SAMHD1 Negatively Regulates the Innate Immune Responses to Inflammatory Stimuli and Viral Infection
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| Language: | English |
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The Ohio State University / OhioLINK
2020
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=osu1587587968104986 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu1587587968104986 |
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English |
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Immunology Molecular Biology Virology SAMHD1 Innate immune responses NF-KB pathway Type I interferon pathway dNTPase activity Nuclear localization |
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Immunology Molecular Biology Virology SAMHD1 Innate immune responses NF-KB pathway Type I interferon pathway dNTPase activity Nuclear localization Qin, Zhihua SAMHD1 Negatively Regulates the Innate Immune Responses to Inflammatory Stimuli and Viral Infection |
| author |
Qin, Zhihua |
| author_facet |
Qin, Zhihua |
| author_sort |
Qin, Zhihua |
| title |
SAMHD1 Negatively Regulates the Innate Immune Responses to Inflammatory Stimuli and Viral Infection |
| title_short |
SAMHD1 Negatively Regulates the Innate Immune Responses to Inflammatory Stimuli and Viral Infection |
| title_full |
SAMHD1 Negatively Regulates the Innate Immune Responses to Inflammatory Stimuli and Viral Infection |
| title_fullStr |
SAMHD1 Negatively Regulates the Innate Immune Responses to Inflammatory Stimuli and Viral Infection |
| title_full_unstemmed |
SAMHD1 Negatively Regulates the Innate Immune Responses to Inflammatory Stimuli and Viral Infection |
| title_sort |
samhd1 negatively regulates the innate immune responses to inflammatory stimuli and viral infection |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2020 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1587587968104986 |
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AT qinzhihua samhd1negativelyregulatestheinnateimmuneresponsestoinflammatorystimuliandviralinfection |
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1719457222798344192 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu15875879681049862021-08-03T07:14:39Z SAMHD1 Negatively Regulates the Innate Immune Responses to Inflammatory Stimuli and Viral Infection Qin, Zhihua Immunology Molecular Biology Virology SAMHD1 Innate immune responses NF-KB pathway Type I interferon pathway dNTPase activity Nuclear localization Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is the first demonstrated human deoxynucleoside triphosphohydrolase (dNTPase) that reduces intracellular deoxynucleoside triphosphate (dNTP) levels. Homozygous mutations of SAMHD1 can cause autoimmune diseases, such as Aicardi-Goutieres syndrome (AGS). These patients have increased levels of type I interferon (IFN-I) or interferon-induced gene expression, suggesting that SAMHD1 is a negative regulator of innate immunity. IFN-I induction is also reported in SAMHD1 knockout (KO) mice, which further indicates the negatively regulatory function of SAMHD1 in interferon responses. SAMHD1 is also demonstrated to act at the replication fork to restart DNA replication, resulting in the prevention of IFN-I induction. However, the exact function and role of SAMHD1 in regulating the innate immune responses to viral infections and inflammatory stimuli remain unknown. Here, we report that SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by interacting with key proteins in both NF-kB and IFN-I pathways such as interferon regulatory factor 7 (IRF7); we also identify that the HD domain of SAMHD1 is required for its suppression of IFN-I induction, and that the dNTPase activity but not nuclear localization of SAMHD1 is important for its inhibition of innate immunity in differentiated monocytes.To investigate the role of SAMHD1 in regulating innate immune responses, we conducted siRNA-mediated SAMHD1 knockdown (KD) in human primary monocyte-derived macrophages (MDM), and infected the cells with Sendai virus (SeV). TNF-a, IL-6, IFN-a and IFN-b mRNA levels were quantified, and we found that SAMHD1 KD increased TNF-a, IL-6, IFN-a and IFN-b mRNA levels after SeV infection in MDMs, suggesting that SAMHD1 suppresses NF-kB activation and IFN-I induction to viral infection. Moreover, we demonstrated that in HEK293T cells the suppression of IFN-I induction by SAMHD1 is specific to IRF7 but not IRF3, and that SAMHD1 inhibits IkappaB kinase epsilon (IKKe)-mediated phosphorylation of IRF7. We showed that SAMHD1 interacts with IRF7 in both MDMs and HEK293T cells. By generating SAMHD1 mutants and conducting co-immunoprecipitation (co-IP) assays, we demonstrated that the HD domain of SAMHD1 is responsible for its interaction with IRF7, and suppression of IRF7-mediated IFN-I induction.The dNTPase activity is a major characteristic of SAMHD1, which leads us to evaluate its importance in SAMHD1’s suppression of innate immune responses to viral infections and inflammatory stimuli. We generated monocytic U937 cells, with no detectable endogenous wildtype SAMHD1, stably expressing a SAMHD1 mutant defective for dNTPase activity - HD/RN. After phorbol 12-myristate 13-acetate (PMA) treatment to differentiate U937 cells and to induce the expression of SAMHD1, we stimulated cells with lipopolysaccharides (LPS) to activate the NF-kB pathway. TNF-a and IL-6 mRNA levels were tested, and we found that SAMHD1 mutant HD/RN is unable to inhibit LPS-induced TNF-a and IL-6 mRNA levels, suggesting that the dNTPase activity of SAMHD1 is important for its suppression of the NF-kB pathway in response to inflammatory stimulation in differentiated U937 cells. Moreover, HD/RN could not reduce TNF-a, IFN-a and IFN-b mRNA levels after SeV infection in differentiated U937 cells relative to wildtype SAMHD1, indicating that the dNTPase activity of SAMHD1 is also significant for inhibiting the innate immune responses to viral infection in differentiated monocytes. Consistent results were obtained in THP-1 SAMHD1 KO (THP-1/KO) cells with reconstituted HD/RN expression, affirming the importance of the dNTPase activity of SAMHD1 in its suppression of innate immune responses in differentiated monocytic cells. SAMHD1 is reported to be localized in both the nucleus and the cytoplasm of the host cell. To investigate whether nuclear localization of SAMHD1 correlates with its suppression of innate immunity, we mutated the nuclear localization signal (NLS) of SAMHD1 and generated a SAMHD1 mutant defective for nuclear localization - mNLS. U937 cells stably expressing mNLS were produced. After differentiation with PMA, we found that mNLS reduced LPS-stimulated TNF-a mRNA levels, and SeV-infection-induced IFN-b mRNA levels relative to wildtype SAMHD1. These all indicated that nuclear localization of SAMHD1 is not required for its suppression of innate immunity in differentiated U937 cells. Furthermore, reconstitution of mNLS in THP-1/KO cells decreased TNF-a mRNA levels after LPS stimulation, and TNF-a, IFN-a and IFN-b mRNA levels after SeV infection, which validated the results we obtained from U937 cells. Overall, we demonstrated that nuclear localization of SAMHD1 is not required for its suppression of innate immune responses in differentiated monocytes.These studies define the negatively regulatory function of SAMHD1 in innate immune responses to inflammatory stimuli and viral infection. Our results help to better understand SAMHD1’s role in regulating the innate immune responses in differentiated monocytic cells. These findings not only fill the knowledge gap of SAMHD1’s role in regulating innate immunity, but also lay the foundation for future research on the underlying mechanisms. 2020-09-30 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1587587968104986 http://rave.ohiolink.edu/etdc/view?acc_num=osu1587587968104986 restricted--full text unavailable until 2025-05-13 This thesis or dissertation is protected by copyright: some rights reserved. It is licensed for use under a Creative Commons license. Specific terms and permissions are available from this document's record in the OhioLINK ETD Center. |