Thread by @KroganLab

Officially out in @CellCellPress: Systematic comparison of SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta, & Omicron) using global proteomic and genomic analyses during infection to understand the molecular responses driving viral evolution 🧵👇 cell.com/cell/fulltext/…

We quantitatively compared the host response to infection in Calu-3 cells using abundance proteomics, phosphoproteomics, APMS, and transcriptomics. We observed equivalent replication kinetics for all VOCs except for Omicron BA.1, showing lower replication.

We observed drastic differences in the production of viral structural and accessory RNA and proteins between the VOCs during infection. Alpha and Beta upregulated Orf6 while Omicron BA.1 (somehow) suppressed its translation.

Alpha and Delta strongly upregulated Orf9b. Interestingly, although Alpha upregulated Orf9b RNA and protein, Delta only upregulated Orf9b protein. We pinpointed several mutations upstream of Orf9b that control Orf9b RNA and protein production, validated using reverse genetics.

Global phosphoproteomics identified stark differences in phosphorylation of N between W1 virus & VOCs (except for Gamma). 12 human kinases identified to phosphorylate these sites are predicted to have greater activity than those previously reported to control N phosphorylation.

We used affinity purification mass spectrometry (AP-MS) to quantify changes in virus-host protein-protein interactions between VOC and early-lineage SARS-CoV-2 protein forms. This network comprises 1746 high-confidence interactions of 39 variant forms across 16 viral proteins.

16% of interactions were altered. Strikingly, we found N*, a truncated version of N, to specifically bind to the human PAF complex, which was completely absent from binding to full-length N. This may impact variant-specific transcriptional responses to infection.

An integrative computational analysis of the host response revealed conserved and divergent pathways regulated during infection across VOCs. Translation and cell cycle regulation were most conserved; innate immune and inflammatory pathways were most divergent.

Modulation of the host inflammatory response was most divergent. Alpha and Beta, but not Omicron BA.1, antagonized ISGs, a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated pro-inflammatory genes compared to other VOCs.

A comparison of Omicron subvariants (BA.1, BA.2, BA.4, and BA5) revealed BA.5 evolved enhanced innate immune suppression, which also correlated with changes in Orf6 expression, effects dampened in BA.4 due to a mutation that disrupts the Orf6-nuclear pore protein interaction.

We propose that having achieved the antigenic shift required to escape widespread adaptive immune responses to Spike (BA.1), Omicron, like previous VOCs, subsequently experienced the next strongest selective pressure, resulting in the enhancement of innate immune evasion (BA.5).

Our integrative analyses describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.

Read more about the function of Orf6 and its effects in vivo from our accompanying manuscript by @LisaMiorin and Adolfo Garcia-Sastre: Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis. cell.com/cell-host-micr…

Big thanks to the collaborative QCRG network that fueled this work from @QBI_UCSF, @UCLInfectImm, @IcahnMountSinai, @GladstoneInst, @UofGlasgow, @sangerinstitute, @txbiomed, @TelAvivUni, @Synthego, @PhrmMar, @MIT, and @TheCrick.

Thanks to the amazing supervisors: @towerslab, @LorenaZAlvarez, @Jolly_lab, Adolfo Garcia-Sastre, @dlswaney, Kris White, @LisaMiorin, Massimo Palmarini, Arvind Patel, @MartinezSobrido, @doudna_lab, Roded Sharan, @pedrobeltrao, Paula Bofanti, Klim Verba, @ig_ech, & @manoliskellis.

Massive shout out to the first authors who drove this home: @DoctorBou, @akreuschl, @bpolacco, @lucygthorne, and @mrummadi!

For the open access version of our paper: authors.elsevier.com/c/1hoENL7PXmdc5

For the open access version of our Cell Host & Microbe accompanying manuscript: authors.elsevier.com/sd/article/S19…