Periodic Reporting for period 4 - RetroChrom (Deciphering the molecular mechanisms of HIV DNA nuclear import and the impact of 3D genome organization on integration site selection)
Berichtszeitraum: 2024-03-01 bis 2024-08-31
Chromosomes of eukaryotes adopt highly dynamic and complex hierarchical structures in the nucleus. The three-dimensional (3D) organization of chromosomes profoundly affects DNA functions, primarily transcription. During retroviral infection, histone-free viral DNA copy is synthesized from viral genomic RNA. vDNA will ultimately integrate into the host genome to ensure its maintenance and expression. Understanding retrovirus-host interactions at the genomic level, and the peculiar mechanisms by which lentiviruses, including HIV-1, and their related gene transfer vectors, are imported into the nucleus, loaded with nucleosomes, integrate in, and interact with, the human genome will provide valuable information about HIV-1 replication and viral reservoir establishment and maintenance. Beyond HIV-1 biology, it will reveal important features required to establish the basis for the development of safer and more efficacious lentiviral vectors for human gene therapy.
Our objectives were: 1-To identify and characterize cellular proteins associated with the HIV-1 Pre-Integration Complex, and determine their roles in nuclear import and/or integration. 2-To explore the role of the epigenome of unintegrated vDNA in HIV-1 gene expression from unintegrated and integrated vDNA. 3- To determine the impact of nuclear organization on integration site selection and on viral and host transcription. We applied state of the art technologies to achieve these challenging objectives. Knowledge gained from this proposal will certainly have a major impact on the design of effective therapy aiming at targeting HIV reservoir to achieve cure. Beyond HIV, this project will make an outstanding contribution for the development of safe lentiviral vectors for gene therapy.
Our objectives were: 1-To identify and characterize cellular proteins associated with the HIV-1 Pre-Integration Complex, and determine their roles in nuclear import and/or integration. 2-To explore the role of the epigenome of unintegrated vDNA in HIV-1 gene expression from unintegrated and integrated vDNA. 3- To determine the impact of nuclear organization on integration site selection and on viral and host transcription. We applied state of the art technologies to achieve these challenging objectives. Knowledge gained from this proposal will certainly have a major impact on the design of effective therapy aiming at targeting HIV reservoir to achieve cure. Beyond HIV, this project will make an outstanding contribution for the development of safe lentiviral vectors for gene therapy.
RetroChrom ambition to understand the interaction between HIV and its host at the genomic level. Our objectives are:
2- To explore the role of the epigenome of UninvDNA in HIV-1 gene expression from unintegrated and integrated vDNA.
UninvDNA are transcriptionally silent by mechanisms involving host chromatin silencing factors. Identifying the host factors silencing uniHIV-1 DNA is key towards understanding their role during viral replication. Here, we applied the proteomics of isolated chromatin segments approach (PICh)4 to purify linear uniHIV-1 DNA and its associated viral and host factors, and identified 456 candidates selected for their specific binding to HIV-1 DNA. siRNA screen using HIV-1 integrase mutant (IND116A) identified 46 factors as potential repressors of uniHIV-1 DNA. Knockdown (KD) and Knockout (KO) experiments revealed POLE3 as transcriptional repressor of uniHIV-1 DNA with no effect on transcription from integrated provirus. KD of POLE3 had a marginal effect on unintegrated Murine Leukemia Virus (MLV). POLE3 is a subunit of the POLE holoenzyme (POLE3/POLE4), a histone H3-H4 chaperone that maintains chromatin integrity during DNA replication. Depletion of POLE3 resulted in increased RNAPII loading, enhanced H3ac active mark and loss of the repressive linker histone H1 on the uniHIV-1 DNA. POLE3 and the recently identified host factors silencing unintegrated HIV-1 DNA CAF1 and SMC5/SMC6/SLF2 show specificity towards different forms of unintegrated HIV-1 DNA. Loss of POLE3 impaired HIV-1 replication, suggesting that repression of linear unintegrated HIV-1 DNA is important for optimal replication of the virus. POLE3 depletion reduces HIV-1 integration efficiency. Remarkably, POLE3, by maintaining a repressive chromatin structure of unintegrated HIV-1 DNA, ensures HIV-1 escape from innate immune sensing in primary CD4 T cell. (Houssier-Thenin et al. 2023). Such innate immune response is dependent on cGAS sensor. Moreover, cGAS inhibition partially restored viral replication in POLE3 KO cells. Remarkably, chromatin immunoprecipitation experiments revealed that uvDNA recruits cGAS through its binding to uvDNA-associated nucleosomes, a mechanism known to inhibit cGAS activation (Jahan et al. PNAS 2025). Such findings prompted us to explore the mechanism and the regulation of cGAS tethering to chromatin. For this purpose, we developed a method allowing the solubilisation of cGAS bound to nucleosome. This method allowed us to immunopurify cGAS-nucleosome complexes et to determine their protein composition by mass spectrometry. Biochemical, coimmunoprecipitation and functional analysis allowed us de identify key host factors responsible for cGAS tethering to chromatin and regulators of its activity. Remarkably, our experiments revealed an unexpected function for cGAS in chromatin organization and function (Houssier-Thenin et al. Submitted). A manuscript resuming our findings is currently under evaluation (Houssier-Thenin et al. 2025).
2) To determine the impact of nuclear organization in integration site selection and on viral and host transcription
We performed Chromosomal Conformation combined with high throughput sequencing (Hi-C) to identify global inter- and intra-chromosomal interactions and PCHi-C (promoter capture Hi-C) which identify the interactions between promoters and their regulatory elements within the genome. Hi-C data were used to rank genomic fragments according to their 3D interaction levels with distant sites. ISs identified from in vitro infected primary CD4 T cells isolated from uninfected individuals associated with productive infection (PIC) and from cART were enriched in highly connected genomic fragments. In contrast, we found that ISs from ECs and those associated with latent infection (LIC) were highly enriched in genomic regions with lowest connection to distant genomic sites. Next, we analyzed the interactions between ISs and enhancers/superenhancer using PCHi-C. Remarkably, while PIC and cART ISs are frequently connected to enhancers and superenhancers both in quiescent and active CD4 T cell, LIC and EC ISs are not. Taken together, these experiments identified two chromatin-associated features allowing to distinguish EC IS from those in non-EC (Heng Chang Chen et al. Submitted).
2- To explore the role of the epigenome of UninvDNA in HIV-1 gene expression from unintegrated and integrated vDNA.
UninvDNA are transcriptionally silent by mechanisms involving host chromatin silencing factors. Identifying the host factors silencing uniHIV-1 DNA is key towards understanding their role during viral replication. Here, we applied the proteomics of isolated chromatin segments approach (PICh)4 to purify linear uniHIV-1 DNA and its associated viral and host factors, and identified 456 candidates selected for their specific binding to HIV-1 DNA. siRNA screen using HIV-1 integrase mutant (IND116A) identified 46 factors as potential repressors of uniHIV-1 DNA. Knockdown (KD) and Knockout (KO) experiments revealed POLE3 as transcriptional repressor of uniHIV-1 DNA with no effect on transcription from integrated provirus. KD of POLE3 had a marginal effect on unintegrated Murine Leukemia Virus (MLV). POLE3 is a subunit of the POLE holoenzyme (POLE3/POLE4), a histone H3-H4 chaperone that maintains chromatin integrity during DNA replication. Depletion of POLE3 resulted in increased RNAPII loading, enhanced H3ac active mark and loss of the repressive linker histone H1 on the uniHIV-1 DNA. POLE3 and the recently identified host factors silencing unintegrated HIV-1 DNA CAF1 and SMC5/SMC6/SLF2 show specificity towards different forms of unintegrated HIV-1 DNA. Loss of POLE3 impaired HIV-1 replication, suggesting that repression of linear unintegrated HIV-1 DNA is important for optimal replication of the virus. POLE3 depletion reduces HIV-1 integration efficiency. Remarkably, POLE3, by maintaining a repressive chromatin structure of unintegrated HIV-1 DNA, ensures HIV-1 escape from innate immune sensing in primary CD4 T cell. (Houssier-Thenin et al. 2023). Such innate immune response is dependent on cGAS sensor. Moreover, cGAS inhibition partially restored viral replication in POLE3 KO cells. Remarkably, chromatin immunoprecipitation experiments revealed that uvDNA recruits cGAS through its binding to uvDNA-associated nucleosomes, a mechanism known to inhibit cGAS activation (Jahan et al. PNAS 2025). Such findings prompted us to explore the mechanism and the regulation of cGAS tethering to chromatin. For this purpose, we developed a method allowing the solubilisation of cGAS bound to nucleosome. This method allowed us to immunopurify cGAS-nucleosome complexes et to determine their protein composition by mass spectrometry. Biochemical, coimmunoprecipitation and functional analysis allowed us de identify key host factors responsible for cGAS tethering to chromatin and regulators of its activity. Remarkably, our experiments revealed an unexpected function for cGAS in chromatin organization and function (Houssier-Thenin et al. Submitted). A manuscript resuming our findings is currently under evaluation (Houssier-Thenin et al. 2025).
2) To determine the impact of nuclear organization in integration site selection and on viral and host transcription
We performed Chromosomal Conformation combined with high throughput sequencing (Hi-C) to identify global inter- and intra-chromosomal interactions and PCHi-C (promoter capture Hi-C) which identify the interactions between promoters and their regulatory elements within the genome. Hi-C data were used to rank genomic fragments according to their 3D interaction levels with distant sites. ISs identified from in vitro infected primary CD4 T cells isolated from uninfected individuals associated with productive infection (PIC) and from cART were enriched in highly connected genomic fragments. In contrast, we found that ISs from ECs and those associated with latent infection (LIC) were highly enriched in genomic regions with lowest connection to distant genomic sites. Next, we analyzed the interactions between ISs and enhancers/superenhancer using PCHi-C. Remarkably, while PIC and cART ISs are frequently connected to enhancers and superenhancers both in quiescent and active CD4 T cell, LIC and EC ISs are not. Taken together, these experiments identified two chromatin-associated features allowing to distinguish EC IS from those in non-EC (Heng Chang Chen et al. Submitted).
Thus, several major discoveries were made:
1) revealing the mechanism and the biological significance of unintegrated HIV-1 transcriptional silencing.
2) The discovery of a nucleosome loaded on the unintegrated HIV-1: implication in the establishment of latent provirus. This discovery calls for revisiting our approaches aiming at targeting the latent HIV-1 reservoir to achieve cure.
3) The interplay between chromatin and innate immune evasion mechanism evolved by HIV-1. The discovery of host factors regulating cGAS tethering to chromatin and a novel function for cGAS in chromatin organisation and function.
4) CD4 T cell isolated from HIV-1 elite controllers have an intrinsic property to target viral integration into chromatin regions that are less permissive for transcription.
1) revealing the mechanism and the biological significance of unintegrated HIV-1 transcriptional silencing.
2) The discovery of a nucleosome loaded on the unintegrated HIV-1: implication in the establishment of latent provirus. This discovery calls for revisiting our approaches aiming at targeting the latent HIV-1 reservoir to achieve cure.
3) The interplay between chromatin and innate immune evasion mechanism evolved by HIV-1. The discovery of host factors regulating cGAS tethering to chromatin and a novel function for cGAS in chromatin organisation and function.
4) CD4 T cell isolated from HIV-1 elite controllers have an intrinsic property to target viral integration into chromatin regions that are less permissive for transcription.