The Mechanism And Significance Of SAMHD1 N-terminal Fragment Inhibiting LINE-1 Retroelements

Interferon(IFN)is well-known as an important antiviral factor and immune regulator.Typically,pattern recognition receptors recognize the specific pathogen-associated molecular patterns and promote the expression of IFN.Although the IFN expression exerts beneficial antiviral effects by promoting the expression of interferon-stimulated genes,impropriate activation of IFN can trigger immune disorders and lead to corresponding pathogenic effects.Recent studies have described the increased IFN levels in cases with interstitial lung disease(ILD),and the application of the IFN signaling pathway inhibitor could improve the prognosis.In addition,persistent improper activation of IFN after viral infection was reported to be associated with poor prognosis in some studies of respiratory viral infection models such as respiratory syncytial virus.Therefore,it is important to find potential triggers of improper IFN activation,which helps the exploration of therapeutic approaches for abnormal IFN activation.Type 1 long interspersed elements 1(LINE-1s or L1s)are the only known autonomous active retrotransposons.They can trigger the activation of the IFN system through both DNA and RNA sensing pathways,which makes it one of the important endogenous triggers of impropriate IFN activation.Moreover,increased L1 activity and the corresponding IFN activation were reported in some ILD cases.Therefore,it might provide new treatment options for IFN activation-related diseases to explore approaches to L1 suppression.In fact,there are multiple host restriction factors that can effectively inhibit L1 activity,including the sterile-?-motif and histidine/aspartic acid domain containing protein 1(SAMHD1).SAMHD1 is the only known d NTPase in human cells,while it is also an important restriction factor against retrovirus and retroelements.The deficiency of SAMHD1 will lead to increased L1 activity and the corresponding pathological effects.However,most studies focused on its C-terminal fragment.Research showed that the SAMHD1 109-626 mutant has intact d NTPase activity and a more effective L1 inhibiting ability.This seems to imply that SAMHD1 1-108 does not play a positive role in L1 suppression.Here,we find that SAMHD1 1-108 alone remained effective in L1 suppression and that the protein splicing phenomenon is likely to release protein fragments whose relative molecular mass is similar to SAMHD1 1-108.This suggests the protein splicing mechanism might achieve more effective control of the IFN signaling pathway by releasing the L1 suppression ability of the N-terminal and C-terminal fragments of the SAMHD1.Therefore,we designed experiments to further investigate the mechanism and significance of L1 suppression by SAMHD11-108.This will contribute to further understanding of the biological function of SAMHD1 and provide clues to the pathogenic mechanisms of improper IFN activation-related diseases.In this study,we use various experimental methods of molecular biology and cell biology to investigate the molecular mechanism and biological significance of the L1 inhibition by SAMHD1 1-108,with the following results.1.SAMHD1 1-108 significantly inhibits L1 activity.Firstly,we establish the LINE-1 retrotransposition assays model with SAMHD1 WT,1-108,and 109-626 constructs.After excluding the effects of toxic effects,we demonstrated that both SAMHD1 1-108 and 109-626 can inhibit L1 activity efficiently.Considering that SAMHD1 1-108 does not contain any known functional region,the existing mechanism cannot explain its inhibition of L1 activity.Therefore,we designed experiments to further investigate the mechanism behind this phenomenon2.SAMHD1 1-108 inhibits L1 activity through a mechanism different from SAMHD1 WT.Current studies reveal that SAMHD1 inhibits L1 activity by suppressing the expression of L1-open reading frame 2 protein(L1-ORF2p)and promoting the relocation to stress granule of L1-open reading frame 1 protein(L1-ORF1p).However,our results show that SAMHD1 109-626 downregulates ORF2 p levels and promotes ORF1 p relocalization to stress granules,while SAMHD1 1-108 does not possess either of these functions.This suggests that the current knowledge of SAMHD1 to inhibit L1 activity is mainly performed by its 109-626 region and that SAMHD11-108 achieves inhibition of L1 activity by a novel mechanism different from that of the SAMHD1 WT.3.SAMHD1 1-108 suppresses LINE-1 retrotransposition by targeting LINE-1ribonucleoprotein particles,and damages the interaction between L1-m RNA,ORF1 p,and ORF2 p.To further investigate the mechanism of L1 inhibition by SAMHD1 1-108,we successively examine the effects of SAMHD1 1-108 on various processes of L1 activity.First,we demonstrated that SAMHD1 1-108 does not regulate L1 promoter activity,does not affect exogenous or endogenous L1-m RNA levels,does not down-regulate L1-ORF1 p expression levels nor does it interfere with the nucleoplasmic distribution of ORF1 p.Accordingly,we hypothesized that SAMHD11-108 might achieve the restriction of L1 activity by affecting the normal function of the L1 ribonucleoprotein particle(RNP),the basic unit of the L1 retrotransposon.To test this hypothesis,we targeted two important proteins in L1-RNP,ORF1 p and ORF2 p,respectively,purified L1-RNP by immunoprecipitation,and examined the interactions within L1 RNP under near-physiological conditions.Such model effectively verified that SAMHD1 1-108 disrupted the interaction between ORF1 p,ORF2p,and L1-m RNA,thus explaining the mechanism by which SAMHD1 1-108 inhibits L1 activity.4.Restriction of L1 activity by SAMHD1 1-108 prevents improper IFN activation caused by L1 through both DNA and RNA sensing pathways.The study above explains the mechanism by which SAMHD1 1-108 inhibits L1 activity,and the next question is whether this restriction of L1 activity has biological significance in avoiding improper activation of the interferon signaling pathway.Here,we explored both DNA and RNA sensing pathways separately.L1 activates the DNA sensing pathway mainly by DNA fragments generated from genome damage by L1 activity.As such,we indirectly demonstrated that SAMHD1 1-108 can avoid L1-induced DNA sensing pathway-dependent IFN activation.Furthermore,in a DNA sensing pathway-deficient cell model,we directly demonstrated that SAMHD1 1-108 can effectively prevent the activation of IFN promoter and increased the level of IFN-m RNA by L1 through the RNA sensing pathway.Thus,our results reveal that the inhibition of L1 by SAMHD1 1-108 possesses the biological significance of avoiding the improper activation of the IFN system.In summary,in this study,we found that SAMHD1 1-108 inhibits retrotransposon element L1 activity,and examined its mechanism and biological significance in detail.We found that SAMHD1 1-108 inhibits L1 activity by destabilizing L1 RNP,the basic unit of the L1 retrotransposon,thereby avoiding the impropriate activation of the subsequent IFN signaling pathway system.Our study deepens the understanding of the biological function of SAMHD1,explores new ideas to limit the pathogenic effects of L1,and provides a molecular target and theoretical basis for the development of therapeutic approaches for diseases related to improper activation of the IFN signaling pathway.