Wenfu Ma

The cGAS-STING pathway is a central component of innate immunity. Upon activation, the endoplasmic reticulum (ER)-resident protein STING is packaged into COPII vesicles and transported to the Golgi apparatus to initiate downstream signaling. Despite the importance of this process, the molecular mechanisms governing STING sorting into COPII vesicles remain incompletely understood. Here, an optimized workflow is presented for the in vitro reconstitution of COPII vesicles to facilitate controlled analysis of cargo selection. A scalable method is described for preparing high-concentration cytosol (30-40 mg/mL) from HEK-293F suspension cells, combined with a straightforward procedure for generating semi-permeabilized HEK-293T cells as a defined membrane source. Compared to traditional adherent cell-based systems, this approach improves scalability, reduces cost, and enhances reproducibility. Using STING as a model cargo, COPII vesicle budding and cargo incorporation are validated by Western blot analysis, with a basal packaging efficiency of approximately 20% under defined conditions. This system allows controlled manipulation of biochemical parameters, including nucleotide stimulation, to assess their effects on cargo incorporation. This protocol provides a robust and adaptable platform for investigating COPII-mediated cargo sorting and can be extended to study additional transmembrane proteins and regulatory factors involved in ER export.

The host cell cytoskeleton plays a critical role in the SARS-CoV-2 life cycle, though the underlying mechanisms remain poorly understood. This study investigates the interaction between the SARS-CoV-2 spike (S) protein and the cytoskeleton-associated ezrin-radixin-moesin (ERM) proteins through biochemical and structural characterization. A previously unidentified ERM-binding motif on the SARS-CoV-2 S protein is identified, revealing that S-ERM interactions are specifically conserved among highly pathogenic coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2. Functionally, these interactions facilitate S packaging into virions by directing it to assembly sites, utilizing ERM’s affinity for negatively curved membranes, akin to its role in cell surface protrusions. Silencing ERM expression significantly reduces SARS-CoV-2 titer, highlighting its essential role in viral propagation. Additionally, leveraging the established role of COPI-mediated trafficking in S localization, a compound is developed to disrupt S-COPI binding, promoting S secretion to the cell surface and effectively reducing viral titers. Our findings revealed a critical host–pathogen interaction that drives S incorporation into virions and identified ERM proteins as key facilitators of coronavirus assembly. Furthermore, our study suggests an antiviral strategy by targeting the S-COPI trafficking pathway. These insights advanced our understanding of coronavirus–host interactions and provided a potential therapeutic approach against SARS-CoV-2 and other highly pathogenic coronaviruses.

Despite advances in understanding the STING signaling pathway, mechanisms governing cyclic GMP‐AMP (cGAMP)‐induced STING trafficking out of the endoplasmic reticulum (ER) remain unclear. This study reveals that STING localization is regulated by the balance between coat protein II (COPII)‐ and coat protein I (COPI)‐mediated trafficking, maintaining ER residency in the inactive state or promoting transport to the cis ‐Golgi via enhanced COPII‐mediated export upon activation. Two novel TANK‐binding kinase 1 (TBK1)‐regulated phosphorylated COPII sorting signals on STING—a conserved pSGME motif and a primate‐specific pFS motif—are biochemically and structurally identified. These cGAMP‐induced signals drive activated STING toward the ER‐Golgi intermediate compartment (ERGIC) and the cis ‐Golgi complex. Using a cell‐free COPII vesicle reconstitution system, TBK1 activation is shown to occur on COPII vesicles, while IRF3 phosphorylation is confined to the ERGIC or the cis ‐Golgi complex post‐uncoating, due to the competitive binding of COPII Sec24 and IRF3 to phosphorylated STING. A class of compounds is also identified that attenuates IRF3 phosphorylation by inhibiting phosphorylated STING packaging into COPII vesicles. These findings elucidate STING trafficking mechanisms and offer therapeutic potential for diseases linked to dysregulated STING activation.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus-like particle (SC2-VLP) method offers a powerful and accessible tool for studying the SARS-CoV-2 life cycle without the need for biosafety level 3 (BSL-3) laboratories. This system effectively mimics critical stages of the viral life cycle, including assembly, genome packaging, and egress, using a luciferase reporter fused to the T20 signal for sensitive and precise detection of viral particle production. SC2-VLPs are generated by co-expressing SARS-CoV-2 structural proteins, including membrane (M), nucleocapsid (N), envelop (E), and spike (S), along with the RNA packaging signal in HEK-293T cells. Unlike traditional virus-like particle systems, the SC2-VLP method ensures accurate quantification and greater fidelity to the natural viral life cycle. Furthermore, compared to lentiviral pseudotyping methods, which are limited to studying viral entry through the incorporation of S protein into HIV-based lentiviral particles, the SC2-VLP system provides a more comprehensive platform for exploring multiple stages of SARS-CoV-2 biology. While this method bypasses the risks of handling live virus and expands accessibility. The SC2-VLP method represents a significant advancement in antiviral research and the development of therapeutic strategies against SARS-CoV-2.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus-like particle (SC2-VLP) method offers a powerful and accessible tool for studying the SARS-CoV-2 life cycle without the need for biosafety level 3 (BSL-3) laboratories. This system effectively mimics critical stages of the viral life cycle, including assembly, genome packaging, and egress, using a luciferase reporter fused to the T20 signal for sensitive and precise detection of viral particle production. SC2-VLPs are generated by co-expressing SARS-CoV-2 structural proteins, including membrane (M), nucleocapsid (N), envelop (E), and spike (S), along with the RNA packaging signal in HEK-293T cells. Unlike traditional virus-like particle systems, the SC2-VLP method ensures accurate quantification and greater fidelity to the natural viral life cycle. Furthermore, compared to lentiviral pseudotyping methods, which are limited to studying viral entry through the incorporation of S protein into HIV-based lentiviral particles, the SC2-VLP system provides a more comprehensive platform for exploring multiple stages of SARS-CoV-2 biology. While this method bypasses the risks of handling live virus and expands accessibility. The SC2-VLP method represents a significant advancement in antiviral research and the development of therapeutic strategies against SARS-CoV-2.

The gut microbiota has emerged as a key modulator of host health, particularly because of its capacity to metabolize dietary glycosides. While C-glycosides demonstrate superior chemical stability and resistance to enzymatic cleavage compared to O-glycosides, specialized gut bacteria can selectively metabolize them, improving their bioavailability. Since the identification of microbial C-glycosidase enzymes, the precise reaction mechanisms remain incompletely understood, and standardized methodologies for studying these processes are still not well-established. To address this gap, we introduced an integrated multidisciplinary approach combining structural biology, enzymology, and analytical techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR), to systematically characterize these enzymatic activities. This comprehensive approach enables precise characterization of C-glycoside metabolism at molecular, enzymatic, and metabolic levels.

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.