iiinova — where your digital self keeps evolving

I’m incredibly proud of our team’s work to unravel these complexities, especially Shahrzad Ezzatpour, Jennifer Hayashi, and Chengjin Ye. Special thanks to members of the Luis Martinez-Sobrido, Hector Aguilar-Carreno, and @theottlab.bsky.social labs for their contributions and helpful discussions.

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Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6

This discovery highlights the virus's adaptability. That is, while Spike evolves for immune evasion, mutations like L260F in NSP6 enhance RNA replication, balancing the evolutionary trade-offs. For the full details, check out our preprint here: www.biorxiv.org/content/10.1...

biorxiv.org

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating acr...

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As of today, this mutation has somewhat disappeared from dominant lineages post-XBB.1.16, but it is still detected at low levels and was certainly present in variants prior to BQ.1.1. JN.1-derived lineages may be content with NSP6 R252K for now. See outbreak.info report

Interestingly, the NSP6 L260F mutation was enriched in the lungs of experimentally infected mink (insight.jci.org/articles/vie...) suggesting that it alters viral tropism (lung > upper respiratory tract) and may explain the decreased pathogenesis of the revertant virus in our study.

We validated our results in vivo using the K18-hACE2 mouse model 🐁and found that while replication was lower in the NSP6 F260L virus, pathogenesis was much lower indicating a role for this mutation in pathogenesis as well.

To verify our results in the context of replication competent virus, we generated revertant XBB.1.16 and BA.5 viruses (i.e. NSP6 F260L) that are hypothesized to have LOWER replication. Indeed, both revertant viruses replicated lower in infected cells.

We see this phenotype if we only express NSP6 in cells as well.

In the current study, we find that L260F rescues the reduced lipid droplet consumption and RNA replication of BA.2 NSP6 (i.e. ΔSGF).

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We validated that SARS-CoV-2 consumes lipids during infection, and we found that Omicron BA.1 mutations in NSP6 reduce the lipid droplet consumption function and this correlates with reduced viral RNA replication.

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We used replicons to safely validate that the enhanced replication in these variants is due to NSP6 L260F. Indeed, reversion of this mutation to wt decreases replication significantly. New NSP6 R252K mutation in JN.1.1.1 enhanced replication as well but not as much as L260F.

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For more insight into this mutation, see posts from @jbloomlab.bsky.social and @ryanhisner.bsky.social x.com/jbloom_lab/s...

x.com

We found that within a variant lineage (e.g. BA.5 or XBB) strains emerged with higher entry but evolved towards higher replication. Two high RNA replication strains, BQ.1.1 and XBB.1.16, had independently acquired NSP6 L260F. This mutation has been observed in chronic infections.

Using Spike-defective replicons, we decoupled viral entry and RNA replication. We observed a fascinating trend: as Spike function decreased under immune pressure, RNA replication increased as a compensatory mechanism. 📈

We showed using replicons that the SARS-CoV-2 Omicron BA.1 variant has attenuated RNA replication due to mutations in NSP6, namely ΔLSG and I189V.

We have previously developed a reverse genetics platform to construct natural variant genomes rapidly. Using this technology, we also developed a replicon that reports on RNA replication and lacks the Spike coding sequence. www.nature.com/articles/s41...

While many BA.2-derived mutations (shown in red) have been fixed in subsequent variants, mutations continued to accumulate across the genome.

Phenotypic evolution of SARS-CoV-2 spike during the COVID-19 pandemic - Nature Microbiology

The impact of the ~40 nonsynonymous mutations OUTSIDE of Spike is less well understood. It's difficult to study these mutations using replication competent viruses due to the confounding differences in Spike function across variants. See recently published paper: www.nature.com/articles/s41...

nature.com

Systematic comparison of SARS-CoV-2 spike proteins from pre- and post-Omicron variants in cell lines, primary respiratory epithelial cells and Syrian hamsters show distinct phenotypic trajectories in ...

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First, let's take a look at how SARS-CoV-2 variants evolved in the past ~4 years. 🧬🔬 The virus has accumulated over 60 nonsynonymous mutations in Spike and research efforts focused on defining the role of these mutations in spread, pathogenesis, and immune evasion.

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