Though the full mutational pattern is very diverse, its strongest & most distinctive aspects are three:
#1. Extraordinarily high rate of E mutations
#2. Specific mutations in NSP4
#3. A marked *absence* of spike RBD, NTD, & especially RBM mutations
11/
#1. Extraordinarily high rate of E mutations
#2. Specific mutations in NSP4
#3. A marked *absence* of spike RBD, NTD, & especially RBM mutations
11/
September 2, 2025 at 3:16 AM
Though the full mutational pattern is very diverse, its strongest & most distinctive aspects are three:
#1. Extraordinarily high rate of E mutations
#2. Specific mutations in NSP4
#3. A marked *absence* of spike RBD, NTD, & especially RBM mutations
11/
#1. Extraordinarily high rate of E mutations
#2. Specific mutations in NSP4
#3. A marked *absence* of spike RBD, NTD, & especially RBM mutations
11/
There was clearly a tight connection between mutations in E (esp. T30I), M (esp. H125Y), and specific sites in NSP4. As I noticed that several were bronchoalveolar lavage (BAL) samples. In fact, *every single BAL sequence on my list* had some form of this mut pattern.
7/
bsky.app/profile/ryan...
7/
bsky.app/profile/ryan...
I want to make clear just how extreme the E mutation rate is in known BAL chronic sequences. Below is the ratio of mutation density in each SARS-CoV-2 protein in known, labeled BAL seqs vs high-quality circulating sequences (HCQS).
The wider BAL-pattern graph (~400 seqs) looks very similar. 15/
The wider BAL-pattern graph (~400 seqs) looks very similar. 15/
June 26, 2026 at 9:08 PM
There was clearly a tight connection between mutations in E (esp. T30I), M (esp. H125Y), and specific sites in NSP4. As I noticed that several were bronchoalveolar lavage (BAL) samples. In fact, *every single BAL sequence on my list* had some form of this mut pattern.
7/
bsky.app/profile/ryan...
7/
bsky.app/profile/ryan...
Interestingly, B.1.637 is the only lineage with ORF1a:K3142E (another NSP4 mutation), which is *also* in this Kazakhstan dog sequence. Outside of B.1.637, ORF1a:K3142E is extremely rare, and occurred almost exclusively early in the pandemic. 16/
April 10, 2025 at 12:48 AM
Interestingly, B.1.637 is the only lineage with ORF1a:K3142E (another NSP4 mutation), which is *also* in this Kazakhstan dog sequence. Outside of B.1.637, ORF1a:K3142E is extremely rare, and occurred almost exclusively early in the pandemic. 16/
New work from Salic Lab @harvardcellbio.bsky.social & Mitchison lab reports a ER-derived vesicle harboring coronovirus RNA polymerase & an Nsp3/4 pore that allows export of newly synthesized coronovirus RNA to to the cytoplasm. Super cool biology! www.science.org/doi/10.1126/...
A coronaviral pore-replicase complex links RNA synthesis and export from double-membrane vesicles
Coronaviral nsp4 is the key organizer of double-membrane vesicle pores and forms a complex with the active viral replicase.
www.science.org
November 16, 2024 at 2:48 PM
New work from Salic Lab @harvardcellbio.bsky.social & Mitchison lab reports a ER-derived vesicle harboring coronovirus RNA polymerase & an Nsp3/4 pore that allows export of newly synthesized coronovirus RNA to to the cytoplasm. Super cool biology! www.science.org/doi/10.1126/...
EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase https://www.biorxiv.org/content/10.64898/2026.09.26.754626v1
September 29, 2026 at 4:16 AM
EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase https://www.biorxiv.org/content/10.64898/2026.09.26.754626v1
I tried to ask the regular Claude interface how to download sarbecovirus NSP4 genomes (and similar other requests) from Genbank, and it said the request was too dangerous, so I’d have to use an older version of Claude.
Unlike Theo, I’m not sympathetic.
Unlike Theo, I’m not sympathetic.
April 15, 2026 at 10:57 AM
I tried to ask the regular Claude interface how to download sarbecovirus NSP4 genomes (and similar other requests) from Genbank, and it said the request was too dangerous, so I’d have to use an older version of Claude.
Unlike Theo, I’m not sympathetic.
Unlike Theo, I’m not sympathetic.
New preprint alert! 🦟 <-> 🦠 <-> 🧑
I am thrilled to share our latest study on alphavirus host adaptation. More specifically, on how a single codon helps blunt alphavirus-induced host innate immune responses in mosquito and human cells. Continue reading for more!
www.biorxiv.org/content/10.1...
I am thrilled to share our latest study on alphavirus host adaptation. More specifically, on how a single codon helps blunt alphavirus-induced host innate immune responses in mosquito and human cells. Continue reading for more!
www.biorxiv.org/content/10.1...
The Sindbis virus nsP3 opal codon protects viral RNA and fitness by maintaining replication spherule integrity
Most alphaviruses encode an in-frame opal stop codon between nsP3 and nsP4 in their nsP ORF. This opal stop codon mediates a temperature-dependent balance between viral polymerase production and prote...
www.biorxiv.org
September 29, 2025 at 11:23 AM
New preprint alert! 🦟 <-> 🦠 <-> 🧑
I am thrilled to share our latest study on alphavirus host adaptation. More specifically, on how a single codon helps blunt alphavirus-induced host innate immune responses in mosquito and human cells. Continue reading for more!
www.biorxiv.org/content/10.1...
I am thrilled to share our latest study on alphavirus host adaptation. More specifically, on how a single codon helps blunt alphavirus-induced host innate immune responses in mosquito and human cells. Continue reading for more!
www.biorxiv.org/content/10.1...
Houston researchers found that rotavirus protein NSP4 disrupts cell calcium, spreading signals to uninfected cells and worsening disease.
Similar to SARS-CoV-2’s E protein.
www.science.org/doi/10.1126/...
Similar to SARS-CoV-2’s E protein.
www.science.org/doi/10.1126/...
Viroporin activity is necessary for intercellular calcium signals that contribute to viral pathogenesis
The NSP4 viroporin initiates an innate immune response and intercellular calcium waves that contribute to pathogenesis.
www.science.org
January 18, 2025 at 11:22 PM
Houston researchers found that rotavirus protein NSP4 disrupts cell calcium, spreading signals to uninfected cells and worsening disease.
Similar to SARS-CoV-2’s E protein.
www.science.org/doi/10.1126/...
Similar to SARS-CoV-2’s E protein.
www.science.org/doi/10.1126/...
Interesting tidbit: in chronics there’s a very tight connection between mutations at E:5-42 (esp ∆V14 & T30I) & a region of NSP4 (ORF1a:3049-3089 plus—oddly—ORF1a:2972).
*All* S:L1186F chronics have ≥1 mut at E:5-36 & 8/11 have ≥1 in the NSP4 region.
31/64
*All* S:L1186F chronics have ≥1 mut at E:5-36 & 8/11 have ≥1 in the NSP4 region.
31/64
December 23, 2024 at 6:09 PM
Interesting tidbit: in chronics there’s a very tight connection between mutations at E:5-42 (esp ∆V14 & T30I) & a region of NSP4 (ORF1a:3049-3089 plus—oddly—ORF1a:2972).
*All* S:L1186F chronics have ≥1 mut at E:5-36 & 8/11 have ≥1 in the NSP4 region.
31/64
*All* S:L1186F chronics have ≥1 mut at E:5-36 & 8/11 have ≥1 in the NSP4 region.
31/64
Allegedly, the addition of this mutation in *NSP4* of all places, caused both WT & Delta viruses to acquire all consensus Omicron mutations—in three separate replicates!—while the T3255 viruses "only" acquired a few Omicron mutations. Just transparent fraud if you ask me. Supplementary table below.
April 26, 2026 at 7:34 PM
Allegedly, the addition of this mutation in *NSP4* of all places, caused both WT & Delta viruses to acquire all consensus Omicron mutations—in three separate replicates!—while the T3255 viruses "only" acquired a few Omicron mutations. Just transparent fraud if you ask me. Supplementary table below.
....namely, that a nearly identical copy of the entire Omicron genome is reproduced on repeated Calu3 passaging of both WT and Delta isolates—but only when a specific NSP4 mutation (ORF1a:T3255I, found in most Delta as well as Omicron) is present.
June 5, 2026 at 2:42 PM
....namely, that a nearly identical copy of the entire Omicron genome is reproduced on repeated Calu3 passaging of both WT and Delta isolates—but only when a specific NSP4 mutation (ORF1a:T3255I, found in most Delta as well as Omicron) is present.
Everything, but E, M, & NSP4 the most. I posted various predictions elsewhere, but I'll throw my top E/M/NSP4 here again:
ORF1a:S2972F/P, T3058I, A3070V, G3072C, H3076Y
(or if it goes Cryptic, then: V2943F, I2961F, C3059S)
E:V5A/F, G10V/C, T9I, ∆V14, S16N, L27S, T30I, Y42C
M:S4P, S94N/R, H125Y
ORF1a:S2972F/P, T3058I, A3070V, G3072C, H3076Y
(or if it goes Cryptic, then: V2943F, I2961F, C3059S)
E:V5A/F, G10V/C, T9I, ∆V14, S16N, L27S, T30I, Y42C
M:S4P, S94N/R, H125Y
December 2, 2025 at 2:32 AM
Everything, but E, M, & NSP4 the most. I posted various predictions elsewhere, but I'll throw my top E/M/NSP4 here again:
ORF1a:S2972F/P, T3058I, A3070V, G3072C, H3076Y
(or if it goes Cryptic, then: V2943F, I2961F, C3059S)
E:V5A/F, G10V/C, T9I, ∆V14, S16N, L27S, T30I, Y42C
M:S4P, S94N/R, H125Y
ORF1a:S2972F/P, T3058I, A3070V, G3072C, H3076Y
(or if it goes Cryptic, then: V2943F, I2961F, C3059S)
E:V5A/F, G10V/C, T9I, ∆V14, S16N, L27S, T30I, Y42C
M:S4P, S94N/R, H125Y
University of California Study Finds That SARS-CoV-2 NSP4 Protein Alters Endoplasmic Reticulum Structure
www.thailandmedical.news/news/univers...
www.thailandmedical.news/news/univers...
University of California Study Finds That SARS-CoV-2 NSP4 Protein Alters Endoplasmic Reticulum Structure - Thailand Medical News
www.thailandmedical.news
December 11, 2024 at 1:39 AM
University of California Study Finds That SARS-CoV-2 NSP4 Protein Alters Endoplasmic Reticulum Structure
www.thailandmedical.news/news/univers...
www.thailandmedical.news/news/univers...
Threads take me a really long time to do, & I've had no mental energy to do anything the past few days, so probably not going to do a thread. This is the relevant figure. eWT-I is the 90-day Calu-3 passaged WT with ORF1a:T3255I/NSP4:T492I, while eWT-T has T3255.
www.nature.com/articles/s41...
www.nature.com/articles/s41...
April 26, 2026 at 7:28 PM
Threads take me a really long time to do, & I've had no mental energy to do anything the past few days, so probably not going to do a thread. This is the relevant figure. eWT-I is the 90-day Calu-3 passaged WT with ORF1a:T3255I/NSP4:T492I, while eWT-T has T3255.
www.nature.com/articles/s41...
www.nature.com/articles/s41...
This is the paper. Among other things, it claims that by passaging WT and Delta viruses in Calu-3 cells, they produced almost exact replicas of Omicron. www.nature.com/articles/s41...
A single mutation may contribute to accelerated evolution of SARS-CoV-2 toward Omicron - Nature Communications
Drivers of viral evolution in SARS-CoV-2 are insufficiently understood. In this study, the authors show how a key SARS-CoV-2 mutation, NSP4 T492I, is potentially responsible for accelerating genome ev...
www.nature.com
April 26, 2026 at 3:09 PM
This is the paper. Among other things, it claims that by passaging WT and Delta viruses in Calu-3 cells, they produced almost exact replicas of Omicron. www.nature.com/articles/s41...
Viroporin activity is necessary for intercellular calcium signals that contribute to viral pathogenesis www.science.org/doi/10.1126/...
Viroporin activity is necessary for intercellular calcium signals that contribute to viral pathogenesis
The NSP4 viroporin initiates an innate immune response and intercellular calcium waves that contribute to pathogenesis.
www.science.org
January 20, 2025 at 2:55 PM
Viroporin activity is necessary for intercellular calcium signals that contribute to viral pathogenesis www.science.org/doi/10.1126/...
A structural roadmap for the formation of the coronavirus nsp3/nsp4 double membrane vesicle pore and its implications for polyprotein processing and replication/transcription https://www.biorxiv.org/content/10.1101/2025.04.10.648184v1
April 12, 2025 at 4:19 AM
A structural roadmap for the formation of the coronavirus nsp3/nsp4 double membrane vesicle pore and its implications for polyprotein processing and replication/transcription https://www.biorxiv.org/content/10.1101/2025.04.10.648184v1
Accumulation of mutations in nsp4, E, and the S2 subunit underlies mammalian cell tropism expansion and virulence attenuation of avian coronavirus
journals.plos.org/plospathogen...
journals.plos.org/plospathogen...
Accumulation of mutations in nsp4, E, and the S2 subunit underlies mammalian cell tropism expansion and virulence attenuation of avian coronavirus
Author summary Infectious bronchitis virus (IBV), a member of the γ-coronavirus genus, is primarily controlled via live attenuated vaccines. However, the production of these vaccines depends on embryo...
journals.plos.org
April 15, 2026 at 2:24 PM
Accumulation of mutations in nsp4, E, and the S2 subunit underlies mammalian cell tropism expansion and virulence attenuation of avian coronavirus
journals.plos.org/plospathogen...
journals.plos.org/plospathogen...
A structural roadmap for the formation of the coronavirus nsp3/nsp4 double membrane vesicle pore and its implications for polyprotein processing and replication/transcription https://www.biorxiv.org/content/10.1101/2025.04.10.648184v1
April 12, 2025 at 4:19 AM
A structural roadmap for the formation of the coronavirus nsp3/nsp4 double membrane vesicle pore and its implications for polyprotein processing and replication/transcription https://www.biorxiv.org/content/10.1101/2025.04.10.648184v1
A coronaviral pore-replicase complex links RNA synthesis and export from double-membrane vesicles www.science.org/doi/10.1126/...
A coronaviral pore-replicase complex links RNA synthesis and export from double-membrane vesicles
Coronaviral nsp4 is the key organizer of double-membrane vesicle pores and forms a complex with the active viral replicase.
www.science.org
November 11, 2024 at 3:07 PM
A coronaviral pore-replicase complex links RNA synthesis and export from double-membrane vesicles www.science.org/doi/10.1126/...
EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase https://www.biorxiv.org/content/10.64898/2026.09.26.754626v1
September 29, 2026 at 4:16 AM
EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase https://www.biorxiv.org/content/10.64898/2026.09.26.754626v1
Riveting fuckin' watch, this: www.youtube.com/watch?v=NSP4...
100 Vintage Story Players Simulate Civilization REACTION | Lightly Cut
YouTube video by Angeweenie VTuber
www.youtube.com
March 1, 2026 at 10:50 PM
Riveting fuckin' watch, this: www.youtube.com/watch?v=NSP4...
Molecular architecture of coronavirus double-membrane vesicle pore complex
www.nature.com/articles/s41...
www.nature.com/articles/s41...
Molecular architecture of coronavirus double-membrane vesicle pore complex - Nature
A study details the molecular architecture of the double-membrane-spanning pore formed by the proteins nsp3 and nsp4 in double-membrane vesicles of SARS-CoV-2.
www.nature.com
August 15, 2024 at 12:56 AM
Molecular architecture of coronavirus double-membrane vesicle pore complex
www.nature.com/articles/s41...
www.nature.com/articles/s41...
EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase bioRxivpreprint
EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase
Mosquito-transmitted alphaviruses, including chikungunya (CHIKV), Mayaro, Eastern equine encephalitis, and Venezuelan equine encephalitis viruses, cause severe arthritic or neurologic disease in humans. There are currently no approved antiviral treatments against alphavirus infection. Here, we report that the purine ribonucleoside EIDD-3525 is a potent inhibitor of alphavirus replication in vitro with broad spectrum antiviral activity against the Alphavirus genus. Using a trans-replicase assay, we find that EIDD-3525 blocks alphavirus RNA replication. Mutational profiling suggests that EIDD-3525 targets the alphavirus nsP4 RNA-dependent RNA polymerase that is conserved across medically important alphaviruses. In a pre-clinical mouse model of CHIKV infection and disease, oral treatment with EIDD-3525 reduced viral tissue burdens and the severity of virus-induced disease. Collectively, our findings support the continued development of the purine ribonucleoside EIDD-3525 as an orally available antiviral therapeutic for use against a broad group of alphaviruses.
dlvr.it
September 30, 2026 at 11:32 AM
EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase bioRxivpreprint
pGBW-m4134196
Depositor: Ginkgo Bioworks
Purpose: Mammalian expression plasmid for SARS-CoV-2 nsp4
www.addgene.org/152506/
Depositor: Ginkgo Bioworks
Purpose: Mammalian expression plasmid for SARS-CoV-2 nsp4
www.addgene.org/152506/
September 20, 2024 at 8:00 PM
pGBW-m4134196
Depositor: Ginkgo Bioworks
Purpose: Mammalian expression plasmid for SARS-CoV-2 nsp4
www.addgene.org/152506/
Depositor: Ginkgo Bioworks
Purpose: Mammalian expression plasmid for SARS-CoV-2 nsp4
www.addgene.org/152506/