Silencing of Unintegrated Retroviral DNAs

Goff, Stephen P.

doi:10.3390/v13112248

Cited by 11 publications

(9 citation statements)

References 96 publications

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“…Thus, we have acquired fresh perspectives on the mechanisms governing retroviral suppression mediated by Smarcad1. From the point of MLV transduction to cells, in approximately 24 hours, the newly incoming retroviral sequence is being integrated into the cell genome (38). Trim28 is recruited to the MLV provirus via direct interaction with ZFP809 (4), YY1 (9) and EBP1 (39).…”

Section: Discussionmentioning

confidence: 99%

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The role of Smarcad1 in retroviral repression in mouse embryonic stem cells

Bren,

Strauss,

Schlesinger

2023

Preprint

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The replication of Moloney murine leukemia virus (MLV) is suppressed in mouse embryonic stem cells (ESCs) by the Trim28-SETDB1 complex. The chromatin remodeler Smarcad1 interacts with Trim28 and was suggested to enable the deposition of histone variant H3.3. However, the role of Trim28, H3.3, and Smarcad1 in the repression of MLV in ESCs is not fully understood. In this study, we investigate the role of Smarcad1 in the silencing of incoming MLV retroviruses, specifically focusing on the role of the chromatin structure. We found that Smarcad1 binds to proviral DNA and that Smarcad1 depletion leads to up-regulation of retroviral expression. Additionally, our findings demonstrate that Smarcad1 is necessary to properly recruit Trim28 and deposition of H3.3 on the MLV provirus and other Trim28-repressed genomic loci. Finally, we also show that the mutual depletion of Smarcad1 and Trim28 results in enhanced de-repression of the MLV provirus, indicating that these two proteins may also function independently to maintain repressive chromatin states. Our results provide evidence for the crucial role of Smarcad1 in the silencing of retroviral elements in embryonic stem cells. Further research is needed to fully understand how Smarcad1 and Trim28 cooperate and their implications for gene expression and genomic stability.

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Section: Discussionmentioning

confidence: 99%

“…We begin at the point of MLV transduction to the cells. From this point, in about 24 hour, the newly incoming retroviral sequence is being integrated into the cells' genome (Goff, 2021). we examine the recruitment of Smarcad1 to the incoming MLV retroviral sequence.…”

Section: Discussionmentioning

confidence: 99%

The role of Smarcad1 in retroviral repression in mouse embryonic stem cells

Bren,

Strauss,

Schlesinger

2023

Preprint

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“…In the absence of functional IN, unintegrated HIV-1 proviruses accumulate repressive epigenetic marks, including trimethylation of lysine 9 on histone H3 (H3K9me3), and are depleted of activating marks, such as H3 acetylation (H3Ac) 4 , 5 . While the epigenetic silencing of transcription from unintegrated HIV-1 DNA probably represents a host defence against foreign DNA, the underlying mechanisms and cellular factors that mediate this effect remain incompletely defined 6 , 7 .…”

Section: Mainmentioning

confidence: 99%

Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency

2022

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After viral entry and reverse transcription, HIV-1 proviruses that fail to integrate are epigenetically silenced, but the underlying mechanism has remained unclear. Using a genome-wide CRISPR/Cas9 knockout screen, we identified the host SMC5/6 complex as essential for this epigenetic silencing. We show that SMC5/6 binds to and then SUMOylates unintegrated chromatinized HIV-1 DNA. Inhibition of SUMOylation, either by point mutagenesis of the SMC5/6 component NSMCE2—a SUMO E3 ligase—or using the SUMOylation inhibitor TAK-981, prevents epigenetic silencing, enables transcription from unintegrated HIV-1 DNA and rescues the replication of integrase-deficient HIV-1. Finally, we show that blocking SMC5/6 complex expression, or inhibiting its SUMOylation activity, suppresses the establishment of latent HIV-1 infections in both CD4+ T cell lines and primary human T cells. Collectively, our data show that the SMC5/6 complex plays a direct role in mediating the establishment of HIV-1 latency by epigenetically silencing integration-competent HIV-1 proviruses before integration.

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“…Another feature, similar to plasmid-based expression in mammalian cells is the possibility to enhance expression levels with the addition of histone deacetylase inhibitors [ 22 ]. Foreign transfected or transduced DNA is silenced by modification via host histone deacetylases as a self-defense mechanism in mammalian cells [ 23 , 24 , 25 ]. Previous reports already demonstrated the effect of trichostatin A (TSA) or sodium butyrate, which are potent histone deacetylase inhibitors, on different cell types.…”

Section: Introductionmentioning

confidence: 99%

Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells

Toth,

Reithofer,

Dutra

et al. 2024

Viruses

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(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.

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Silencing of Unintegrated Retroviral DNAs

Cited by 11 publications

References 96 publications

The role of Smarcad1 in retroviral repression in mouse embryonic stem cells

The role of Smarcad1 in retroviral repression in mouse embryonic stem cells

Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency

Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells

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