Biological Functions Of Cellular Factor SAMHD1

SAM and HD domain containing protein(SAMHD1) is a very unique protein. It is so far the only known enzyme that regulates cellular d NTP levels directly through hydrolysis, the antiviral factor that suppresses the infection of many viruses including retroviruses, the associated protein that is related to autoimmune diseases like AGS and FSL. All these demonstrate the very importance of SAMHD1 in human cells.Yet, the functions of SAMHD1 have not been fully understood. For example, there were not enough evidences confirming the involvement of d NTPase in SAMHD1-mediated retroviral suppression. Also, as an interferon-induced protein, the role of SAMHD1 in interferon-induced antiviral mechanism was not clear. Furthermore, considering the high similarity between retroviral infection and retrotransposition, it was to be tested whether SAMHD1 could potently suppresses the activity of endogenous retroelements. All these questions were meant to be answered in this study.Through systematic research, this study has revealed the following findings on the biological functions of SAMHD1:1. SAMHD1 may not participate in interferon-induced antiviral activityInterferon induces strong antiviral activity in PBMC. Similar effects could be observed on monocyte THP-1 in vitro. We in this study have discovered that, both IFNα and PMA induces antiviral activity in THP-1 cells, which IFNα is significantly powerful. Interestingly, neither IFNα nor PMA increases SAMHD1 levels in THP-1. Subsequent reports have indicated that PMA grants SAMHD1 with antiviral potency, while Vpx induces the degradation of SAMHD1. Therefore, wild type SIV that expresses Vpx is able to breakthrough the antiviral defense in PMA-treated THP-1 cells. However, HIV-1, SIV, or SIVΔvpx cannot overcome the antiviral effects induced by IFNα. In fact, SIV fails in reducing SAMHD1 in IFNα-treated THP-1 cells. This on one hand indicates that in IFNα-treated THP-1 cells, retroviruses no longer could inject the core into cells through membrane fusion. On the other hand, it also suggests that, other immune pathways have been activated by IFNα to prevention retroviral infection in THP-1 cells, which precedes to the function of SAMHD1. Meanwhile, we provided evidence suggesting that, SAMHD1 acts against retroviral infection even in cycling cells.2. SAMHD1 is a novel retroelement regulatorRetroelements are the only known DNA elements in eukaryotes that utilize reverse transcription during replication. The activity of retroelements has been associated with hemophilia A and cancer. Recent evidences also suggested that, retrotransposition may also be linked to the development of autoimmune diseases. As an associated protein of AGS, SAMHD1 was likely to suppress retrotransposition and regulate cellular immune activation, through d NTPase to inhibit endogenous reverse transcription. As a result, SAMHD1 represses the activities of multiple retroelements, which is indeed achieved by suppressing reverse transcription. However, detailed study indicated that, SAMHD1 regulate retrotransposition by specifically reducing protein levels of LINE1 ORF2 p instead of lowering endogenous d NTP. Tests on SAMHD1 from different species demonstrated that LINE1 suppression is a conserved feature among mammalian SAMHD1 proteins. AGSassociated mutations weaken the potency of SAMHD1 suppressing LINE1, indicating that AGS-associated protein, retroelements, and cellular immune activation are potentially linked.3. Tetramer formation is essential for the biological function of SAMHD1Differences were observed for LINE1 suppression by SAMHD1 N-terminal truncations 109-626 and 120-626, which triggered our interests in finding structural differences between the two, and finally led to the discovery of SAMHD1 tetramer formation. We in this study subsequently tested the essentialness of tetramer formation on SAMHD1’s functions. And we found that, monomer mutant D137 A maintained only 5% potency in d NTP hydrolysis. In U937 cells, D137 A barely suppressed retroviral infection. And in HEK293 T cells, both monomer D137 A and dimer 120-626 failed in LINE1 inhibition. These data suggested that, tetramer formation is essential for SAMHD1 to perform its biological functions. More surprisingly, tetramer is also a pre-requirement for Vpx-induced SAMHD1 degradation. Therefore, tetramer formation is extremely important for SAMHD1 protein.Over above, results from this study further tested the biological function of SAMHD1 in multiple angles, revealed that SAMHD1 as a novel retroelement regulator, indicated that SAMHD1 is involved in protein stability regulation, and verified that tetramer formation is a prerequisite for all SAMHD1’s functions. All these findings will certainly provide more information on understanding the activity of SAMHD1 in human cells, and more evidences on SAMHD1 as an antiviral factor. They are also important to uncover the mechanism of SAMHD1 regulating cellular immune activation, and set up new molecular mechanism and targets for developing treatments against AIDS and autoimmune diseases.