This was a pretty cool collab between our @memprotlabuv.bsky.social led by @ismaelmingarro.bsky.social at @uv.es and @rongellerlab.bsky.social at @i2sysbio.es

And thanks to all authors for the effort put into this!

However, in order to allow fusion and infection, big rearrangements of the Spike protein are needed, so it makes sense that the opposite face is also essential for viral entry.

That might be why Spike TMD is the only fully-conserved TMD across all SARS-CoV-2 VOCs...

Strutural modeling of the TMD trimer also favored this motif to be responsible for stabilising the trimer, as it orients it to the inner core of the trimer in almost all models.

This motif might favor a transient oligomeric state in specific steps of the membrane fusion process.

Another controversy that existed was if the TMD can oligomerize by itself thus aiding Spike trimerization, since there are no structures of full-length pre-fusion Spike.

We showed that it does, and the only way to disrupt the oligomer is to disrupt the GxxAG motif.

As for viral entry, scrambling the TMD sequence completely disrupted Spike cell-cell fusion activity...

and mutating the AG from the GxxAG motif also did! This residues might be playing an essential role in fusing the viral and host membranes, but how?

To investigate how these structural motifs allow viral entry, we applied different functional approaches:

First, we showed that all mutations that blocked viral entry (except one) were able to reach the cell surface, so the infectivity deficiency was not due to a defect in protein trafficking...

Point mutations on both sides of the TMD helix had an influence on pseudovirus entry, especially through a small GxxAG motif but also other specific hydrophobic residues from the opposite, bulkier side.

It was shocking to see how almost any change that you apply to this region abolishes viral entry of Spike-pseudotyped VSV particles.

Any whole-turn scan and insertion on the N-terminal and core regions of the TMD blocked infectivity, implying essential structural motifs within the helix.

There was controversy in the literature since SARS-CoV-1 about if the Spike protein TMD was relevant for protein funcion and viral infectivity, so we decided to design a broad panel of mutations, insertions and whole-turn scans to adress this problem thoroughly.