The Role Of Ca²⁺ Dependent Pathway In HIV-1 Vpu Antagonism Of Tetherin

Tetherin is a type II trans-membrane protein which possess a unique topology.Tetherin has an N-terminal cytoplasmic tail,followed by a trans-membrane helix,a coiled-coil ecto domain,and a C-terminal GPI anchor?or potentially second membrane anchor region?.Tetherin has been found to incorporate its two membrane embedding regions into host cell plasma membrane and nascent viruses membrane thus form a physical tether between the virions and the host cell so as to prevent the release of the virions.HIV-1 accessory protein Vpu is an 81 amino acids type I trans-membrane protein,with a single N-terminal transmembrane helix,and a C-terminal cytoplasmic domain containing two amphipathic helices.Early study indicated that Vpu has two major functions during the spread of HIV in vivo.The first is the down-regulation of CD4 receptors from the cell surface,through the interaction between their cytoplasmic domains and subsequently inducing CD4 degradation.The second is to enhance the release of progeny HIV-1 virionsby eliminating the tether effect of host restriction factor Tetherin.Similarly with its CD4 down-regulation activity,originally Vpu was proposed to eliminate the physical tether effect of host restriction factor Tetherin,by binding to Tetherin via trans-membrane helix interaction and leading to its surface down-regulation and degradation through ubiquitination and proteasome system.However,later research pointed out that Vpu can enhance the progeny virions release without degradation and cell surface down-regulation of Tetherin.Further study showed that Vpu can interfere with the recruitment of Tetherin to the virions budding site.However,some other report also indicated that BST2 still can be detected in the virions even in the presence of Vpu,which suggest that some other mechanisms are likely still waiting to be found.Vpu might form oligomers and possess weak selective monovalent cation channel activity.Vpu could interact with host cell background potassium channel TAS?,which is widely expressed and responsible for maintaining the resting membrane potential.Previously,we had proposed and found that Vpu could induce a slight augmentation of intracellular calcium level.In this thesis,we tried to further confirm whether the rise of intracellular calcium concentration induced by Vpu is due to the triggering of voltage dependent calcium channels and how this triggering happen.And what kind of role of calcium-dependent downstream pathways could play to mediate HIV-1 Vpu antagonism of Tetherin,so as to provide new insight into the mechanism for Vpu antagonism of Tetherin.We first investigate the effect of Vpu on intracellular Ca²⁺ and the relationship with voltage dependent calcium channel in 293 T cells which stable expressing Vpu.The charge of intracellular calcium concentration was detected by inverted microscope,fluorescent-spectrophotometer and flow cytometry.The result shows that Vpu could up-regulate the level of Ca²⁺ in cells.We aslo detected the change of intracellular calcium concentration by VDCC antagonist and RNA silencingmethod,to determine the change of calcium concentration is mediated by voltage dependent calcium channel.Second,in this thesis,we further studied the effect of calcium ion mediated downstream pathway on Vpu antagonism of Tetherin.We used transmission electron microscope to observe whether Tetherin identify progeny virus budding site caused by its special topology,and interference of Vpu to this process and the effect of calcium ions on it.At the same time,we also used the Co-ip to verify whether Tetherin and Gag physical interaction,and whether Vpu has an impact on the process.The results showed that Vpu did not interfere with the localization of Tetherin in budding sites.In addition,we tried to explore the possible role of Vpu induced intracellular Ca²⁺ concentration on inhibiting the phosphorylation of Hem ITAM,and the further impact on the NF-?B signaling activity of Tetherin.In the end,we did some preliminary studies on the possible mechnism that Vpu might counteract Tetherin though activating certain cutting enzymes via calcium signaling.We explored the role of calcium ions on the activityand expression of matrix metalloproteinases and Furin attranscription and protein level.Although in contrast to our hypothesis,our result indicated that Furin does not cut Tetherin,we found some intersting phenomena that Tetherin might inhibit Furin.We found that SPP may also play a role in cutting the Tetherin through the prediction of the action site software,so we made a preliminary study on it.Through this thesis study,we achieved a more detailed understanding of the effect of Vpu on intracellular calcium concentration,and the possible downstream mechanism involved in Vpu's counteraction of Tetherin,provided a new direction for the study of themechanism of Vpu antagonism to Tetherin.In the future,we will integrate the above preliminary exploration of the different pathways in to a more comprehensive understanding to reveal the Vpu antagonism to Tetherin mediated by calcium channel.