WEBVTT Kind: captions Language: en 00:00:07.600 --> 00:00:11.040 Hello everybody and thank  you for the organizers. So,   00:00:11.040 --> 00:00:18.720 my topic is about the SARS-CoV-2 fusion peptide  which is but- more like a bio physical study. 00:00:19.280 --> 00:00:20.440 00:00:23.040 --> 00:00:31.600 So, this is the- how the SARS virus enter  the host cells. So basically, it is a common   00:00:35.920 --> 00:00:40.800 process shared by a lot of envelope  virus. So, the virus first attaches   00:00:40.800 --> 00:00:46.160 to the cell membrane receptors and then  they will possibly go through two pathways.   00:00:46.160 --> 00:00:51.360 The first one is to go through the plasma  membrane. So, there is a fusion between the   00:00:52.240 --> 00:00:58.720 virus envelope and the plasma membrane. And then  the second one is the virus first engulfed by the   00:00:59.440 --> 00:01:04.720 host cell to go into the endosome and  then the fusion between the endosomal   00:01:04.720 --> 00:01:09.920 membrane and the viral envelope will release  the genetic material into the host cell. 00:01:09.920 --> 00:01:16.080 So, during this process, the spike protein   00:01:16.080 --> 00:01:22.240 or other called- similar proteins or another  virus are very important. So basically, the spike   00:01:22.240 --> 00:01:32.480 protein is a trimer of the S1-S2 heterodimers. So,  the S1 is basically responsible for the binding   00:01:33.040 --> 00:01:41.360 of the receptors and then the S2 subunit is  responsible for the membrane fusion. So, as we can   00:01:41.360 --> 00:01:48.800 see here, there is a very small part which we call  the fusion peptide that's actually inserted into   00:01:48.800 --> 00:01:54.640 the membrane, and this insertion is very important  because it will initiate the membrane fusion and   00:01:54.640 --> 00:02:00.960 this is a required step in the viral infection.  So that's why we need to find out the mechanism   00:02:00.960 --> 00:02:05.840 of how distribution peptide interacts with the  membrane and how they initiate membrane fusion. 00:02:05.840 --> 00:02:07.360 00:02:07.360 --> 00:02:11.840 The method we mainly use is the- we call ESR  [erythrocyte sedimentation rate] which is   00:02:14.640 --> 00:02:25.840 magnetic spectrometer method. So basically, that  we want to put some spin- a free spin labeled   00:02:26.400 --> 00:02:34.480 lipids onto the membrane as we're showing here  and then this beam will locate our different   00:02:34.480 --> 00:02:40.720 position in the membrane. And then we mix the  fusion peptide with the membrane and detect the   00:02:40.720 --> 00:02:44.960 change of the structure of the membrane,  which is reflected by the spectrum. So,   00:02:44.960 --> 00:02:51.280 we collect this spectrum and then we repeat this  experiment in the condition of different peptide   00:02:51.280 --> 00:02:59.520 concentration. And then, from this spectrum, we do  a simulation and then we extract the parameters.   00:02:59.520 --> 00:03:05.600 So, one of the most important parameters in this  study is called the order parameters. As you can   00:03:05.600 --> 00:03:12.480 show in this figure, if the concentration of  the peptide increased, and then the- this x not   00:03:12.480 --> 00:03:18.800 very increased as well so there is the x type of  jump. We have repeated this experiment for a very-   00:03:18.800 --> 00:03:26.480 a wide range of virus fusion peptides and what  we find is that the active fusion peptide will   00:03:26.480 --> 00:03:34.000 induce this as junk- as shape junk- while  the mutants and then the non-active mutants   00:03:34.000 --> 00:03:41.600 cannot induce this kind of junk. So, what we  think is that this membrane ordering effect   00:03:42.480 --> 00:03:45.840 is a prerequisite for the membrane  fusion in the viral entry process. 00:03:45.840 --> 00:03:52.400 So, when it goes to the SARS-CoV glycoprotein, the   00:03:52.400 --> 00:03:59.920 S protein, then the- it's a little bit difficult  to determine which part is the fusion peptide   00:03:59.920 --> 00:04:07.360 because the for the SARS COVID, the S protein  has several distinctive cleavage sites. So,   00:04:07.360 --> 00:04:14.640 the most important are tool that- what we call  is the S1H2 side and then the S2 prime side which   00:04:14.640 --> 00:04:22.000 looking here. So, the first thing we need to do is  to determine which one is the real fusion peptide.   00:04:22.000 --> 00:04:30.080 So, and actually for- we have several candidates  and then for these candidates if we put it in the   00:04:30.640 --> 00:04:38.080 artificial system, they can induce a kind of  artificial membrane fusion. So, it's very-   00:04:39.040 --> 00:04:44.400 it's not very effective. They use the traditional  method. That's why we think the membrane ordering   00:04:44.400 --> 00:04:49.600 can be a criterion to distinguish which  one to identify the real fusion peptide. 00:04:49.600 --> 00:04:53.360 So, what we find here is that the FP1,   00:04:54.000 --> 00:05:00.000 it can show a very significant jump while the  other two candidates cannot have this kind of   00:05:01.600 --> 00:05:07.360 certain degree of increase. So, and then  we finally can identify- determine that   00:05:07.360 --> 00:05:15.520 the FP1 that which we immediately after the S2  prime position cleavage site is the real fusion   00:05:15.520 --> 00:05:22.560 peptide. And during this process, we also found a  very interesting thing that is quite not uncommon   00:05:24.080 --> 00:05:29.760 in the virus- that is dependent on  the calcium. So, as you can show here,   00:05:29.760 --> 00:05:37.360 without the calcium there is no big jump. And then  in here if we fix the concentration of the fusion   00:05:37.360 --> 00:05:41.360 peptide and increase the concentration  of the calcium, we can see the calcium   00:05:41.360 --> 00:05:47.120 significantly increase the ability of- for the  fusion peptide to induce the membrane ordering. 00:05:47.120 --> 00:05:50.560 And then when it goes to the SARS-2 fusion   00:05:50.560 --> 00:05:56.240 peptide, we first compare with the SARS the  sequence with SARS-1, SARS-2 and then the MERS and   00:05:56.240 --> 00:06:02.640 we identify the homologous sequence of the SARS-2.  And then we think that is the SARS-2 fusion   00:06:02.640 --> 00:06:08.720 peptide. And then we do this experiment and  we really found that it can also induce the   00:06:10.480 --> 00:06:17.680 membrane ordering and in the condition of the  calcium. And then we compare the ability to induce   00:06:17.680 --> 00:06:24.160 the membrane ordering between the SARS-1, SARS-2,  and the MERS, and we found that the SARS-2 has a   00:06:24.160 --> 00:06:34.160 higher activity. And we also have detected  the calcium dependency of the SARS-2 fusion   00:06:34.160 --> 00:06:40.240 peptide is very specific as we showed here.  The other ions do not have- cannot induce the   00:06:41.680 --> 00:06:47.840 membrane ordering as much as the calcium. So,  and with some other method, we can also know that   00:06:48.400 --> 00:06:53.280 one SARS-2 fusion peptide binds two calcium  ions and then the interaction between the   00:06:53.280 --> 00:07:03.840 SARS-2 fusion peptide and calcium is stronger  than those- this last one is MERS fusion peptide. And we also go one step forward   00:07:03.840 --> 00:07:09.760 that form a separate fusion peptide to the host  by protein trimer. So, remember that the fusion   00:07:09.760 --> 00:07:16.560 peptide is only part of the whole protein and we  need to know whether the whole protein- the fusion   00:07:16.560 --> 00:07:23.200 peptide domain of the whole protein functions as  the separate fusion peptide. So, what we use is   00:07:23.200 --> 00:07:31.040 a pseudotype virus particle which we call pp which  express the spike protein trimers on the membrane.   00:07:31.040 --> 00:07:38.240 And we use this to interact with the SUV which  have the spin label on the membrane and then we   00:07:38.240 --> 00:07:47.440 detect this- the increase of the membrane ordering  in real time. So, as we can shown here, after we   00:07:47.440 --> 00:07:56.240 trigger with the calcium we can find out the jump,  and then if we use some other ions then we cannot   00:07:56.240 --> 00:08:01.600 observe that. So that means that the whole S  protein trimer anchor of the membrane also induced   00:08:01.600 --> 00:08:12.320 the membrane ordering as the fusion peptide and  also is a very specific calcium-dependent factor. So, this is a conclusion then. The   00:08:12.320 --> 00:08:19.040 SARS-2 fusion peptide locates in the downstream of  the S2 prime cleavage site and it can induce the   00:08:19.040 --> 00:08:24.720 membrane ordering in a calcium-dependent fashion  and it binds to the calcium in specifically in   00:08:24.720 --> 00:08:32.880 one peptide to two calcium ratio and has a higher  fighting affinity and stronger membrane ordering   00:08:32.880 --> 00:08:40.400 effect than the SARS-1 fusion peptide. And then  we also notice that the SARS S2- S protein trimer   00:08:40.400 --> 00:08:46.960 also induced the membrane ordering as the separate  fusion peptide. So, this study will help us to   00:08:46.960 --> 00:08:52.720 understand the mechanism of the viral entry in  the whole cell and also indicate how- give some   00:08:52.720 --> 00:08:58.800 hints to how to develop a drug and vaccines.  So, yeah, that's my talk. Thank you so much.