Aug 2026· Entomological Research· Vol 56· 0 citations· 45 references
TL;DR
It is demonstrated that precise control of transcriptional stoichiometry and structural configuration is critical for high‐yield CVA16 VLP production, providing a robust platform for multivalent HFMD vaccine development.
Abstract
Coxsackievirus A16 (CVA16) is a major causative agent of hand, foot, and mouth disease (HFMD), yet efficient virus‐like particle (VLP)–based vaccine development remains challenging due to enteroviral structural complexity and the cytotoxicity of the 3CD protease required for polyprotein processing. In this study, we established an optimized production strategy for CVA16 VLPs using the baculovirus expression vector system (BEVS) by balancing structural protein expression and proteolytic cleavage. To mitigate 3CD‐mediated cytotoxicity, we applied an attenuation strategy using the modified p10‐BS5 promoter, which reduced transcriptional strength by ~90% and delayed protease expression, thereby extending the production window. We further examined the impact of P1 structural protein conformation and expression levels on VLP assembly. Unlike Enterovirus 71 (EV71), which favors high‐level expression of full‐length P1, CVA16 assembly was highly sensitive to expression dynamics. Optimal VLP formation was achieved using a standard expression vector (pAcPol) combined with a VP4‐deficient P1 construct (ΔVP4). Under optimized conditions (multiplicity of infection 1, 7 days postinfection), the rAcPol‐CV‐ΔVP4‐BS5 construct yielded the highest level of assembled particles. Transmission electron microscopy confirmed uniform, spherical VLPs (30–35 nm) resembling native CVA16 virions. These findings demonstrate that precise control of transcriptional stoichiometry and structural configuration is critical for high‐yield CVA16 VLP production, providing a robust platform for multivalent HFMD vaccine development.
Marek's disease virus (MDV), an oncogenic alphaherpesvirus, induces severe immunosuppression and T-cell lymphomas in chickens, posing a major threat to poultry production. Viral morphogenesis in alphaherpesviruses depends on coordinated membrane remodeling processes mediated by conserved viral membrane proteins. Although MDV encodes a homolog of UL20 protein, its functional role remains unknown. In this study, we employed an integrated approach combining bioinformatic analysis, bacterial artificial chromosome (BAC)-based mutagenesis, and ultrastructural characterization to elucidate the role of MDV UL20 in viral replication and morphogenesis. Sequence analysis revealed that MDV UL20 is a conserved four-pass transmembrane protein with a membrane topology similar to that of UL20 homologs in other alphaherpesviruses. To assess its functional relevance, a UL20 deletion mutant (Md5BACΔUL20) was generated, and its biological properties were evaluated in chicken embryonic fibroblasts. Deletion of UL20 completely abolished the production of infectious virus, resulting in the absence of plaque formation and impaired cell-to-cell spread. In contrast, genetic reconstitution of UL20 fully restored viral replication to wild-type levels. Transmission electron microscopy demonstrated that UL20 deletion did not affect nuclear capsid assembly but markedly reduced the number of cytoplasmic capsids, secondary envelopment intermediates, and extracellular virion accumulation. UL20 re-expression assays confirmed that these defects were specifically attributable to the loss of UL20. Collectively, these findings demonstrate that MDV UL20 is dispensable for nuclear capsid formation but essential for cytoplasmic secondary envelopment and infectious virion production, providing new insights into the membrane-associated mechanisms underlying MDV morphogenesis.
Assembly of flaviviruses such as Zika virus (ZIKV), dengue virus, and West Nile virus in the host cell endoplasmic reticulum is driven by the structural envelope (E) and premembrane (prM) proteins. The formation of an infectious virion requires cleavage of prM by the host furin protease during a maturation step that is dependent on a conformational change in virion structure. Here, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM protein or proteolytic activation. The expression of E protein preceded by a truncated version of prM (M-E) was sufficient for the formation of noninfectious ZIKV subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response in macaques that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher-quality and more broadly applicable approach for vaccination against flaviviruses.
Kimberly A. Dowd, Michelle Schroeder, Egan Sanchez et al.· Science Translational Medici...· 0 citations
Foot-and-mouth disease virus (FMDV) causes substantial economic losses in global livestock production; however, the key host factors supporting its early infection process remain poorly characterized.
In this study, we performed an unbiased genome-wide CRISPR/Cas9 knockout screening using porcine cells to screen and identify host factors involved in FMDV infection.
We identified that the E3 ubiquitin ligase RNF24 supports efficient FMDV entry. RNF24 depletion inhibits viral entry and replication, whereas its overexpression enhances viral infectivity. Mechanistically, RNF24 preferentially promotes K27-linked non-degradative polyubiquitination of leupaxin (LPXN) at lysine 162, driving LPXN’s trafficking to the plasma membrane. At the membrane, LPXN assembles a ternary integrin-LPXN-VP1 complex that strengthens virus-receptor interactions and promotes viral adsorption and entry. Disruption of this ubiquitination event via the LPXN K162R mutation impairs complex formation and compromises viral entry.
Together, our study reveals a ubiquitin-dependent RNF24-LPXN regulatory axis that supports FMDV entry, highlights the role of non-degradative ubiquitination in viral pathogenesis, and proposes this interface as a potential target for antiviral intervention.
Jinyan Zhang, Hailong Liu, Jian Du et al.· Frontiers in Cell and Develo...· 0 citations
Zika virus (ZIKV) nonstructural protein 1 (NS1) represents a valuable diagnostic target due to its early secretion during infection and reduced cross-reactivity with other flaviviruses. Optimization of NS1 expression conditions is essential for obtaining large-scale protein production. The use of bacterial expression systems as Escherichia coli offers a cost-effective platform. We developed two molecular cloning strategies to express full-length ZIKV NS1 (1053 bp) from Cuban strain 9661/2016 using pQE30/XL1-Blue and pET50b(+)/BL21(DE3) expression systems. The ns1 gene cloned via pGEM-T intermediate vector, and subcloned into expression vectors by using directional restriction-based strategy. The effects of culture medium and IPTG concentration on bacterial growth and protein accumulation was evaluated in order to optimizing the NS1 expression conditions. Both expression systems successfully produced recombinant ZIKV NS1. However, the BL21(DE3)/pET50b(+) system yielded soluble NS1, whereas the XL1-Blue/pQE30 system predominantly produced insoluble protein. Culture medium exerted a greater effect on protein expression than IPTG concentration, with LB medium and 0.1 mM IPTG providing the most favorable conditions for recombinant NS1 production. Notably, XL1-Blue demonstrated competency for protein expression. The pET50b(+)-ns1/BL21(DE3) system offers decisive advantages through soluble expression, eliminating refolding requirements for downstream purification process. This work establishes optimized protocols for ZIKV NS1 expression applicable to obtaining a recombinant protein for developing an ELISA system and broadly transferable to other flavivirus antigens.
Aliana Saenz de la Torre Leal, Thalía Morán Ávila, M. Muné et al.· Protein Expression and Purif...· 0 citations
Human noroviruses (HuNoVs) are one of the main causative agents of non-bacterial gastroenteritis. The viral capsid protein VP1 is the main target for diagnostics. Although eukaryotic expression systems can produce correctly assembled VP1, they are associated with high costs and complexity. The Escherichia coli prokaryotic expression system is attractive for large-scale expression; however, VP1 often aggregates into insoluble inclusion bodies in conventional strains (BL21 (DE3)). The ArcticExpress (DE3) strain co-expresses psychrophilic chaperonins to improve protein folding at low temperatures enhancing the soluble yield of difficult-to-express targets. In this study, we demonstrated that E. coli ArcticExpress (DE3) under low-temperature induction enabled the high-yield production of soluble full-length VP1 for HuNoVs GII.4 and GII.17. In BL21 (DE3), the mentioned antigens were aggregated into insoluble inclusion bodies. Using ArcticExpress(DE3), VP1 yield was identified as approximately 18-93 mg/L across the tested genotypes. In BL21 (DE3), the protein outcome was genotype-dependent: GII.4 could be recovered only via insoluble-fraction processing, GII.17 remained undetectable or present only at trace levels under the conditions tested. The ArcticExpress-derived viral antigen retained antigenic properties and was recognized by cross-reactive anti-GII antibodies. To our knowledge, this study provides evidence that ArcticExpress (DE3) enable the soluble production of full-length HuNoVs VP1 suitable for downstream immunochemical analysis.
I. Kazlouski, N. Paklonskaya, I. Belskaya et al.· FEMS Microbiology Letters· 0 citations
Zika virus (ZIKV) is a mosquito-transmitted orthoflavivirus that caused an epidemic in 2015–2016 in the Americas and raised serious global health concerns due to its association with congenital brain anomalies when infections occur during pregnancy. Various viruses can form compartments within the cell to facilitate viral replication and assembly, referred to as viroplasms, replication organelles, or virus factories depending on the type of virus. ZIKV assembles virus particles in virus-generated compartments adjacent to the nucleus, referred to here as a replication compartment (RC), which is formed by remodeling the host cell endoplasmic reticulum (ER). How the viral proteins control RC assembly remains unknown. Here we show that the ZIKV non-structural protein 3 (NS3), a dual-function protease and RNA helicase, is sufficient to drive the assembly of a replication compartment-like structure (RCLS) in human cells. While sufficient to generate the RCLS, NS3 is less efficient in several aspects compared to ZIKV-induced RC assembly. Nonetheless, the RCLS is similar to the ZIKV RC in its assembly at the nuclear periphery, its recruitment of ER, association with the Golgi and centrosome, and the arrangement of microtubules at its surface. Moreover, NS3 expression results in activation of the unfolded protein response (UPR), but attenuates expression of the downstream transcription factor CHOP, mirroring the manipulation of the different aspects of the UPR by ZIKV infection. We further show that the helicase domain and not the protease domain is required for optimal RCLS formation and organelle recruitment, yet each domain affects different control over the UPR. Overall, these findings advance our understanding of the mechanism of RC assembly by ZIKV, its involvement in hijacking the UPR, and the central role of NS3 in the process.
T. Sultana, Chunfeng Zheng, Jenna Jones et al.· Viruses· 0 citations
