subnanometer-resolution-structural-determination-hemagglutinin-from

Subnanometer-Resolution Structural Determination of Hemagglutinin from Cryo-Electron Tomography of Influenza Viruses

Cryo-electron tomography is a powerful tool to visualize heterogeneous samples, with one major application being structural characterization of ...

University of Massachusetts Chan Medical School

Influenza viruses pose severe public health threats globally. Influenza viruses are extensively pleomorphic, in shape, size, and organization of viral proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships and aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we leveraged convolutional neural networks and cryoET to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza morphology, glycoprotein density, and conducted subtomogram averaging of influenza glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.

Quantitative structural analysis of influenza virus by cryo-electron tomography and convolutional neural networks.

Distinguishing infectious pathogens from harmless microorganisms is essential for animal health. The mechanisms used to identify infectious microbes are not fully understood, particularly in metazoan hosts that eat bacteria as their food source. Here, we characterized a non-canonical pattern-recognition system in Caenorhabditis elegans (C. elegans) that assesses the relative threat of virulent Pseudomonas aeruginosa (P. aeruginosa) to activate innate immunity. We discovered that the innate immune response in C. elegans was triggered by phenazine-1-carboxamide (PCN), a toxic metabolite produced by pathogenic strains of P. aeruginosa. We identified the nuclear hormone receptor NHR-86/HNF4 as the PCN sensor in C. elegans and validated that PCN bound to the ligand-binding domain of NHR-86/HNF4. Activation of NHR-86/HNF4 by PCN directly engaged a transcriptional program in intestinal epithelial cells that protected against P. aeruginosa. Thus, a bacterial metabolite is a pattern of pathogenesis surveilled by nematodes to identify a pathogen in its bacterial diet.

Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans.

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (M) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 M, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 M. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.

Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses.

Influenza viruses are enveloped, negative-sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80 to 90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of purified receptor-free and receptor-bound HA, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryoelectron tomography to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even interglycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and the formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8 to 10 Å reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.

Article	Year
Quantitative structural analysis of influenza virus by cryo-electron tomography and convolutional neural networks. Structure (London, England : 1993)\| PubMed ID: 35290796	2022
Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans. Immunity\| PubMed ID: 36804958	2023
Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses. Viruses\| PubMed ID: 36992489	2023
Virion-associated influenza hemagglutinin clusters upon sialic acid binding visualized by cryoelectron tomography. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 40244672	2025

Qiuyu J. Huang

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