Chao Wu

The multidrug efflux pump EmrE is one of the smallest known active transporters and has become a model system for studying multidrug recognition and transport. While recent high-resolution structures have illuminated its dynamic substrate binding pocket, the conformations of its interhelical loops and C-terminal tail, regions critical for controlling proton coupling and gating, remain poorly characterized. Here, we report the high-resolution structure of protonated S64V EmrE determined using solution and solid-state NMR data. This new structural model shows the C-terminal tail occluding the open face of the transport pore, providing a structural basis for how EmrE minimizes proton leak in the absence of substrate. These findings support growing evidence that relatively simple model transporters must leverage an occluded state during alternating access to avoid physiologically unfavorable proton leak.

Respiratory syncytial virus (RSV) poses a significant global health challenge, particularly affecting infants, the elderly, and immunocompromised individuals. Despite recent progress in the development of vaccines and monoclonal antibodies, effective antiviral therapies remain limited. To advance the discovery of antiviral drugs, we have developed a dual-reporter RSV minigenome system, providing a safe and robust platform for antiviral evaluation. This system incorporates NanoLuc luciferase and superfolder GFP (sfGFP) linked by a self-cleaving P2A peptide, allowing for the simultaneous detection of orthogonal signals. Validation with L polymerase inhibitors confirmed the system’s reliability for screening small-molecule inhibitors. The linear correlation observed between the reporter signals enhances the assay’s reliability for antiviral assessment. This dual-reporter minigenome system advances targeted therapeutic strategies against RSV.

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-β), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms.

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic and Reston (RESTV) causing no severe disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with reduced inhibition of IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation caused almost complete inhibition of genome replication and transcription suggesting the role of these mutations in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha affect ebolavirus pathogenicity by IFN-I-dependent and independent mechanisms.

The SARS-CoV-2 Omicron variant has demonstrated enhanced transmissibility and escape of vaccine-derived immunity. Although first-generation vaccines remain effective against severe disease and death, robust evidence on vaccine effectiveness (VE) against all Omicron infections, irrespective of symptoms, remains sparse. We used a community-wide serosurvey with 5,310 subjects to estimate how vaccination histories modulated risk of infection in infection-naive Hong Kong during a large wave of Omicron BA.2 epidemic in January–July 2022. We estimated that Omicron infected 45% (41–48%) of the local population. Three and four doses of BNT162b2 or CoronaVac were effective against Omicron infection 7 days after vaccination (VE of 48% (95% credible interval 34–64%) and 69% (46–98%) for three and four doses of BNT162b2, respectively; VE of 30% (1–66%) and 56% (6–97%) for three and four doses of CoronaVac, respectively). At 100 days after immunization, VE waned to 26% (7–41%) and 35% (10–71%) for three and four doses of BNT162b2, and to 6% (0–29%) and 11% (0–54%) for three and four doses of CoronaVac. The rapid waning of VE against infection conferred by first-generation vaccines and an increasingly complex viral evolutionary landscape highlight the necessity for rapidly deploying updated vaccines followed by vigilant monitoring of VE.

The SARS-CoV-2 Omicron variant has demonstrated enhanced transmissibility and escape of vaccine-derived immunity. While current vaccines remain effective against severe disease and death, robust evidence on vaccine effectiveness (VE) against all Omicron infections (i.e. irrespective of symptoms) remains sparse. We addressed this knowledge-gap using a community-wide serosurvey with 5,310 subjects by estimating how vaccination histories modulated risk of infection in Hong Kong (which was largely infection naïve) during a large wave of Omicron epidemic during January-July 2022. We estimated that Omicron infected 45% (41-48%) of the Hong Kong population. Three and four doses of BNT162b2 or CoronaVac were effective against Omicron infection (VE of 47% (95% credible interval 34-68%) and 70% (43-99%) for three and four doses of BNT162b2 respectively; VE of 31% (1-73%) and 59% (10-99%) for three and four doses of CoronaVac respectively) seven days after vaccination, but protection waned with half-lives of 15 (3-47) weeks for BNT162b2 and 5 (1-37) weeks for CoronaVac. Our findings suggest that booster vaccination can temporarily enhance population immunity ahead of anticipated waves of infections.

The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible ‘loop 2’ region in LAG3 domain 1. We also defined the LAG3–FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.