| BST-2(bone marrow stromal antigen 2)was identified as the host restriction factor of HIV-1(Human Immunodeficiency Virus type 1)in 2008.By its unique topological structure,it binds to the cell membrane,and the other end binds to the virus envelope,thereby preventing the release of HIV-1 nascent virus particles.The HIV-1 accessory protein Vpu(viral protein U)counteracts this process.It is generally considered that Vpu conteracts BST-2 by direct interaction with BST-2 which leads to BST-2’s lysosomal degradation or at least removal from the virion budding site.However,this current explanations of these molecular mechanisms are incompleted.In 2010,several studies reported the detection of intact BST-2 and small fragments of BST-2 in nascent virions obatined in the presence of Vpu.This suggests that Vpu does not completely exclude BST-2 from the virion budding site,and Vpu might use certain kind of decomposing enzyme inside the cell to cleave BST-2,thereby inhibiting the function of BST-2.We analyzed the amino acid sequence of BST-2 and found that the BST-2 protein has potential furin and signal peptidase(SP)cleavage sites.Therefore,we propose the first hypothesis: Furin and SP may cleave BST-2.However,preliminary experiments using site-directed mutagenesis on potential Furin and SP cleavage sites,and co-transfection using Furin or SP with BST-2,did not surport this first hypothesis.Nevertheless,during these preliminary studies,we found that mutation on potential furin cleavage site acturally decreased the BST-2’s restriction activity,which imply that BST-2 might exert its restrction function through this "furin-cleave site like" motif.Since Furin has already been reported to play improtant roles in the processing of HIV Env,we proposed second hypothesis that BST-2 might inhibit Furin by this "substrate like" motif,through which it can inhibit the processing of HIV-1 Env and therefore have another virion restriction mechanism other that direct tether.Based on this hypothesis,we designed experiments to examine BST-2’s impact on Furin’s function from four different aspects: its protein expression level,post-translation maturation,enzyme activity,and its cleavage of HIV-1Env.Our results indicated that heterologously expression of BST-2 in 293T cells and Hela cells(Hela cellsendogenously express Furin,while the amount of endogenous Furin in 293T cells is small,which is relatively difficult to detect.Hela cells endogenously express BST-2 protein.)reduce the overall protein expression level of Furin.It can also be found that the percentage of mature form protein of Furin decreased.In addition,BST-2 can also inhibit Furin enzyme activity in a dose-dependent manner.We then determined BST-2’s impact on HIV-1 Env’s processing.We found when that the amount of BST-2 expression increasing,whether in the cell or in the supernatant virus particles,the percentage of gp120 in the total amount of gp120 and gp160 gradually decreased.At the same time,the virus infectivity of the cell supernatant also gradually decreased.Thereafter,This study further explored the molecular mechanism of BST-2 inhibiting Furin’s processing of Env.We found that BST-2 does not seem to have a direct interaction with Furin,but BST-2 can immunoprecipitate with gp160.However,gp120 and gp41 cannot co-precipitate with BST-2.Therefore,We speculate that BST-2 may prevent the binding of Furin to gp160 by binding to gp160.And BST-2 may inhibit the expression of HIV-1 Env.In summary,We concluded that the host restriction factor BST-2 can reduce Furin enzyme activity by reducing Furin expression level and inhibiting Furin maturation in addition to the classic antiviral mechanism reported in the literature.Through these mechanism,Furin’s ability to process HIV-1 Env will be inhibited by BST-2.Finally,BST-2 can also bind to Furin’s substrate gp160.Therefore,the packaging of Env mature body gp120 and gp41 in the virus particles will be affected,and eventually this could significantly reduce the infectivity of HIV-1 virus particles. |
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