Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY ADAM10 is a membrane-anchored endopeptidase that cleaves a range of membrane proteins at membrane- proximal external sites, and thus causes the shedding of their ectodomains. We now find that HIV-1 virions selectively incorporate considerable amounts of the active form of ADAM10. Our data indicate that ADAM10 cleaves the HIV-1 transmembrane protein gp41 within the membrane-proximal external region (MPER), which leaves behind a C-terminal fragment (CTF) and causes the shedding of the gp41 ectodomain from virions. Interestingly, Nef protects gp41 from cleavage by ADAM10, and thus increases the amount of virion- associated Env glycoprotein. Furthermore, Nef-deficient HIV-1 replicates strikingly faster in the absence of ADAM10, including in primary human CD4+ T cells. Additionally, we find that ADAM10 potently inhibits the propagation of Nef-deficient strains bearing primary Envs from different HIV-1 clades, and that ADAM10 inhibits HIV-1 replication through its catalytic activity. To understand the role of ADAM10 in HIV-1 spreading, we propose to examine its effects on virus infectivity and transmission, including the possibility that cellular ADAM10 substrates are involved. We also propose to examine whether the suppression of gp41 cleavage by ADAM10 is a core function of Nef, and whether it depends on the engagement of the clathrin adaptor AP-2. Interestingly, we have obtained a revertant with a point mutation in the gp41 MPER, the putative site of ADAM10 cleavage, that fully rescued a Nef-deficient HIV-1 in the presence of ADAM10. We propose to test the hypothesis that the MPER mutation protects gp41 from cleavage by ADAM10 because it alters the N- capping propensity of the mutated residue. We also intend to examine whether the sensitivity of gp41 to cleavage by ADAM10 correlates with the accessibility of the MPER in the unliganded Env spike. ADAM10 is regulated by TSPANC8 tetraspanins, which form complexes with ADAM10 and are thought to determine its substrate specificity. We thus plan to determine the roles of individual TSPANC8 family members in the cleavage of gp41 by ADAM10, and to explore whether Nef protects gp41 by altering the repertoire of TSPANC8/ADAM10 complexes. We also plan to explore the possibility that Nef suppresses the ADAM10-mediated cleavage of gp41 by reducing the exposure of phosphatidylserine, which is known to activate ADAM10. Interestingly, this possibility is suggested by recent evidence that the Nef targets SERINC3 and SERINC5 are lipid scramblases that promote phosphatidylserine exposure on virions. The proposed studies have the potential to yield fundamental new insights into what constitutes a novel mode of HIV-1 neutralization through the incorporation of a host protease. Of particular significance would be the identification of specific TSPANC8/ADAM10 complexes as being responsible for the cleavage of gp41, as this would offer the prospect of inactivating HIV-1 through agonists that selectively target these complexes.
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