Molecular Mechanisms Underlying HIV-1-induced Damage Of Mucosal Cell Junctions

Acquired immunodeficiency syndrome(AIDS)is a disease caused by infection with the human immunodeficiency virus(HIV).The HIV-1 is the main causative strain.The main routes of transmission of HIV-1 include mother-to-child transmission,blood transmission and sexual transmission.During sexual transmission,mucosal epithelial contact is the key step in HIV-1 infection.HIV-1facilitates its transmission by increasing the mucosal permeability and disrupting the mucosal epithelial barrier.The cell junction complexes at the epithelial cell layer of the mucosa form the mucosal barrier.Researchers including us have demonstrated that the dynamic assembly and stability regulation of microtubules play an important role in the maintenance of mucosal epithelial barrier homeostasis.The microtubule dynamics are regulated by microtubule-binding proteins,including the microtubule-depolymerizing protein stathmin.Our previous studies found that the HIV-1 envelope protein gp120 induced degradation of stathmin protein,leading to microtubule hyperstabilization,which in turn disrupted the cell junction complexes and ultimately contributed to HIV-1 breaking through the mucosal epithelial barrier and invading the human immune system.However,the molecular basis of this process is still unclear and needs to be further investigated.In this thesis,we preliminarily explore the molecular mechanism by which HIV-1 disrupts the mucosal epithelial barrier to initiate its infection cycle.We treated mucosal epithelial cells with pseudotyped HIV-1 particles,results showed that HIV-1 infection resulted in the destruction of tight junctions and adhesion junctions,the hyperstabilization of microtubules and a decrease in the protein level of stathmin.By immunoprecipitation combined with mass spectrometry,we identified PKG1(protein kinase c GMP-dependent 1)and ADCK3(aar F domain containing kinase 3)as interacting proteins of stathmin.Further,We performed an in-depth screening of PKG1 and ADCK3,the experimental results showed that si RNA knockdown of PKG1 protein could rescue HIV-1-damaged cell junction,while ADCK3 knockdown could not.Therefore,it is likely that PKG1 plays an important role in bridging the gap between HIV-1 infection of mucosal epithelium and stathmin.We hypothesized that the protein kinase PKG1 is likely to play a role in HIV-1 infection by phosphorylating stathmin.PKG1 interacts with stathmin both in vivo and in vitro by immunoprecipitation assays.Overexpression of PKG1 increased the phosphorylation level of stathmin and was accompanied by a decrease of stathmin protein level.The results of phosphorylation mass spectrometry analysis about stathmin showed that the serine sites at positions 38 and 63 of stathmin were phosphorylated and modified under HIV-1 gp120 treatment.Further,the immunoblotting results showed an increased phosphorylation level at serine 63 of stathmin.The level of serine phosphorylation at serine 63 of stathmin was reduced after co-treatment of cells with HIV-1 gp120 and inhibitor of PKG1.More importantly,both PKG1 inhibition and stathmin-GFP overexpression could rescue HIV-1 gp120-induced disruption of cell junction,while overexpression of a serine phosphorylation-deficient mutant at serine 63 of stathmin could better restore the integrity of cell junction.In summary,we found that HIV-1 exposure activates PKG1,leading to phosphorylation modification at serine 63 of stathmin,hyperstabilization of microtubule and disassembly of cell junction complexes,ultimately resulting in damage to the mucosal epithelial barrier.Our results deeply revealed the molecular mechanism of HIV-1 infection of the mucosal epithelium,which can help to develop effective prevention strategies for HIV-1 infection.