Highlights d Identification of DGGGP synthase as a member of the UbiA superfamily d Structures determination of Apo-and lipid bound states of DGGGPase d Functional and structural insights into lipid modifying enzyme in three kingdoms d Archaeal lipid biosynthesis, remodeling, and membrane adaptation in cell membrane

The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg-bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.

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In the originally published version of this article, the supplemental information file containing Figures S1-S7 and Table S1 was inadvertently removed. The complete supplemental information file is now included with the paper online.The production team regrets this error.

Background The RNA-dependent RNA polymerase (RdRp) is a crucial element in the replication and transcription of RNA viruses. Although the RdRps of lethal human coronaviruses SARS-CoV-2, SARS-CoV, and MARS-CoV have been extensively studied, the molecular mechanism of the catalytic subunit NSP12, which is involved in pathogenesis, remains unclear. Results In this study, the biochemical and cell biological results demonstrate the interactions between SARS-CoV-2 NSP12 and seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8), suggesting that the polymerase activity and stability of SARS-CoV-2 RdRp were affected by them to varying degrees. Furthermore, the entry efficacy of SARS-CoV-2 pseudovirus considerably decreased when SLU7 or PPIL3 was knocked out, indicating that abnormal splicing of the host genome was responsible for this occurrence. In addition, NSP12 and its homologues from SARS-CoV and MARS-CoV suppressed thealternative splicing (AS) of cellular genes, which were influenced by the three splicing factors. Conclusions Overall, our research illustrates that SARS-CoV-2 NSP12 can engage with various splicing factors, thereby impacting virus entry, replication, and gene splicing. This not only improves our understanding of how viruses cause diseases but also lays the foundation for the development of antiviral therapies.