| Sterile alpha motif and HD-containing protein 1(SAMHD1)is a restricting factor that limits the replication of retroviruses in mammalian myeloid cells,and possesses deoxyribonucleotide triphosphate(dNTPs)hydrolase(dNTPase).SAMHD1 utilizes its dNTPase activity to maintain low levels of intracellular dNTP in non-dividing myeloid cells to limit the replication of retroviruses.SAMHD1 has been shown to play an important role in the retroviral life cycle.Due to HIV-1(Human Immunodeficiency Virus Type 1)is unable to overcome the restriction of SAMHD1,HIV-1 replication in macrophages is kinetically suppressed.However,HIV-2 and some SIV(Simian Immunodeficiency Virus)strains are able to replicate rapidly in macrophages,because their accessory protein Vpx can counteract human SAMHD1(huSAMHD1)by directly loading huSAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1,where it is polyubiquitylated and subsequently degraded.SAMHD1 limits the replication of diverse retroviruses,but currently only HIV-2 and some SIV strains have been revealed to have mechanisms against SAMHD1.EIAV has the smallest and the genetically simplest genome.In addition to three major structural genes gag,pol and env,EIAV contains three additional open reading frames that encode three accessory proteins(Tat,Rev,and S2).More importantly,unlike retroviruses that can infect both non-dividing and dividing myeloid cells,EIAV(Equine Infectious Anemia Virus)is the sole member of the lentivirus family that primarily infects equine macrophages in vivo.Therefore,EIAV,a macrophage tropism non-primate lentivirus with simple genome,could overcome the restriction of SAMHD1 is largely unknown.This study aims to explore whether EIAV can counteract SAMHD1 and the mechanisms of EIAV against SAMHD1,in order to broaden our vision of the interaction,adaptation and evolution between lentivirus and host.We first clarify the antiviral effect of eqSAMHD1 on EIAV replication in equine monocyte-derived macrophages(eMDMs),and found that depletion of eqSAMHD1 expression enhance EIAV infection in equine macrophages.Furthermore,we found that it is the dNTPase activity of eqSAMHD1 that is required for its antiviral effect,as the mutant lacking the HD domain loses its antiviral activity.To investigate whether EIAV can counteract eqSAMHD1,we first measured the expression of endogenous eqSAMHD1 after EIAV infected equine macrophages,and the results showed that EIAV down-regulate the expression of eqSAMHD1.By overexpressing the viral protein of EIAV and eqSAMHD1,it was determined that EIAV can down-regulate the expression of SAMHD1 through its accessory protein Rev.The interaction of SAMHD1 and Rev was confirmed using co-immunoprecipitation(Co-IP),and the interaction is independent of mRNA.Through indirect immunofluorescence assays(IFA)and bimolecular fluorescence complementation(BiFC)assays,it was found that EIAV Rev can relocate SAMHD1 to the cytoplasm,forming a spot-like focus.Through treatment with protein degradation inhibitor and analysis the co-localization between SAMHD1-Rev complex and lysosomes,we found that Rev degrades SAMHD1 via the Class PI3K Ⅲ-dependent lysosomal pathway.These results indicate that Rev encoded by EIAV antagonizes the limiting effect by degrading SAMHD1 through the lysosomal pathway,and the strategy counteract SAMHD1 employ by EIAV is diferent from HIV-2.In the early stage,Beclinl was found to specifically interact with Rev using yeast two hybrid screening.Beclinl is the core member of the Class Ⅲ PI3K complex,so we explored whether Beclinl is involved in Rev-mediated degradation of SAMHD1.First,we confirmed that Beclinl interacts with Rev using Co-IP assays,and their interaction is independent of mRNA;depletion of eqBeclinl blocked EIAV-induced eqSAMHD1 degradation;Co-IP assays revealed that SAMHD1 utilize Rev to form an immunoprecipitation complex with Beclinl;it was found that Beclinl can be recruited to the SAMHD1-Rev complex to form a ring or "Ω" structure using IFA and BiFC assays.These results indicate that Beclinl is involved in Rev-mediated degradation of SAMHD1.Beclinl is a multifunctional protein,and its most well-known function is to regulate the nucleation of autophasome.Therefore,we monitored the autophagy flux after EIAV infection and overexpression of EIAV proviral DNA and Rev,the results showed that EIAV prompts the degradation of SAMHD1 through the lysosome pathway without inducing autophagy.To further verify the role of autophagy in eqSAMHD1 degradation mediated by EIAV Rev,a series of autophagy-related(ATG)gene knockout(KO)HEK293T cell lines,ATG3 KO,ATG5 KO,ATG7 KO,Beclinl KO,and PIK3C3 KO,were generated using CRISPR/Cas9 technology,we found that knockout of PIK3C3 or Beclinl,but not ATG3,ATG5 or ATG7,was able to completely rescue the expression of eqSAMHD1 in the presence of EIAV Rev.Finally,we performed structure-function analysis to map the key domain of Rev responsible for eqSAMHD1 degradation,and found that the ability of Rev to shuttle between the nucleus and cytoplasm is important for Rev-mediated eqSAMHD1 degradation the nuclear export activity of Rev;the Beclinl-binding deficient Rev mutant bearing 122-148 aa deletion in the non-essential domain of Rev failed to target SAMHD1 for lysosomal degradation.In addition,we also found that Rev degrades SAMHD1 in a species-specific manner.EIAV Rev is unable to degrade huSAMHD1,and HIV-1 Rev fails to degrade eqSAMHD1.In summary,this study demonstrated for the first time that EIAV infection can degrade SAMHD1 in EIAV target cells,and clarified the mechanism by which EIAV antagonizes SAMHD1:EIAV Rev degrades SAMHD1 via a Beclinl-dependent lysosomal pathway.We found that Rev has the function of binding Beclinl and antagonizing SAMHD1,and this function is species-specific,dependent on non-essential regions of Rev.Therefore,this study reveals a new mode of non-primate lentivirus in overcoming an intrinsic viral defense process.These findings may expand our understanding of the evolutionary conflicts between lentiviruses and restriction factors. |
