Studies On The Effect Of Calcium Ion And Proteases On HIV-1Vpu Antagonism Of Tetherin

Tetherin (BST2, CD317) is an IFN-α induced host restriction factor. Tetherininhibits a wide range of enveloped viruses release from the host cells, which relies onits unusual topology. Tetherin is a type II transmembrane protein consisting of an N-terminal cytoplasmic (CT) domain, a single transmembrane (TM) domain, a coiled-coil ectodomain and C-terminal glycosyl-phosphatidyl inositol (GPI) anchor. In orderto escape from the host cells, HIV-1encodes Vpu to antagonize the anti-viral activityof Tetherin. Vpu is a type I transmembrane protein including a short ectodomain, atransmembrane hydrophobic helical domain and an α-helical cytoplasmic domain.HIV-1Vpu antagonism of host restriction factor Tetherin is essential to the HIV-1viral particles release. It is still under debate how Vpu counteracts with Tetherin.Many researches focus on degradation and surface down-regulation of tetherininduced by Vpu. However, some resent results show that degradation of Tetherin wasnot essential for Vpu to enhance virus release. The mechanism of Vpu antagonism ofTetherin remains to be determined.Vpu could homo-oligomerize and form cation-selective ion channel. It isreported that Vpu interfere the function of background potassium channel TASK.However, it remains unclear whether the ion channel activity of Vpu could facilitateHIV-1budding and release. The ion channel activity of Vpu suggests that Vpu mightinfluent cell membrane protential and the equilibrium potentials for some essentialions, such as calcium. Since Tetherin functions through physical association withHIV-1virions, we hypothesize that Vpu might promote virus release by activatingsome proteases to hydrolyze Tetherin. In this thesis, we tried to identify other possiblemechanisms of the interaction between Tetherin and Vpu from the three aspects above,provide new insight in HIV research field and discover a potential target for AIDStherapy.First of all, we explored the effect of Vpu on the calcium concentration of cells.We also analyzed whether the change of calcium concentration is associated with the ion channel activity of Vpu. We loaded Vpu expressing cells with the calciumindicator fluo-4AM and used flow cytometry and fluorescence microscope to monitorchanges in fluorescence. The result shows that Vpu could up-regulate the level of Ca2+in cells.Second, we studied the influence of the variation of cellular calciumconcentration on Vpu antagonism of Tetherin, and whether voltage-dependent calciumchannels (VDCC) are involved. We discovered that α1subunit of both L-type calciumchannel and T-type calcium channel endogenous express in Hela and293T cells byreal time quantitative PCR. The method of RNA inference and blockade of calciumchannel by antagonists were utilized to detect the influence of VDCC on virus release.Our result indicates that T type VDCC might involve in Vpu antagonism of Tetherin.Also, we found that VDCC might not participate in Vpu induced down-regulation ofthe level of Tetherin on cell surface. These results suggested that Vpu might regulatecalcium concentration in host cells by activating T type VDCC, and calcium mightmodulate Vpu counteraction of Tetherin by other unclarified mechanisms such asinhibiting the colocalization of Tetherin to virus budding sites or activating somespecific kinds of protease to hydrolyze Tetherin.Moreover, we attempted to explore the potential protease that might hydrolyzeTetherin and participate in Vpu antagonism of Tetherin. Blocking the activity ofproteases by specific inhibitors, we found that phospholipase C (PLC), phospholipaseD (PLD) and matrix metalloproteinases (MMP) were not involved in counteraction ofTetherin by Vpu. However, MMP might promote HIV-1release. We also found thatTetherin has a potential Furin cleave site, but our study basically ruled out thepossibility of Furin cleavage of Tetherin.From the above experiments, we explored the possible factors that mightmodulate the antagonism of Tetherin by Vpu. Through our study, we have a deeperunderstanding on the mechanism of the interaction between Vpu and Tetherin, andadjust the directions for future research. We will continue investigating themechanism of the level of Ca2+in cytoplasm mediated by Vpu regulating the Vpuantagonism of Tetherin and the role played by T type VDCC in this process. Moreover,we will have a preliminary study on the potential effect of membrane curvature on counteraction of Tetherin by Vpu. From the exploration, we hope to discover a newapproach of calcium-mediated antagonism of Tetherin by Vpu.