to target gene PCR, and in vitro transcription is critical.Appropriate annealing during target gene PCR of HIV-1 RRE RNA temperature can obtain the PCR product of a single target gene and prepare the transcribed target gene template for in vitro transcription. In the in vitro transcription, the optimal RNA polymerase successfully transcribed a single band of HIV-1 RRE RNA product. HIV-1 Rev was over-expressed and purified in vitro, and a single band of HIV-1 Rev monomer was obtained. The stable preparation of these samples laid a good foundation for the project to carry out RNA-Protein interactions, and successfully verified the different binding ratios of HIV-1 RRE RNA and Rev by EMSA experiments, which provided a reliable molecular basis for screening drug targets. Therefore, in the long run, the basic research of this project will be a powerful assistant to end HIV disease.

Although the whole process of this project is mainly an in vitro experiment, we still require the team members involved in wet experiments to strictly do personal protection in accordance with the standard requirements of biological and chemical laboratories and other related kits, which include wearing nitrile gloves, lab coats, medical masks, and goggles when using hazardous chemicals, etc., and strictly prohibit all personnel from eating and drinking in the experimental sites. In each experiment, according to the requirements of experiments and waste classification, waste is classified and disposed of according to the requirements and safety protocols, etc., in order to ensure that the experimental sites are clean and the waste is properly disposed of in order to meet the safety and cleanliness standards.

[1] Kenzui Taniue, Nobuyoshi Akimitsu. The Functions and Unique Features of LncRNAs in Cancer Development and Tumorigenesis. Int J Mol Sci. 2021. 22(2):632.

[2] Jason W. Rausch, Stuart F. J. Le Grice (2015) HIV Rev Assembly on the Rev Response Element (RRE): A Structural Perspective, Viruses. 7, 3053-3075.

[3] Carmen Catherine-Ann Swanepoel, Beverley Ann Van Rooyen, Emmanuel Akinola Abayomi. The human immunodeficiency virus, (HIV-1), pandemic: cellular therapies, stem cells and biobanking. 2013. Transfus Apher Sci. 49(1):9-11.