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SAMHD1 Inhibits LINE-1 Retrotransposition by Promoting Stress Granule Formation.

Hu S, Li J, Xu F, Mei S, Le Duff Y, Yin L, Pang X, Cen S, Jin Q, Liang C, Guo F - PLoS Genet. (2015)

Bottom Line: The SAM domain and HD domain containing protein 1 (SAMHD1) inhibits retroviruses, DNA viruses and long interspersed element 1 (LINE-1).This function of SAMHD1 enhances sequestration of LINE-1 RNP in stress granules and consequent blockade to LINE-1 retrotransposition.In support of this new mechanism of action, depletion of stress granule marker proteins G3BP1 or TIA1 abrogates stress granule formation and overcomes SAMHD1 inhibition of LINE-1.

View Article: PubMed Central - PubMed

Affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P. R. China.

ABSTRACT
The SAM domain and HD domain containing protein 1 (SAMHD1) inhibits retroviruses, DNA viruses and long interspersed element 1 (LINE-1). Given that in dividing cells, SAMHD1 loses its antiviral function yet still potently restricts LINE-1, we propose that, instead of blocking viral DNA synthesis by virtue of its dNTP triphosphohydrolase activity, SAMHD1 may exploit a different mechanism to control LINE-1. Here, we report a new activity of SAMHD1 in promoting cellular stress granule assembly, which correlates with increased phosphorylation of eIF2α and diminished eIF4A/eIF4G interaction. This function of SAMHD1 enhances sequestration of LINE-1 RNP in stress granules and consequent blockade to LINE-1 retrotransposition. In support of this new mechanism of action, depletion of stress granule marker proteins G3BP1 or TIA1 abrogates stress granule formation and overcomes SAMHD1 inhibition of LINE-1. Together, these data reveal a new mechanism for SAMHD1 to control LINE-1 by activating cellular stress granule pathway.

No MeSH data available.


Related in: MedlinePlus

ImageStream flow cytometry was utilized to monitor the formation of stress granules and co-localization of SAMDH1 and ORF1p.HeLa cells were co-transfected with ORF1-tRFP and wild type EGFP-SAMHD1 DNA or the SAMHD1-H233A mutant DNA. Twenty-four hours post transfection, cells were stained with anti-G3BP1 antibody. Automated quantification of stress granule formation on gated cells was performed using the ImageStream technology. (A) Gating of cells. Single cells were first gated in R1, then cells in focus were selected in R2. EGFP and G3BP1 positive cells were selected in R3. tRFP, EGFP and G3BP1 positive cells were finally gated in R4 for imaging and further analysis. (B) Representative bright-field and fluorescence emission images for individual cells from each sample shown. The fluorescent granule inside each cell is clearly visible. (C) A number of 1x104 cells were examined and the frequency of cells with different numbers of G3BP1-positive puncta is presented in the graph. (D) The degree of colocalization of G3BP1 and ORF1p was calculated as the bright detail similarity score. EGFP was utilized as the control. Cells with SAMHD1 overexpression have higher similarity values as a result of greater co-localization of G3BP1 and ORF1p.
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pgen.1005367.g002: ImageStream flow cytometry was utilized to monitor the formation of stress granules and co-localization of SAMDH1 and ORF1p.HeLa cells were co-transfected with ORF1-tRFP and wild type EGFP-SAMHD1 DNA or the SAMHD1-H233A mutant DNA. Twenty-four hours post transfection, cells were stained with anti-G3BP1 antibody. Automated quantification of stress granule formation on gated cells was performed using the ImageStream technology. (A) Gating of cells. Single cells were first gated in R1, then cells in focus were selected in R2. EGFP and G3BP1 positive cells were selected in R3. tRFP, EGFP and G3BP1 positive cells were finally gated in R4 for imaging and further analysis. (B) Representative bright-field and fluorescence emission images for individual cells from each sample shown. The fluorescent granule inside each cell is clearly visible. (C) A number of 1x104 cells were examined and the frequency of cells with different numbers of G3BP1-positive puncta is presented in the graph. (D) The degree of colocalization of G3BP1 and ORF1p was calculated as the bright detail similarity score. EGFP was utilized as the control. Cells with SAMHD1 overexpression have higher similarity values as a result of greater co-localization of G3BP1 and ORF1p.

Mentions: We then used an automated method to quantify subcellular distribution of ORF1p by examining a large group of cells. The ImageStream system (Amnis) combines flow cytometry with intracellular imaging to calculate localization of fluorescent molecules [48]. A large cell population (104 cells) was analyzed to score the SAMHD1-induced ORF1p foci and the co-localization of ORF1p with G3BP1. A 3-fold increase in the frequency of G3BP1/ORF1p granule formation was observed as a result of SAMHD1 overexpression, the H233A mutant did not exert any effect (Fig 2).


SAMHD1 Inhibits LINE-1 Retrotransposition by Promoting Stress Granule Formation.

Hu S, Li J, Xu F, Mei S, Le Duff Y, Yin L, Pang X, Cen S, Jin Q, Liang C, Guo F - PLoS Genet. (2015)

ImageStream flow cytometry was utilized to monitor the formation of stress granules and co-localization of SAMDH1 and ORF1p.HeLa cells were co-transfected with ORF1-tRFP and wild type EGFP-SAMHD1 DNA or the SAMHD1-H233A mutant DNA. Twenty-four hours post transfection, cells were stained with anti-G3BP1 antibody. Automated quantification of stress granule formation on gated cells was performed using the ImageStream technology. (A) Gating of cells. Single cells were first gated in R1, then cells in focus were selected in R2. EGFP and G3BP1 positive cells were selected in R3. tRFP, EGFP and G3BP1 positive cells were finally gated in R4 for imaging and further analysis. (B) Representative bright-field and fluorescence emission images for individual cells from each sample shown. The fluorescent granule inside each cell is clearly visible. (C) A number of 1x104 cells were examined and the frequency of cells with different numbers of G3BP1-positive puncta is presented in the graph. (D) The degree of colocalization of G3BP1 and ORF1p was calculated as the bright detail similarity score. EGFP was utilized as the control. Cells with SAMHD1 overexpression have higher similarity values as a result of greater co-localization of G3BP1 and ORF1p.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4489885&req=5

pgen.1005367.g002: ImageStream flow cytometry was utilized to monitor the formation of stress granules and co-localization of SAMDH1 and ORF1p.HeLa cells were co-transfected with ORF1-tRFP and wild type EGFP-SAMHD1 DNA or the SAMHD1-H233A mutant DNA. Twenty-four hours post transfection, cells were stained with anti-G3BP1 antibody. Automated quantification of stress granule formation on gated cells was performed using the ImageStream technology. (A) Gating of cells. Single cells were first gated in R1, then cells in focus were selected in R2. EGFP and G3BP1 positive cells were selected in R3. tRFP, EGFP and G3BP1 positive cells were finally gated in R4 for imaging and further analysis. (B) Representative bright-field and fluorescence emission images for individual cells from each sample shown. The fluorescent granule inside each cell is clearly visible. (C) A number of 1x104 cells were examined and the frequency of cells with different numbers of G3BP1-positive puncta is presented in the graph. (D) The degree of colocalization of G3BP1 and ORF1p was calculated as the bright detail similarity score. EGFP was utilized as the control. Cells with SAMHD1 overexpression have higher similarity values as a result of greater co-localization of G3BP1 and ORF1p.
Mentions: We then used an automated method to quantify subcellular distribution of ORF1p by examining a large group of cells. The ImageStream system (Amnis) combines flow cytometry with intracellular imaging to calculate localization of fluorescent molecules [48]. A large cell population (104 cells) was analyzed to score the SAMHD1-induced ORF1p foci and the co-localization of ORF1p with G3BP1. A 3-fold increase in the frequency of G3BP1/ORF1p granule formation was observed as a result of SAMHD1 overexpression, the H233A mutant did not exert any effect (Fig 2).

Bottom Line: The SAM domain and HD domain containing protein 1 (SAMHD1) inhibits retroviruses, DNA viruses and long interspersed element 1 (LINE-1).This function of SAMHD1 enhances sequestration of LINE-1 RNP in stress granules and consequent blockade to LINE-1 retrotransposition.In support of this new mechanism of action, depletion of stress granule marker proteins G3BP1 or TIA1 abrogates stress granule formation and overcomes SAMHD1 inhibition of LINE-1.

View Article: PubMed Central - PubMed

Affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P. R. China.

ABSTRACT
The SAM domain and HD domain containing protein 1 (SAMHD1) inhibits retroviruses, DNA viruses and long interspersed element 1 (LINE-1). Given that in dividing cells, SAMHD1 loses its antiviral function yet still potently restricts LINE-1, we propose that, instead of blocking viral DNA synthesis by virtue of its dNTP triphosphohydrolase activity, SAMHD1 may exploit a different mechanism to control LINE-1. Here, we report a new activity of SAMHD1 in promoting cellular stress granule assembly, which correlates with increased phosphorylation of eIF2α and diminished eIF4A/eIF4G interaction. This function of SAMHD1 enhances sequestration of LINE-1 RNP in stress granules and consequent blockade to LINE-1 retrotransposition. In support of this new mechanism of action, depletion of stress granule marker proteins G3BP1 or TIA1 abrogates stress granule formation and overcomes SAMHD1 inhibition of LINE-1. Together, these data reveal a new mechanism for SAMHD1 to control LINE-1 by activating cellular stress granule pathway.

No MeSH data available.


Related in: MedlinePlus