Influence Of The Nuclear Localization Signal Of SAMHD1 From Different Species On Anti-virus Activity

A host restriction factor is a kind of host protein which has anti-virus capacity.The host restriction factors involved in the inhibition of Human Immunodeficiency Virus(HIV)include APOBEC3,Trim5?,BST-2 and SAMHD1,etc.SAMHD1 was identified as a host restriction factor in 2011 by Laguette,et al.,and is a protein located in the nucleus and composed of a SAM domain and a HD domain.The HD domain has the activity as a d NTPase which can metabolize a d NTP into a deoxyribose nucleoside and a free triphosphoric acid group.The activity of SAMHD1 as a d NTPase can regulate cellular d NTP level,thereby inhibiting the reverse transcription of HIV-1,consequently,to restrict the virus.However,viral accessory protein Vpx expressed by HIV-2 and Simian Immunodeficiency Virus(SIV)can degrade SAMHD1 to restore the replication of the viruses.Research showed that after deletion of the nuclear location signal(NLS)of SAMHD1,it was relocated into the cytoplasm and could antagonize the degradation by Vpx and enhance its anti-virus activity,suggesting that the NLS of SAMHD1 has an influence on its anti-virus activity.In addition,the interaction study of SAMHD1 from primates and Vpx found that the alternation of some amino acids of SAMHD1 affects the degradation effect of Vpx.For the non-primate animals which are distant relatives of human,there may be more factors that can affect the interaction between SAMHD1 and Vpx.Until now,the structure and function of SAMHD1 from the species other than human is rarely described,especially from two non-primate species,feline and bovine.Feline is often used as an animal model to mimic HIV infection,while bovine is a well studied model of its APOBEC3G(A3G)and HIV-1 in our previous study that the interaction mechanism of bovine A3 G with HIV-1 Vif was found to be different from that of homo A3 G.Therefore,in this study,SAMHD1 from feline and bovine were chosen as the research objects to explore the mechanism of the influence of the NLS of SAMHD1 from different species on anti-virus capacity,aiming to further understand the interaction pattern of SAMHD1 and Vpx and the anti-virus mechanism of SAMHD1.In this study,we first analyzed the amino acid sequences of human(homo),feline and bovine SAMHD1,and found the NLSs between the homo,feline and bovine SAMHD1 were conservative.A cellular localization experiment showed that SAMHD1 from the three species were relocated into the cytoplasm.After transfection of SAMHD1 from the three species into TZM-bl cells,we found that all of the wild type(wt)SAMHD1s from the three species have anti-virus activity and the TZM-bl cells harboring NLS-deleted homo and feline SAMHD1 were less vulnerable to be infected by HIV-2 and SIVmac239.For bovine SAMHD1,the deletion of NLS could only enhance its anti-HIV-2 activity but had no obvious influence on its anti-SIVmac239 activity.To investigate whether the differences of anti-virus activity between wt and NLS-deleted SAMHD1 was a result from the diffenrent degree of degradation of them by Vpx,degradation experiments were conducted in HEK293 cells and the results confirmed that Vpx derived from SIVmac239 could significantly degrade homo SAMHD1,partially degrade bovine SAMHD1,and hardly degrade feline SAMHD1.At the presence of a Vpx Q76 A mutant(a key site to the function of Vpx for degrading SAMHD1)from SIVmac239,the degradation of homo and bovine SAMHD1 could be restored compared with at the presence of wt Vpx.For the NLS-deleted homo and bovine SAMHD1,the degradation by Vpx was also restored.For the consideration that the C terminal domain(CTD)of homo SAMHD1 affects its combination with Vpx derived from SIVmac strains and with the sequence alignment result of SAMHD1 between different species,we drew a conclusion that the difference of some individual amino acids between the CTD of feline and homo SAMHD1 was a possible reason to explain the difficulty in degrading feline SAMHD1 by Vpx.Although bovine SAMHD1 did not has a CTD compared with homo SAMHD1,it still could be degraded by Vpx.Therefore,we next invesgated whether the wt and NLS-deleted SAMHD1 from the three species could interact with Vpx.The results showed that similar to homo SAMHD1,feline and bovine SAMHD1 could interact with Vpx,and this interaction was not influenced even the NLSs of feline and bovine SAMHD1 were deleted.Moreover,with the consideration that the deletion of SAMHD1 NLS may affect its enzyme activity and the location where it functions as a d NTPase and then influence its anti-virus activity,we studied whether the d NTPase function of SAMHD1 from the three species affected its anti-virus capacity.The HD domain is closely related to the d NTPase function of SAMHD1.By sequence alignment,we found the amino acid H233(a key site to the d NTPase function of SAMHD1)of homo SAMHD1 was conservative in the sequences of feline and bovine SAMHD1 and confirmed the anti-virus capacity of SAMHD1 was associated with its d NTPase activity in the TZM-bl cell line.Further experiments will focus on whether the NLS affects the d NTPase activity of SAMHD1 and intracellular level of d NTPs.In conclusion,the influence of the NLS of SAMHD1 from different species on anti-virus capacity was investigated in this study.The difference of anti-virus capacity reflected at the presence and absence of the NLS of SAMHD1 from different species was analyzed and interpreted from the aspects of the antagonism and interaction domain between SAMHD1 and Vpx and d NTPase activity of SAMHD1.This study will be helpful to further understand the specific mechanism of the anti-virus capacity of SAMHD1 and the mechanism of interaction with Vpx.