NB.1.8.1+Orf3a:W193R(33 seqs, 9 places, 30% Hong Kong, 6 with Orf3a:I20T)

[xz-keg]

From sars-cov-2-variants/lineage-proposals#2559

NB.1.8.1 seems to be getting the SARS-1 Orf3a:193R mutation.

According to @ryhisner,

ORF3a:W193 is a key residue. The SARS-CoV-1 ORF3a seems to function very differently than the SARS-CoV-2 ORF3a, and at least two studies identified S171 (E in SARS-1) and W193 (R in SARS-1) as being the most important differences in causing the change in function.

NB.1.8.1+T25969C(Orf3a:W193R)

GISAID query: T25969C, G22865T, -16456(may add more -s)
Query for Orf3a:I20T branch: T25969C, G22865T,T25451C
No. of seqs: 19(Canada 1(ON) Germany 2 Hong Kong 8 Singapore 5 South Korea 1 USA 2(OH,GBW from Japan))

First: EPI_ISL_19794372, Hong Kong, 2025-3-9
Latest: EPI_ISL_19860736, Canada, 2025-4-22

usher

[ryhisner]

ORF3a:W193R is the residue in SARS-CoV-1's ORF3a. It's in the vicinity of the only exposed, instructured loop in the entire ORF3a protein. It's thought to interact with VPS39, which is part of the HOPS membrane complex that fuses late endosomes/lysosomes with autophagosomes. Coronaviruses exit the cell via the lysosomal pathway, and increasing the pH (lowering acidity) of lysosomes (or preventing their pH from decreasing) is essential for viral egress.

Partially, this task is performed by the E protein, which opens up an ion channel in lysosomes/late endosomes, allowing positively charged ions to escape to the cytosol. But ORF3a also contributes to the task by preventing the fusion of late endosomes/autophagosomes with lysosomes, as well as likely blocking the import of lysosomal hydrolases (which increase pH and break down a multitude of proteins & other molecules) from the Golgi complex.

. .
.
SARS-CoV-1's ORF3a seems to be an outlier among sarbecoviruses in that it does not interact with VPS39 or seemingly prevent autophagosome/endosome fusion with lysosomes. One preprint (linked to below) found that ORF3a fosters the creation of what they call 3DBs, which may be involved in shuttling Spike and M proteins to the site of assembly in the ERGIC:

Here we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs have unusual electron-dense and dynamic inner structures, and their formation is driven by the accessory protein ORF3a via hijacking a specific subset of the trans-Golgi network (TGN) and early endosomal membranes. 3DB formation is conserved in related bat and pangolin coronaviruses yet lost during the evolution to SARS-CoV.

They found that W193R interfered with 3DB formation in SARS-CoV-2 and that switching the SARS-1 AA residues at 171 and 193 with SARS-2's enabled 3DB formation by the SARS-1 ORF3a. Similarly, several other studies have found that 171 and 193 are the key residues for the role of ORF3a in preventing lysosomal fusion with endosomes/autophagosomes.

.

A few great studies on this subject are this one from eLife:
'SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins'
https://elifesciences.org/articles/84477

This one from Developmental Cell:
'ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress'
https://www.cell.com/developmental-cell/fulltext/S1534-5807(21)00807-8

This one from Nature Communications
'SARS-CoV-2 virulence factor ORF3a blocks lysosome function by modulating TBC1D5-dependent Rab7 GTPase cycle'
https://www.nature.com/articles/s41467-024-46417-2

and this fascinating preprint:
'SARS-CoV-2 ORF3a drives dynamic dense body formation for optimal viral infectivity'
https://pmc.ncbi.nlm.nih.gov/articles/PMC11118709/

[FedeGueli]

Thank you @ryhisner great review !!

[xz-keg]

+3 GBW from China

[xz-keg]

+6 Hong Kong, prevalence rises to 30% there.

[xz-keg]

add the Orf3a:I20T sub-branch to the proposal

[Memorablea]

I also found a branch with ORF3a:S171L.GISAID Query:C21812T,C25904T,T26861C

I reviewed some papers, and they mentioned that positions 171 and 193 on ORF3a play a prominent role.

The residues at 171 and 193 are key determinants of the differential function of SARS-CoV-2 and SARS-CoV ORF3a in lysosomal exocytosis and autophagy inhibition.

One of the natural variants, ORF3a (Q57H/S171L), did not co-immunoprecipitate with Vps39 as much as did ORF3a (WT)

These papers, as you mentioned, S171 and W193 are important residues affecting 3DB generation, having a significant impact on the virus's infectivity. This is particularly evident in S171E and W193R. May I ask if S171L has the same or similar effect on SARS-CoV-2?@ryhisner

Reference:
https://pmc.ncbi.nlm.nih.gov/articles/PMC8502680/
https://www.nature.com/articles/s41467-024-46417-2
https://pmc.ncbi.nlm.nih.gov/articles/PMC8502680/

[xz-keg]

+1 South Korea

[ryhisner]

@Memorablea, that ORF3a:S171L branch is super interesting! I don't think it can be a coincidence that ORF3a:S171L has shown up so quickly—perhaps twice even—in this ORF3a:W193R branch.

That section you quoted is the only example I know of where someone actually looked to see if ORF3a:S171L had a phenotype similar to ORF3a:S171E, and it seems like it partially does.

[FedeGueli]

@Memorablea, that ORF3a:S171L branch is super interesting! I don't think it can be a coincidence that ORF3a:S171L has shown up so quickly—perhaps twice even—in this ORF3a:W193R branch.

That section you quoted is the only example I know of where someone actually looked to see if ORF3a:S171L had a phenotype similar to ORF3a:S171E, and it seems like it partially does.

Thank you @ryhisner ! 193 and 171 appeared twice each on NB.1.8.1 , but i dont think together in any branch @xz-keg can you confirm? (I m off from pc now)

[xz-keg]

@Memorablea, that ORF3a:S171L branch is super interesting! I don't think it can be a coincidence that ORF3a:S171L has shown up so quickly—perhaps twice even—in this ORF3a:W193R branch.
That section you quoted is the only example I know of where someone actually looked to see if ORF3a:S171L had a phenotype similar to ORF3a:S171E, and it seems like it partially does.

Thank you @ryhisner ! 193 and 171 appeared twice each on NB.1.8.1 , but i dont think together in any branch @xz-keg can you confirm? (I m off from pc now)

yeah they don't appear together. Given the level of convergence maybe they soon will.

[FedeGueli]

27

[xz-keg]

+1 NY

[xz-keg]

+1 South Korea +2 GBW ( 1 from Japan 1 from South Korea)

[xz-keg]

+1 Australia-NSW

[FedeGueli]

+2 Germany