G Protein Subunit ß1 Facilitates Influenza A Virus Replication by Promoting the Nuclear Import of PB2.

G protein subunit ß1 (GNB1), the beta subunit of the G protein family, plays an important role in regulating transmembrane signal transduction. Although a recent study has demonstrated that GNB1 can bind the matrix protein 1 (M1) to facilitate M1 transport to budding sites and promote the release of progeny influenza A virus (IAV), whether the GNB1 protein has other functions in IAV replication requires further study. Here, we found that GNB1 promoted IAV replication, as virus yield decreased in GNB1 knockdown or knockout cells. GNB1 interacted with polymerase subunits PB2, PB1, and PA. Overexpressed GNB1 facilitated PB2 binding to importin α3, α5, and α7 promoting the nuclear import of PB2, enhancing viral RNA synthesis and polymerase activity. Altogether, our results demonstrated that GNB1 positively regulates virus replication by interacting with polymerase subunits and facilitating the nuclear import of PB2, which provide novel insights into the molecular mechanism of IAV. IMPORTANCE Until now, there has been only one article on the role of GNB1 in IAV budding. No study has investigated the role of GNB1 in IAV replication. In this study, our research demonstrated that GNB1 could increase the interaction between PB2 and the importin α isoform and mediate the nuclear import of PB2. Therefore, GNB1 could promote viral replication and transcription. Our results provide a better understanding of the molecular mechanisms of viral replication and provide potential antiviral drug targets.

A novel mAb broadly neutralizes SARS-CoV-2 VOCs in vitro and in vivo, including the Omicron variants.

Novel immune escape variants have emerged as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. Many of the variants cause breakthrough infections in vaccinated populations, posing great challenges to current antiviral strategies targeting the immunodominance of the receptor-binding domain within the spike protein. Here, we found that a novel broadly neutralizing monoclonal antibody (mAb), G5, provided efficient protection against SARS-CoV-2 variants of concern (VOCs) in vitro and in vivo. A single dose of mAb G5 could significantly inhibit the viral burden in mice challenged with the mouse-adapted SARS-CoV-2 or SARS-CoV-2 Omicron BA.1 variant, as well as the body weight loss and cytokine release induced by mouse-adapted SARS-CoV-2. The refined epitope recognized by mAb G5 was identified as 1148 FKEELDKYF1156 in the stem helix of subunit S2. In addition, a human-mouse chimeric mAb was generated based on the variable region of heavy chain and VL genes of mAb G5. Our study provides a broad antibody drug candidate against SARS-CoV-2 VOCs and reveals a novel target for developing pan-SARS-CoV-2 vaccines.

A nanoluciferase SFTSV for rapid screening antivirals and real-time visualization of virus infection in mice.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that causes severe hemorrhagic fever in humans, but no FDA-approved specific antivirals or vaccines are available to treat or prevent SFTS. METHODS: The plasmids construction and transfection were performed to generate the recombinant SFTSV harboring the nanoluciferase gene (SFTSV-Nluc). Immunostaining plaque assay was performed to measure viral titers, and DNA electrophoresis and Sanger sequencing were performed to evaluate the genetic stability. Luciferase assay and quantitative RT-PCR were performed to evaluate the efficacy of antivirals in vitro. Bioluminescence imaging, titration of virus from excised organs, hematology, and histopathology and immunohistochemistry were performed to evaluate the efficacy of antivirals in vivo. FINDINGS: SFTSV-Nluc exhibited high genetic stability and replication kinetics similar to those of wild-type virus (SFTSVwt), then a rapid high-throughput screening system for identifying inhibitors to treat SFTS was developed, and a nucleoside analog, 4-FlU, was identified to effectively inhibit SFTSV in vitro. SFTSV-Nluc mimicked the replication characteristics and localization of SFTSVwt in counterpart model mice. Bioluminescence imaging of SFTSV-Nluc allowed real-time visualization and quantification of SFTSV replication in the mice. 4-FlU was demonstrated to inhibit the replication of SFTSV with more efficiency than T-705 and without obvious adverse effect in vivo. INTERPRETATION: The high-throughput screening system based on SFTSV-Nluc for use in vitro and in vivo revealed that a safe and effective antiviral nucleoside analog, 4-FlU, may be a basis for the strategic treatment of SFTSV and other bunyavirus infections, paving the way for the discovery of antivirals. FUNDING: This work was supported by grants from the National Key Research and Development Plan of China (2021YFC2300700 to L. Zhang, 2022YFC2303300 to L. Zhang), Strategic Priority Research Program of Chinese Academy of Sciences (XDB0490000 to L. Zhang), National Natural Science Foundation of China (31970165 to L. Zhang, U22A20379 to G. Xiao), the Science and Technology Commission of Shanghai Municipality (21S11903100 to Y. Xie), Hubei Natural Science Foundation for Distinguished Young Scholars (2022CFA099 to L. Zhang).

PDIA4 Is a Host Factor Important for Lymphocytic Choriomeningitis Virus Infection.

Mammalian arenaviruses are rodent-borne zoonotic viruses, some of which can cause fatal hemorrhagic diseases in humans. The first discovered arenavirus, lymphocytic choriomeningitis virus (LCMV), has a worldwide distribution and can be fatal for transplant recipients. However, no FDA-approved drugs or vaccines are currently available. In this study, using a quantitative proteomic analysis, we identified a variety of host factors that could be needed for LCMV infection, among which we found that protein disulfide isomerase A4 (PDIA4), a downstream factor of endoplasmic reticulum stress (ERS), is important for LCMV infection. Biochemical analysis revealed that LCMV glycoprotein was the main viral component accounting for PDIA4 upregulation. The inhibition of ATF6-mediated ERS could prevent the upregulation of PDIA4 that was stimulated by LCMV infection. We further found that PDIA4 can affect the LCMV viral RNA synthesis processes and release. In summary, we conclude that PDIA4 could be a new target for antiviral drugs against LCMV.

Discovery and Mechanism Study of SARS-CoV-2 3C-like Protease Inhibitors with a New Reactive Group.

3CLpro is an attractive target for the treatment of COVID-19. Using the scaffold hopping strategy, we identified a potent inhibitor of 3CLpro (3a) that contains a thiocyanate moiety as a novel warhead that can form a covalent bond with Cys145 of the protein. Tandem mass spectrometry (MS/MS) and X-ray crystallography confirmed the mechanism of covalent formation between 3a and the protein in its catalytic pocket. Moreover, several analogues of compound 3a were designed and synthesized. Among them, compound 3h shows the best inhibition of 3CLpro with an IC50 of 0.322 µM and a kinact/Ki value of 1669.34 M-1 s-1, and it exhibits good target selectivity for 3CLpro against host proteases. Compound 3c inhibits SARS-CoV-2 in Vero E6 cells (EC50 = 2.499 µM) with low cytotoxicity (CC50 > 200 µM). These studies provide ideas and insights to explore and develop new 3CLpro inhibitors in the future.

Sperm-Associated Antigen 9 Promotes Influenza A Virus-Induced Cell Death via the c-Jun N-Terminal Kinase Signaling Pathway.

Upon influenza A virus (IAV) infection, the IAV progeny ribonucleoprotein complex, with a defective viral genome, is sensed by DNA-dependent activator of interferon-regulatory factor (DAI). DAI initiates the recruitment of an array of proteins to form a multiprotein platform (PANoptosome), which triggers apoptosis, necroptosis, and pyroptosis during IAV infection. However, the mechanisms mediating the assembly of the PANoptosome are unclear. Here, we identified a scaffold protein, sperm-associated antigen 9 (SPAG9), which could interact with DAI to promote cell death during IAV infection. We further demonstrated that the cell death enhanced by SPAG9 was achieved through the DAI/SPAG9/c-Jun N-terminal kinase (JNK) axis, which could promote IAV-induced DAI-mediated cell death, including apoptosis, necroptosis, and pyroptosis. Our data further showed that the DAI/SPAG9/JNK signaling pathway enhanced the interactions among receptor-interacting serine/threonine kinase 1 (RIPK1), RIPK3, and DAI, thereby promoting IAV-induced PANoptosome formation. Overall, our study for the first time revealed a feed-forward circuit signaling pathway that enhanced IAV-induced DAI-mediated cell death, provided insights into the molecular mechanisms of cell death, and established therapeutic targets to address infectious and inflammatory diseases. IMPORTANCE Upon influenza A virus (IAV) infection, DAI is activated, recruits downstream proteins to assemble a multiprotein platform (PANoptosome), and then triggers cell death. Until now, the protein composition and assembly mechanism of the PANoptosome during IAV infection had not been elucidated. Using proximity labeling and mass spectrometry technology, we identified SPAG9 as a novel component of the PANoptosome and confirmed that SPAG9 promotes IAV-induced cell death by enhancing the interaction among RIPK1, RIPK3, and DAI. Our study will broaden the knowledge of the molecular mechanisms of cell death.