Project Description
According to statistics from UNAIDS report , 39 million people are infected with HIV globally and 630,000 people will die in 2022 from AIDS-related diseases. The current development of HIV RNA vaccines and their target drugs, among others, does not allow many people to access them for prevention and treatment. We target viral RNA design for exploration. Our project applied env non-coding long chain RRE RNA and Rev proteins from the HIV-1 isolate ARV-2/SF2 viral genome as a means to develop nucleic acid integrase inhibitors and RNA vaccines as well as target drugs. After constructing RRE and Rev sequences separately, we will mainly carry out systematic studies through in vitro transcription of RRE RNA and its possible interaction with Rev protein. Firstly, we will accomplish high quality in vitro transcription of RRE by T7 RNA polymerase, and secondly, we will search for sites of interest for the interaction of high quality RRE RNA obtained by T7 RNA polymerase transcription with the viral protein Rev. The goal of this project is to establish a system for the development of viral long-stranded RNAs that will address the future shortage of viral nucleic acid integrase inhibitors, vaccine development, and targeted drug development through the help of scalable in vitro transcription and viral protein interactions.
A serious infectious disease caused by human immunodeficiency virus (HIV) infection, also known as acquired immuno deficiency syndrome (AIDS) or AIDS. According to the latest UNAIDS report, "2023 Progress Report on the Global AIDS Response - The Road to an End", there are currently 39 million people living with HIV globally, 29.8 million of whom are on antiretroviral treatment, with 1.3 million new HIV infections and 630,000 deaths due to AIDS-related illnesses in 2022[1-2].
After HIV invades and infects the human body, its main pathogenesis are[3]：①The body's immune cells and immune fluids are resistant to HIV after allowing low-level replication of the HIV virus in the early stages of infection；②HIV binds to CD4 receptor-containing CD4+ T-lymphocytes on the cell surface and thus enters the cell to replicate, and some of them also integrate with the cell's chromosomal DNA to become the latent phenotype[4-7]；③Under the action of other factors, latent HIV is activated and replicates in large quantities, extensively invades CD4+ T lymphocytes, which extensively impairs the functions of CD4+ T lymphocytes, monocyte-macrophages, B lymphocytes, CD8+ T lymphocytes, and NK cells, and finally leads to defective immunity in the entire immune function, which ultimately leads to a series of persistent infections and tumorigenesis[8-12].
AIDs may present with a variety of symptoms, and HIV infection is categorized into four stages based on the clinical progression of the disease, with major clinical manifestations at each stage including, but not limited to:
Acute HIV infection: fever, malaise, myalgia, anorexia, nausea, diarrhea, and non-exudative pharyngitis, with some patients presenting with headache, photophobia, and signs of meningeal irritation, oro-esophageal ulcers or candida infections, enlarged lymph nodes, and skin rashes[13];
Asymptomatic HIV infection: a few persistent generalized lymph node enlargement, most common in the cervical, occipital and axillary regions, lymph node diameter >1cm, without conscious pain and pressure[14];
Pre-AIDS: mainly the same as acute HIV infection;
AIDS stage: mainly the same as the symptoms of acute HIV infection, combined with opportunistic infections (bacterial infections, viral infections including herpes simplex virus, varicella zoster virus, EBV, cytomegalovirus and hepatitis virus infections, fungal infections including Pneumocystis carinii, Candida, Cryptococcus, and Histoplasma, protozoan infections, etc.); malignant tumors including Kaposi's sarcoma, lymphomas, invasive cervical carcinoma, etc.; dementia and wasting syndrome.
Current Diagnostics and therapy
The main current clinical and laboratory methods for detecting HIV infection are[15]:（1）Serologic tests to detect HIV antibodies;（2）Tests for direct detection of HIV:（a）p24 antigen detection;（b）Nucleic acid detection and quantification: detection of proviral DNA and HIV-1 RNA;（c）HIV culture, isolation.
Currently, the main drugs used to treat AIDS patients are[16]：Nucleoside reverse transcriptase inhibitors (NRTIs), integrase strand transfer inhibitors (InSTIs), or boosted protease inhibitors (BPIs). Most therapeutic regimens are provided in fixed-dose combinations (FDCs), such as InSTI, or single-tablet regimens (str; e.g., Raltegravir). Non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as efavirenz (Sustiva; Bristol-Myers Squibb (BMS)) and rilpivirine (Edurant; Janssen), Atripla (BMS and Gilead Sciences), the Dolutegravir (Tivicay; ViiV Healthcare), InSTI elvitegravir, Genvoya, BPI darunavir (Prezista; Janssen) and others. To extend the duration of antiretroviral drug therapy and pre-exposure prophylaxis (PrEP), as well as better new combinations, emerging HIV treatment advances are shown in Table 1[16].
Our mission is to provide an enabling cornerstone for HIV testing, effective targeted drug and HIV RNA vaccine development that complements the highly sensitive early detection and termination of HIV-infected individuals. Although there is currently no vaccine against HIV or effective drug for HIV termination, scientists have never stopped working to end the virus. Therefore, our goal is to make the public free from infection and have better treatment options in the future to reduce mortality and extend the life span of infected people.
We investigated the mechanism of action of the HIV-1 virus by examining the RNAs that are transcribed and exported to the cytoplasm or transformed or converted into structural proteins, Rev, or packaged into the genomes of new viral particles when the body is infected with HIV-1 (as shown in Figure 1). In order to evaluate the role of enzymes during HIV-1 RNA transcription and the binding mechanism of HIV-1 RNA to Rev protein, we have conducted studies on HIV-1 RNA viral particle self-replication and the output of complex products mediated by the structural protein Rev based on our experience with retroviral RNA, which will contribute to the development of a future HIV-1 RNA vaccine, a single-molecule high-sensitivity virus assay, and a virus-targeted drug. virus detection and virus-targeted drug development, etc. to provide reliable molecular mechanisms of action.
RNA as a regulator of gene expression[18-20]、Novel drug targets[21-23]、bioengineering applications and synthetic biology tools[24-25]. All are growing in importance. RNA not only links DNA to proteins for the expression of genetic information on proteins, but also serves as the basis for various physiological activities of living organisms. In general, RNA transcripts containing introns are usually retained in the nucleus until they are spliced or degraded[26-28]. In contrast, the nuclear/plasmic export of viral RNA is abnormal, the most typical example of which is HIV-1 (Human immunodeficiency virus-1), which predisposes the infected person to a progressive failure of the immune system leading to the onset of AIDS, and whose development is associated with an imbalance in the regulation of non-coding RNAs.HIV-1 non-coding RNAs are involved in the regulation of many intracellular processes as genetic information involved in the regulation of various intracellular processes, plays an important role in genome defense, RNA generation, modification, splicing, and regulation of gene expression[29]. HIV-1 retrovirus replication and translocation in endogenous reverse transcription factors then utilize unique post-transcriptional regulatory mechanisms for export of partially spliced or unspliced mRNAs into the cytoplasm[29].
HIV-1 RRE RNA structure
HIV-1 Rev response element (RRE) RNA, as a genetic factor for HIV-1 viral genome replication (Figure 2), is structurally (Figure 3): one of the most structurally complex cis-acting elements in the HIV-1 pre-mRNA, with about 350 bases, located in the structural gene env, which is mainly folded to form a stem-loop secondary structure through intra-chain hydrogen bonding, and whose secondary structure mainly contains the central loop I, stem-loop II (IIA, IIB, IIC), and III, IV, and V structures[30].
HIV-1 RRE RNA and Rev Protein Selection
After careful literature research and review, we decided to design the env non-coding long-chain RRE RNA full-length gene (354bp) and the Rev protein full-length gene (116aa) in the HIV-1 isolate ARV-2/SF2 viral genome. Based on HIV-1 RRE RNA and Rev as HIV-1 viral RNA particles replication and interaction with Rev as the research objectives, to realize the reliable molecular mechanism for the future in the development of HIV-1 RNA vaccines, target drugs.
High-quality in vitro transcription of noncoding RNAs is an obstacle to the existence of existing research RNAs. We can efficiently transcribe HIV-1 RNA in vitro by designing the gene and in vitro amplification of HIV-1 RNA, optimizing the modified T7 RNA polymerase, this purpose can be screened for modified RNA polymerase inhibitors, to achieve the inhibition of HIV-1 RNA replication; In addition, in the design of the full-length gene of the Rev protein, the optimization to achieve the high-expression and purity of the Rev full-length monomer protein , preventing Rev self-assembly, affecting the interaction with RRE RNA, and reducing the binding rate with RRE RNA, this purpose is mainly to obtain the binding site of RNA and Rev protein, and provide a reliable molecular mechanism for the screening of targeted drugs.
Throughout our project, the IGEM team consulted and interviewed relevant stakeholders (patients, HIV-1 clinical experts, disease control experts, researchers, advocacy organizations, etc.). Since HIV infected people in varying proportions create a sense of fear and alienation in the minds of the general public, we have taken several steps to publicize and address this issue. We have also taken several steps to publicize and address these inequalities, including the production of a number of science videos and the production of science program materials where we can, in order to disseminate public awareness of HIV prevention and lifestyle awareness.
Through HIV-1 RNA, IGEM is also based on synthetic biology, combining various existing methods of RNA research, including RNA transcription, biochemistry of interactions with viral proteins, and providing basic research for the development and design of viral RNA vaccines and targeted drugs for future viral RNA.
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