Retroviruses integrate into the genomes of infected host cells to form proviruses, a genetic platform for stable viral gene expression. Epigenetic silencing can, however, hamper proviral transcriptional activity. As gammaretroviruses (γRVs) preferentially integrate into active promoter and enhancer sites, the high transcriptional activity of γRVs can be attributed to this integration preference. In addition, long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters by themselves. Here, we investigate the capacity of different γRV LTRs to drive stable expression within a non-preferred epigenomic environment in the context of diverse retroviral vectors. We demonstrate that different γRV LTRs are either rapidly silenced or remain active for long periods of time with a predominantly active proviral population under normal and retargeted integration. As an alternative to the established γRV systems, the feline leukemia virus and koala retrovirus LTRs are able to drive stable, albeit intensity-diverse, transgene expression. Overall, we show that despite the occurrence of rapid silencing events, most γRV LTRs can drive stable expression outside of their preferred chromatin landscape after retrovirus integrations.
- Keywords
- epigenetics, expression, integration site, retrovirus, silencing, vectors,
- MeSH
- Cell Line MeSH
- Gammaretrovirus * genetics MeSH
- Genetic Vectors genetics MeSH
- Virus Integration * MeSH
- Terminal Repeat Sequences * genetics MeSH
- Humans MeSH
- Promoter Regions, Genetic MeSH
- Proviruses * genetics MeSH
- Gene Expression Regulation, Viral MeSH
- Transgenes MeSH
- Gene Silencing MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.
- MeSH
- Cell Line MeSH
- Chromatin metabolism MeSH
- Histones metabolism MeSH
- HIV-1 drug effects genetics metabolism MeSH
- HIV Integrase Inhibitors pharmacology MeSH
- Virus Integration * drug effects MeSH
- Humans MeSH
- Intercellular Signaling Peptides and Proteins MeSH
- Proviruses genetics MeSH
- Gene Expression Regulation, Viral * drug effects MeSH
- RNA, Viral metabolism MeSH
- Gene Silencing * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Chromatin MeSH
- Histones MeSH
- HIV Integrase Inhibitors MeSH
- lens epithelium-derived growth factor MeSH Browser
- Intercellular Signaling Peptides and Proteins MeSH
- RNA, Viral MeSH
It has now been more than two years since we said our last goodbye to Jan Svoboda (14 [...].
