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.
biorxiv.org/content/10.110…
biorxiv.org/content/10.110…
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.
In Coronaviruses they are thought to be located in the ER membrane in the virus producing cells.
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.
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 cells
high expression of Spike however leads to the total disruption of normal ER structure.
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)
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.
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. addgene.org/141347/ over time however you will lose spike expression.
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 fusion
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.
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.
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.
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.
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.
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*
