Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site
ABSTRACT Current antiretroviral treatment fails to cure HIV-1 infection since latent provirus resides in long-lived cellular reservoirs, rebounding whenever therapy is discontinued. The molecular mechanisms underlying HIV-1 latency are complex where the possible link between integration and transcri...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-08-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mbio.00007-22 |
| _version_ | 1797999694629044224 |
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| author | Julie Janssens Flore De Wit Nagma Parveen Zeger Debyser |
| author_facet | Julie Janssens Flore De Wit Nagma Parveen Zeger Debyser |
| author_sort | Julie Janssens |
| collection | DOAJ |
| description | ABSTRACT Current antiretroviral treatment fails to cure HIV-1 infection since latent provirus resides in long-lived cellular reservoirs, rebounding whenever therapy is discontinued. The molecular mechanisms underlying HIV-1 latency are complex where the possible link between integration and transcription is poorly understood. HIV-1 integration is targeted toward active chromatin by the direct interaction with a host protein, lens epithelium-derived growth factor (LEDGF/p75). LEDGINs are small-molecule inhibitors of the LEDGF/p75-integrase (IN) interaction that effectively inhibit and retarget HIV-1 integration out of preferred integration sites, resulting in residual provirus that is more latent. Here, we describe a single-cell branched DNA imaging method for simultaneous detection of viral DNA and RNA. We investigated how treatment with LEDGINs affects the location, transcription, and reactivation of HIV-1 in both cell lines and primary cells. This approach demonstrated that LEDGIN-mediated retargeting hampered the baseline transcriptional state and the transcriptional reactivation of the provirus, evidenced by the reduction in viral RNA expression per residual copy. Moreover, treatment of primary cells with LEDGINs induced an enrichment of provirus in deep latency. These results corroborate the impact of integration site selection for the HIV-1 transcriptional state and support block-and-lock functional cure strategies in which the latent reservoir is permanently silenced after retargeting. IMPORTANCE A longstanding question exists on the impact of the HIV-1 integration site on viral gene expression. This unsolved question has significant implications for the search toward an HIV-1 cure, as eradication strategies set up to reactivate and eliminate HIV-1 depend on the site where the provirus is integrated. The main determinant for integration site selection is the interaction of the HIV-1 integrase (IN) and the host chromatin targeting factor, LEDGF/p75. LEDGINs are small-molecule inhibitors of the LEDGF/p75-IN interaction that inhibit and retarget HIV-1 integration out of preferred integration sites. Using both LEDGINs and branched DNA (bDNA) imaging, we now investigated, in much detail, the impact of integration site selection on the three-dimensional location of the provirus, HIV-1 transcription, and reactivation. Our results provide evidence for a “block-and-lock” functional cure strategy that aims to permanently silence HIV-1 by LEDGIN-mediated retargeting to sites that are less susceptible to reactivation after treatment interruption. |
| first_indexed | 2024-04-11T11:08:47Z |
| format | Article |
| id | doaj.art-a7504e6465124977b558a457b27dfe9a |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-11T11:08:47Z |
| publishDate | 2022-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-a7504e6465124977b558a457b27dfe9a2022-12-22T04:28:05ZengAmerican Society for MicrobiologymBio2150-75112022-08-0113410.1128/mbio.00007-22Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration SiteJulie Janssens0Flore De Wit1Nagma Parveen2Zeger Debyser3Molecular Virology and Gene Therapy, KU Leuven, Leuven, BelgiumMolecular Virology and Gene Therapy, KU Leuven, Leuven, BelgiumDepartment of Chemistry, Indian Institute of Technology, Kanpur, IndiaMolecular Virology and Gene Therapy, KU Leuven, Leuven, BelgiumABSTRACT Current antiretroviral treatment fails to cure HIV-1 infection since latent provirus resides in long-lived cellular reservoirs, rebounding whenever therapy is discontinued. The molecular mechanisms underlying HIV-1 latency are complex where the possible link between integration and transcription is poorly understood. HIV-1 integration is targeted toward active chromatin by the direct interaction with a host protein, lens epithelium-derived growth factor (LEDGF/p75). LEDGINs are small-molecule inhibitors of the LEDGF/p75-integrase (IN) interaction that effectively inhibit and retarget HIV-1 integration out of preferred integration sites, resulting in residual provirus that is more latent. Here, we describe a single-cell branched DNA imaging method for simultaneous detection of viral DNA and RNA. We investigated how treatment with LEDGINs affects the location, transcription, and reactivation of HIV-1 in both cell lines and primary cells. This approach demonstrated that LEDGIN-mediated retargeting hampered the baseline transcriptional state and the transcriptional reactivation of the provirus, evidenced by the reduction in viral RNA expression per residual copy. Moreover, treatment of primary cells with LEDGINs induced an enrichment of provirus in deep latency. These results corroborate the impact of integration site selection for the HIV-1 transcriptional state and support block-and-lock functional cure strategies in which the latent reservoir is permanently silenced after retargeting. IMPORTANCE A longstanding question exists on the impact of the HIV-1 integration site on viral gene expression. This unsolved question has significant implications for the search toward an HIV-1 cure, as eradication strategies set up to reactivate and eliminate HIV-1 depend on the site where the provirus is integrated. The main determinant for integration site selection is the interaction of the HIV-1 integrase (IN) and the host chromatin targeting factor, LEDGF/p75. LEDGINs are small-molecule inhibitors of the LEDGF/p75-IN interaction that inhibit and retarget HIV-1 integration out of preferred integration sites. Using both LEDGINs and branched DNA (bDNA) imaging, we now investigated, in much detail, the impact of integration site selection on the three-dimensional location of the provirus, HIV-1 transcription, and reactivation. Our results provide evidence for a “block-and-lock” functional cure strategy that aims to permanently silence HIV-1 by LEDGIN-mediated retargeting to sites that are less susceptible to reactivation after treatment interruption.https://journals.asm.org/doi/10.1128/mbio.00007-22HIV-1LEDGF/p75LEDGINsintegration site selectionsingle-cell HIV-1 imaging |
| spellingShingle | Julie Janssens Flore De Wit Nagma Parveen Zeger Debyser Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site mBio HIV-1 LEDGF/p75 LEDGINs integration site selection single-cell HIV-1 imaging |
| title | Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site |
| title_full | Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site |
| title_fullStr | Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site |
| title_full_unstemmed | Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site |
| title_short | Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site |
| title_sort | single cell imaging shows that the transcriptional state of the hiv 1 provirus and its reactivation potential depend on the integration site |
| topic | HIV-1 LEDGF/p75 LEDGINs integration site selection single-cell HIV-1 imaging |
| url | https://journals.asm.org/doi/10.1128/mbio.00007-22 |
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