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PLURplus's profile
gerald pao
gerald pao
gerald pao
@PLURplus

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gerald pao

@PLURplus

Biological Scientist @SalkInstitute @Scripps_Ocean dabbling in Bio DataScience, Neuroscience, Cephalopods, AI&HPC using Nonlinear Dynamics & Molecular Biology

La Jolla, CA, USA
deepecoweb.ucsd.edu
Joined March 2011

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    gerald pao‏ @PLURplus 30 Jul 2020

    Our 1. #SARSCoV2 preprint by @junk_junk_junk Collab of @salkinstitute & @RyanLabUSC We show experimental lab confirmation for field observations that D614G is taking over the world. 5X more infectivity. Many thanks to @DanielleFong who funded this work. https://www.biorxiv.org/content/10.1101/2020.07.21.214932v1 …pic.twitter.com/u0LUyTSWbE

    Cells fused by hACE2 and SARS-CoV2 spike protein into syncytia
    11:00 PM - 30 Jul 2020
    • 21 Retweets
    • 53 Likes
    • Sina fanatla (Saint's Fan account/cuenta de fan) Roxana Vuokko Laiho Frosty  444232 rahuldave Karv′ 🇪🇺 tim s #CountryOverParty #GQP
    3 replies 21 retweets 53 likes
      1. New conversation
      2. gerald pao‏ @PLURplus 30 Jul 2020

        So what is spike protein? Spike protein mediates the entry of the #SARSCoV2 virus into cells. Also called S for short. In Coronaviruses they are thought to be located in the ER membrane in the virus producing cells.pic.twitter.com/B7HWxWNRRN

        Coronavirus biogenesis from Fields Virology
        1 reply 2 retweets 8 likes
        Show this thread
      3. gerald pao‏ @PLURplus 30 Jul 2020

        When #SARSCoV2 spike protein is produced in 293T cells it is largely in the ER but not only. It colocalizes with Calnexin and ER marker. But it is also on the plasma membrane and on the nuclear membrane which is contiguous with the ER.pic.twitter.com/xL7wLD7L3M

        1 reply 1 retweet 6 likes
        Show this thread
      4. gerald pao‏ @PLURplus 30 Jul 2020

        To make this more obvious if cells are not permeabilized, you can still observe spike staining, that is Spike is also on the plasma membrane albeit in reduced quanties as shown in these stably infected cellspic.twitter.com/iKfrcf47lN

        non-permeabilized antibody staining of spike stable 293T cells shows some plasma membrane SarsCoV2 Spike glycoprotein
        1 reply 1 retweet 6 likes
        Show this thread
      5. gerald pao‏ @PLURplus 30 Jul 2020

        high expression of Spike however leads to the total disruption of normal ER structure.pic.twitter.com/8eYIxhBoC8

        1 reply 0 retweets 4 likes
        Show this thread
      6. gerald pao‏ @PLURplus 30 Jul 2020

        Another piece of evidence that demonstrates plasma membrane presence of spike is this experiment. Express spike with GFP in some cells, express hACE2 with TdTomato in other cells. Mix. Cells fuse to create massive syncytia (cells sharing cytoplasm after fusion)pic.twitter.com/qcLGW3hhdf

        SARS-CoV2 Spike glycoprotein & hACE2 generated syncytia
        1 reply 0 retweets 5 likes
        Show this thread
      7. gerald pao‏ @PLURplus 30 Jul 2020

        because you are mixing GFP and TdTomato the cells will look yellow under the fluorescent microscope when the ratio of both FPs are balanced. The syncytia can become quite massive. This is good evidence of plasma membrane spike protein not just ER.pic.twitter.com/G5PcHQFWi8

        1 reply 0 retweets 4 likes
        Show this thread
      8. gerald pao‏ @PLURplus 30 Jul 2020

        Surprisingly this was not completely lethal and cells could be stably infected with a lentiviral vector construct. You can get this vector from @Addgene that we deposited some time ago. https://www.addgene.org/141347/  over time however you will lose spike expression.pic.twitter.com/u6vypOKDuG

        1 reply 0 retweets 3 likes
        Show this thread
      9. gerald pao‏ @PLURplus 30 Jul 2020

        you can see large areas of fused syncytia. Ultimately some will round up and become large spheres as you can see happening in this image. Observe also the extensive fusionpic.twitter.com/qX2Xat0eFP

        1 reply 0 retweets 2 likes
        Show this thread
      10. gerald pao‏ @PLURplus 30 Jul 2020

        at higher magnification you can see multiple nuclei but in some cases they seem to degenerate. This fusion happens in normal cell culture conditions so does not require low pH.pic.twitter.com/o7s91y7ng6

        SARS-CoV2 Spike syncytia
        1 reply 0 retweets 3 likes
        Show this thread
      11. gerald pao‏ @PLURplus 30 Jul 2020

        In immunofluorescence you can clearly see that not only you have multiple nuclei in syncytia but the nuclei themselves fuse. So you have plasma membrane fusion events, ER fusion events and nuclear fusion events.pic.twitter.com/5FOglpdJ8m

        1 reply 0 retweets 2 likes
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      12. gerald pao‏ @PLURplus 30 Jul 2020

        If you look more carefully however, it appears to be that the majority of nuclear fusion is only of the outer nuclear membrane. You can still see spike separating nuclei by IF.pic.twitter.com/TBhuc4bhML

        1 reply 1 retweet 4 likes
        Show this thread
      13. gerald pao‏ @PLURplus 30 Jul 2020

        Working with syncytia is difficult. Syncytia are fragile and cannot be FACS sorted. However they will not pass a FACS mesh filter so you can quantify the loss of spike expressing cells. D614G spike had a small but statistically significant (p<0.02) 11% greater loss of GFP+ cells.pic.twitter.com/0qJdLVPHlK

        1 reply 0 retweets 3 likes
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      14. gerald pao‏ @PLURplus 31 Jul 2020

        when comparing Spike 614D and Spike 614G when pseudotyped with GFP expressing lentiviral vectors we observed that the #SARSCoV2 D614G mutant was ~5X more infectious.pic.twitter.com/UBBYi7ILkp

        1 reply 5 retweets 6 likes
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      15. gerald pao‏ @PLURplus 31 Jul 2020

        If you want a lentiviral vector expressing the SARS-CoV2 Spike glycoprotein with the D614G mutation, it should be available through @Addgene soon. This is brought to you by a generous donation of @DanielleFong and the W.M. Keck foundation *End of Thread*pic.twitter.com/zEPQMtqK1J

        2 replies 7 retweets 21 likes
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      16. End of conversation

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