Cell Symposia S5 Notes

multimeric secreted NS1, dengue hemorrhagic fever

Shee-Mei Lok, Duke-NUS Medical School, Singapore

sNS1 hexamer structure

NS1 dimer

Transepithelial-Transendothelial Electrical Resistance (TEER) assay to test the ability of sNS1 and its antibody complex to introduce endothelial cell hyper

sNS1

summary

1. DENV2 sNS1 contains heterogenous oligomeric states with tetramers as the predominant population
2. The structural stability of the stable tetramer is conferred by its N-terminal elongated $\beta$-sheet
3. Structure of Fab 5E3 complex with dimer shows
   • Fab when bound can dismantle loose tetramers and hexamers into dimers
   • Stable tetramer is resistant to Fab binding due to the tight interaction between NS1 dimers - it provides means for immune escape?
4. Combining results from cryoEM and TEER assay
   • suggests stable tetramer has endothelial cell hyperpermeability activity
   • either loose tetramer and hexamer or both are functional

ET study of dengue process

Shee-Mei Lok, Duke-NUS Medical School, Singapore

crystal structure of E protein

Dengue virus structure

entry mechanism of mature dengue virus

virus interactions with different types of liposomes at different pHs

segmentation of volta phase plate cryoET data

visualize the interactions between lipoasomes and dengue virus

unsupervised classification and STA of the E protein densities

fitting of the trimeric E protein structure into the densities

updated fusion process

summary

• At low pH, DENV has affinity towards anionic membrane(mimics endosomal membrane), so that fusion is directed between virus to endosomal membrane instead of virus-to-virus membranes
• At low pH, some E proteins have released their helical stem regions to form extended structure to interact with liposomal membrane
• The extended open trimers could be a result of extended monomers interacting with each other.
• A low resolution E protein open trimer fusion intermediate structure.
• As shown by previous crystal structure of the post-fusion E protein, DIII undergoes drastic movement relative to the rest of the E protein during fusion, our cryoEM structure of the open trimer, shows poor DI density, suggesting DI might facilitate interdomain movements during fusion.
• showed an updated proposed fusion process that suggests one E protein trimer can complete a fusion event.

SARS-CoV-2 resistance to Nirmatrelvir and the Countermeasures

Haitao Yang, ShanghaiTech University, China

the life cycle of CoVs

structure of SARS-CoV Mpro

design of wide-spectrum inhibitors targeting all Mpro

structure of Mpro from SARS-CoV-2

computer aided drug design(CADD), virtual screening(VS), and high-throughput screening(HTS)

Nirmatrelvir targets the SARS-CoV-2 Mpro

different mutations could have different impacts

nirmatrelvir resistance: increase activity, or decrease drug binding

oridonin adopts a novel binding mode

Host protease, TMPRSS2 inhibitors

biospecific compound 212-148

summary

• SARS-CoV-2 could adopt two distinctive routes to benefit itself in the presence of Nirmtrelvir
  • Route1: increase the activity of Mpro
  • Route2: confer drug resistance directly
• SARS-CoV-2 could utilize two routes simultaneously to achieve drug resistance while preserving replicative fitness, e.g. L50F/E166V
• The drug resistance mechanism is universal to an extent, nirmatrelvir resistant mutations also shows cross-resistance to ensitreivir
• Compounds that shows distinct binding mode could potentially be treated as a solution for nirmatrelvir-resistance
• We identified TMPRSS2 inhibitors including nafamostat, camostat and UK-371804 and determined their complex structures respectively
• We solved the structures of CTSB/CTSL in complex with K777 and E64d
• The combinatory usage of nafamostat and K777 showed the synergistic inhibition of SARS-CoV-2 with an unprecedented EC50 value of 1.9 nM
• bispecific compound 212-148 exhibits dual-inhibition effects in the enzymatic activity and cell-based assays, offering an alternative drug discovery strategy

Innate immunity on nucleic acids, signaling and regulation

Pu Gao, Institude of Biophysics, CAS, China

nucleic acid sensing, a central cellular defense process

cGAS-STING pathway: signaling, regulation

different pockets, same channel

ZCCHC3-DNA-cGAS condensates in vitro

phase separation: new strategy for innate immune regulation

S-acylation of gasdermins regulate their function in membrane pore formation and pyroptotic cell death

Tsan Xiao, Case Western Reserve University, Cleveland, USA

Gasdermin D N-terminal domain forms pores

dynamic process of pore formation and pore repair

Dual-site recognition of GSDMD by CASP1

Palmitoylation

Gasdermins are palmitoylated

Type VI secretion system cargo delivery vehicle

Weibo He, Xi’an Jiaotong University, China

Membrane complex, Baseplate, tube-sheath complex

SPA of type VI secretion system

interaction interface between Hcp-VgrG assembly

assembly model for type VI secretion system effector deliverying apparatus