Exploring the role of G-Quadruplexes in maintaining Human Immunodeficiency Virus-1 latency

The human Immunodeficiency Virus-1 (HIV-1) is a human retrovirus, whose genome is constituted by two copies of positive-sense single-stranded RNA (ssRNA). It is responsible for the Acquired Immunodeficiency Syndrome (AIDS), which is characterized by a dramatic decrease in CD4+ T-Lymphocytes. Upon entering the target cell, the viral ssRNA undergoes reverse transcription to double-stranded DNA, which integrates as provirus into host genome, and is replicated by the cellular machinery. The provirus can be transcriptionally active or inactive; in the latter case the virus persists in a so called latent state. People living with HIV-1 require lifelong administration of antiretroviral drugs. Different therapies targeting mainly the virus produced in the blood have been developed to partially recover immune defence; however, complete eradication of the virus is currently hindered by the latent reservoir of infected host cells. According to recent UNAIDS reports, approximately 40 million people worldwide were living with HIV-1 in 2024, and this number is not expected to decrease soon. Therefore, the development of an effective antiretroviral treatment capable of eradicating the virus is urgently needed. To this end, this study focuses primarily on gaining a comprehensive understanding of HIV-1 latency mechanisms, which is essential for achieving viral eradication. In this scenario, the Long Terminal Repeat (LTR) region of HIV-1, corresponding to its unique promoter, plays a crucial role in promoting and modulating proviral transcription. Interestingly, a G-rich sequence within the LTR folds into G-quadruplexes (G4s), non-canonical DNA secondary structures involved in gene regulation. Chromatin Immunoprecipitation (ChIP) using an anti-G4 antibody revealed high G4 enrichment in the LTR during latency in T-lymphocyte cell models, such as J-Lat Tat-GFP A2. LTR G4s decreased significantly upon viral reactivation induced by latency reversing agents (LRAs). This suggests a correlation between LTR G4s and HIV-1 latency, with G4 unfolding associated with viral reactivation. In support of these findings, it has been observed that HIV-1 reactivation is influenced by Werner (WRN) helicase, a member of the RecQ family that binds and unwinds G4s. Reduced or absent WRN functionality correlated with decreased HIV-1 transcription. In this thesis, to validate LTR G4 function in HIV latency, we set up for the first time a protocol to generate a CRISPR/Cas9 mutated latently infected cell population abrogated of LTR G4 folding. We employed a CRISPR/Cas9 strategy to introduce mutations in the LTR of J-Lat Tat-GFP A2 cells, disrupting G4 folding. Genome editing was achieved using single-stranded oligodeoxynucleotides (ssODN) carrying the mutated LTR sequence as a homology-directed repair template following Cas9-mediated double-strand breaks. In addition, we investigated whether WRN is involved in LTR G4 unfolding during LRA-induced viral reactivation. To do this, we performed ChIP followed by qPCR to measure WRN enrichment within the LTR at different post-reactivation timing. The results indicate that WRN is recruited to the LTR in the early phase of viral reactivation. In conclusion, our data support the maintenance of viral latency by LTR G4 structures and their interplay with cellular proteins, offering new perspectives on the complex mechanisms that regulate HIV-1 latency. These findings open new avenues for developing novel antiviral drugs targeting LTR G4 structures.