Induction of translation-suppressive G3BP1+ stress granules and interferon-signaling cGAS condensates by transfected plasmid DNA
Plasmid DNA transfection is one of the fundamental tools of biomedical research. Here, we found that plasmid DNA transfection mediated by liposomes activates multiple innate immune responses in several widely used cell lines. Their activations were visible by the detection of stress granules (SG) an...
Saved in:
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-01-01
|
| Series: | hLife |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2949928324000956 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832590886696910848 |
|---|---|
| author | Vladimir Majerciak Zhi-Ming Zheng |
| author_facet | Vladimir Majerciak Zhi-Ming Zheng |
| author_sort | Vladimir Majerciak |
| collection | DOAJ |
| description | Plasmid DNA transfection is one of the fundamental tools of biomedical research. Here, we found that plasmid DNA transfection mediated by liposomes activates multiple innate immune responses in several widely used cell lines. Their activations were visible by the detection of stress granules (SG) and cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-DNA condensates (cGC) in the transfected cells in a plasmid DNA dose-dependent manner. The elevated levels of phosphorylated eukaryotic translation initiation factor 2 subunit alpha (eIF2α), interferon regulatory factor 3 (IRF3), and signal transducer and activator of transcription 1 (STAT1) were induced in plasmid DNA-transfected cells. The formation of SG but not cGC required active transcription and the formation of double-stranded RNA in transfected cells. Plasmid DNA-induced SG or cGC were mutually exclusive because they triggered two distinct pathways. Knockdown (KD) of protein kinase R (PKR) before plasmid DNA transfection led to abolishing SG without affecting cGC formation. Conversely, cGAS KD could prevent cGC without affecting SG formation. In addition, plasmid DNA-induced SG and cGC formation could be prevented, respectively, by co-expression of Kaposi's sarcoma-associated herpesvirus proteins ORF57 (PKR inhibitor) and ORF52 (cGAS inhibitor). Inhibition of SG formation mediated by PKR KD, but not cGC KD, also led to increased expression of transgenes, indicating that PKR activation represents a major roadblock to gene expression. Together, these data indicate that plasmid DNA triggers innate immune responses in the transfected cells and causes a significant cellular perturbation that should be considered during experiment design and data interpretation. |
| format | Article |
| id | doaj-art-67809c24df024269a7d089717f619f7a |
| institution | Kabale University |
| issn | 2949-9283 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | hLife |
| spelling | doaj-art-67809c24df024269a7d089717f619f7a2025-01-23T05:28:07ZengElsevierhLife2949-92832025-01-01312137Induction of translation-suppressive G3BP1+ stress granules and interferon-signaling cGAS condensates by transfected plasmid DNAVladimir Majerciak0Zhi-Ming Zheng1Correspondence:; Tumor Virus RNA Biology Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Maryland, USATumor Virus RNA Biology Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Maryland, USAPlasmid DNA transfection is one of the fundamental tools of biomedical research. Here, we found that plasmid DNA transfection mediated by liposomes activates multiple innate immune responses in several widely used cell lines. Their activations were visible by the detection of stress granules (SG) and cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-DNA condensates (cGC) in the transfected cells in a plasmid DNA dose-dependent manner. The elevated levels of phosphorylated eukaryotic translation initiation factor 2 subunit alpha (eIF2α), interferon regulatory factor 3 (IRF3), and signal transducer and activator of transcription 1 (STAT1) were induced in plasmid DNA-transfected cells. The formation of SG but not cGC required active transcription and the formation of double-stranded RNA in transfected cells. Plasmid DNA-induced SG or cGC were mutually exclusive because they triggered two distinct pathways. Knockdown (KD) of protein kinase R (PKR) before plasmid DNA transfection led to abolishing SG without affecting cGC formation. Conversely, cGAS KD could prevent cGC without affecting SG formation. In addition, plasmid DNA-induced SG and cGC formation could be prevented, respectively, by co-expression of Kaposi's sarcoma-associated herpesvirus proteins ORF57 (PKR inhibitor) and ORF52 (cGAS inhibitor). Inhibition of SG formation mediated by PKR KD, but not cGC KD, also led to increased expression of transgenes, indicating that PKR activation represents a major roadblock to gene expression. Together, these data indicate that plasmid DNA triggers innate immune responses in the transfected cells and causes a significant cellular perturbation that should be considered during experiment design and data interpretation.http://www.sciencedirect.com/science/article/pii/S2949928324000956plasmid DNAtransfectionstress granulescyclic GMP-AMP synthase (cGAS)Kaposi's sarcoma-associated herpesvirus (KSHV)protein kinase R (PKR) |
| spellingShingle | Vladimir Majerciak Zhi-Ming Zheng Induction of translation-suppressive G3BP1+ stress granules and interferon-signaling cGAS condensates by transfected plasmid DNA hLife plasmid DNA transfection stress granules cyclic GMP-AMP synthase (cGAS) Kaposi's sarcoma-associated herpesvirus (KSHV) protein kinase R (PKR) |
| title | Induction of translation-suppressive G3BP1+ stress granules and interferon-signaling cGAS condensates by transfected plasmid DNA |
| title_full | Induction of translation-suppressive G3BP1+ stress granules and interferon-signaling cGAS condensates by transfected plasmid DNA |
| title_fullStr | Induction of translation-suppressive G3BP1+ stress granules and interferon-signaling cGAS condensates by transfected plasmid DNA |
| title_full_unstemmed | Induction of translation-suppressive G3BP1+ stress granules and interferon-signaling cGAS condensates by transfected plasmid DNA |
| title_short | Induction of translation-suppressive G3BP1+ stress granules and interferon-signaling cGAS condensates by transfected plasmid DNA |
| title_sort | induction of translation suppressive g3bp1 stress granules and interferon signaling cgas condensates by transfected plasmid dna |
| topic | plasmid DNA transfection stress granules cyclic GMP-AMP synthase (cGAS) Kaposi's sarcoma-associated herpesvirus (KSHV) protein kinase R (PKR) |
| url | http://www.sciencedirect.com/science/article/pii/S2949928324000956 |
| work_keys_str_mv | AT vladimirmajerciak inductionoftranslationsuppressiveg3bp1stressgranulesandinterferonsignalingcgascondensatesbytransfectedplasmiddna AT zhimingzheng inductionoftranslationsuppressiveg3bp1stressgranulesandinterferonsignalingcgascondensatesbytransfectedplasmiddna |