Daoyu Profile picture
21 Jul, 10 tweets, 5 min read
Observation 1: RaTG13 RBD, for RBDs that are nested within the ACE2-using clade, had the weakest binding for all ACE2 except for mouse ACE2. It also don’t bind pACE2.
Observation 2: AncSARS1a-alt did not express, SARS-CoV-1 PC4-137 and SARS-CoV-1 GZ0402 was not optimally expressed. (Which explains their low scored on the binding measurements)
Observation 3: all SARS-lengthed RBMs except for SARS-CoV-2 WA1 i.c. (Ancestral ProCoV2) replicated to detectable levels in young BalB/C mouse lungs. All RBDs with this length have an aromatic 498 except for SARS-CoV-2 WA1.
(And other clades of SARS-CoV-2)
Observation 4: All tested ACE2 favors H,Y or F on position 498 over Q498. The only RBD that is better with Q498 is Rs7327. However, it is not relevant to SARS-CoV-2, and carried a F498 by default, indicating that other constraints (folding) is at
Work, or the bat allele was incorrect.
Observation 5: the D501 of RaTG13 is clearly defective. D501N/S/T improve binding by ~10^3-10^4 for all ACE2, including both R.affinis alleles, for RaTg13. Even for mouse, D501T gives ~10^1 improvement for binding. This is why you never see
It in other RBDs/RBMs
Observation 6: the Spike remained uncleaved for the VSV-BtKy72 S.
Observation 7: except for Rs7327, all full-lengthed non-SARS-CoV-2 RBDs bound detectably to mACE2. Even the residual binding spots of the Rs7327-mACE2 interaction is still more numerous than that of SARS-CoV-2-mACE2.

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More from @Daoyu15

20 Jul
The "7896" listed by the RaTG15 "paper" is clearly NOT the same 7896 clade as in the original GenBank deposit. notice that "MMI", which
is found in 7 out of the 8 7896 clade RdRps originally deposited on the "bat coronaviruses in China" paper. This is NOT what you see with Sanger error, which is random and does not repeat itself, as the same mismatch is found in almost all of them.
The same ATGATTC is found in all of them. This is clearly NOT what a sanger error looks like. This mean that the GWH sequence is discontiguous with their deposited RdRp, which indicate that their "samples" for the RaTG15 clade are in fact, NOT the same samples as the 7896 clade.
Read 9 tweets
19 Jul
archive.is/VaV1H A cautionary tale of why you shouldn't swap out the 1st AA residue on your ACE2 by replacing the native signal peptide with a mIgK leader sequence (it adds 2 additional AA (DS1819) in pACE2, and replaces Q18 with D or delete it in RaACE2).
Btw the mIgK sequence is 63nt in length. Far too big for a primer considering that: 1. You need a kozak consensus sequence (6nt) for the beginning, and an additonal 8nt for the BamHI cut site. 2. You need A substantial (19-22nt) sequence after it for ACE2.
This gives at minimal of 96nt for that giant oligo in the beginning of the ACE2 gene, which is far too long to be made into a primer (lop-sided primers tend to not amplify well). The original article for cloning the ACE2 genes cloned 9 genes at once with just PCR. This mean that
Read 8 tweets
19 Jul
@Undergroundsar3 Realized that they had a serious issue with their ACE2 (pangolin). due to an evolutionary quirk, real pangolin ACE2 is cleaved at 1AA later than human ACE2 or bat ACE2. This mean that S19, a residue critical for SARS-CoV-2, GD,GX or RaTG13 infectivity, is absent in native pACE2.
@Undergroundsar3 However, they have chose to ignore this fact and use 19-615 from each ACE2 and append either the mIgK leader signal peptide, which adds an additional DS1819, or a hIFNa1 signal peptide, which forces it to start at S19 in stead of T20 in native mature pACE2. their RaACE2 likely
@Undergroundsar3 had that additional D18 on the N terminus due to the replaced mIgK leader sequence, too. the NH2 and S19 of ACE2 is critical for binding to RBD. by adding that additional D18 with the signal peptide, the electrostatic property of RaACE2 and pACE2 are radically changed, which
Read 19 tweets
18 Jul
@MonaRahalkar @MarionKoopmans RaTG13 intentionally distanced from SARS-CoV-2 by dS injection, SARS-CoV-2 intentionally or unintentionally distanced in the other direction due to recoding or by passage in high mutagenesis conditions in a stable culture.
@MonaRahalkar @MarionKoopmans So much intentional distancing changes, that it rendered the 5’-UTR non-viable.
Read 30 tweets
7 Jul
@BillyBostickson @Topo_Ligio It may have intentionally been made to NOT infect wild mice as a part of attenuation strategy for live vaccine production. hACE2 cells or mice as the passage host, wild mice as test subjects for vaccine efficacy (Aged BalBC a popular SARS model). All it’s ancestors and
@BillyBostickson @Topo_Ligio All of SARS1-lengthed RBMs had the Y/H on 498, and all infect mice except LyRa11 (due to other part of the RBM being too different). No Spike tested before 2020 failed to infect mice for SARS-lengthed CoV S RBMs.
@BillyBostickson @Topo_Ligio Making 498 consensus as either Y or H lead to infection of wild mice.
Read 61 tweets

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