ABSTRACT
Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What's more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131-204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.
Subject(s)
Autophagy , Down-Regulation , Encephalitis Virus, Japanese , Lysosomal-Associated Membrane Protein 2 , Lysosomes , Lysosomes/metabolism , Animals , Encephalitis Virus, Japanese/physiology , Lysosomal-Associated Membrane Protein 2/genetics , Lysosomal-Associated Membrane Protein 2/metabolism , Swine , Lysosomal-Associated Membrane Protein 1/metabolism , Lysosomal-Associated Membrane Protein 1/genetics , Encephalitis, Japanese/virology , Encephalitis, Japanese/veterinary , Cell Line , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Lysosomal Membrane Proteins/metabolism , Lysosomal Membrane Proteins/geneticsABSTRACT
Although a broad range of ligand-functionalized nanoparticles and physico-chemical triggers have been exploited to create stimuli-responsive colloidal systems, little attention has been paid to the reversible assembly of unmodified nanoparticles with non-covalently bound proteins. Previously, we reported that a derivative of green fluorescent protein engineered with oppositely located silica-binding peptides mediates the repeated assembly and disassembly of 10-nm silica nanoparticles when pH is toggled between 7.5 and 8.5. We captured the subtle interplay between interparticle electrostatic repulsion and their protein-mediated short-range attraction with a multiscale model energetically benchmarked to collective system behavior captured by scattering experiments. Here, we show that both solution conditions (pH and ionic strength) and protein engineering (sequence and position of engineered silica-binding peptides) provide pathways for reversible control over growth and fragmentation, leading to clusters ranging in size from 25 nm protein-coated particles to micrometer-size aggregate. We further find that the higher electrolyte environment associated with successive cycles of base addition eventually eliminates reversibility. Our model accurately predicts these multiple length scales phenomena. The underpinning concepts provide design principles for the dynamic control of other protein- and particle-based nanocomposites.
Subject(s)
Carrier Proteins , Nanoparticles , Peptides , Silicon DioxideABSTRACT
Japanese encephalitis virus (JEV) is a major causative agent of neurological infection affecting humans and pigs. Human T Cell Immunoglobulin and Mucin Domain 1 (hTIM-1) enhances the infection of JEV through virion-associated phosphatidylserine (PS) binding. Here, five swine TIM-1 (sTIM-1) gene variants were cloned from pig lung tissues by reverse-transcriptase polymerase chain reaction (RT-PCR). Sequence alignment analysis revealed that the gene homology between the sTIM-1 and hTIM-1 was 42.3-43.8%. Furthermore, ectopic expression of all five sTIM-1 variants in 293 T cells can promote JEV entry and infection. However, sTIM-1 V3 exhibited significantly less potent at promoting virus entry compared to the other four variants. Further studies revealed that the 34th amino acid of sTIM-1is critical for the entry of JEV, which is Pro34 in sTIM-1V3 while Leu34 in other four sTIM-1 variants. Mechanically, leucine at locus 34 was associated with the membrane distribution of sTIM-1, thereby affecting viral entry and infection. In total, our findings provide evidence that the PS receptor sTIM-1 promotes the infection of JEV and that the 34th amino acid position is critical for sTIM-1 to mediate viral infection.
Subject(s)
Encephalitis Virus, Japanese , Encephalitis, Japanese , Swine , Animals , Humans , Encephalitis Virus, Japanese/genetics , Phosphatidylserines , Leucine/genetics , Encephalitis, Japanese/veterinary , Mutation , Immunoglobulins , Mucins/geneticsABSTRACT
At-will tailoring of the formation and reconfiguration of hierarchical structures is a key goal of modern nanomaterial design. Bioinspired systems comprising biomacromolecules and inorganic nanoparticles have potential for new functional material structures. Yet, consequential challenges remain because we lack a detailed understanding of the temporal and spatial interplay between participants when it is mediated by fundamental physicochemical interactions over a wide range of scales. Motivated by a system in which silica nanoparticles are reversibly and repeatedly assembled using a homobifunctional solid-binding protein and single-unit pH changes under near-neutral solution conditions, we develop a theoretical framework where interactions at the molecular and macroscopic scales are rigorously coupled based on colloidal theory and atomistic molecular dynamics simulations. We integrate these interactions into a predictive coarse-grained model that captures the pH-dependent reversibility and accurately matches small-angle X-ray scattering experiments at collective scales. The framework lays a foundation to connect microscopic details with the macroscopic behavior of complex bioinspired material systems and to control their behavior through an understanding of both equilibrium and nonequilibrium characteristics.
Subject(s)
Biomimetic Materials , Nanoparticles , Nanostructures , Biomimetic Materials/chemistry , Humans , Molecular Dynamics SimulationABSTRACT
The Japanese encephalitis serogroup of the neurogenic Flavivirus has a specific feature that expresses a non-structural protein NS1' produced through a programmed -1 ribosomal frameshifting (-1 PRF). Herein, C19orf66, a novel member of interferon-stimulated gene (ISG) products, exhibited significant activity of antagonizing Japanese encephalitis virus (JEV) infection. Overexpression of C19orf66 in 293T cells significantly inhibited JEV replication, while knock-down of endogenous C19orf66 in HeLa cells and A549 cells significantly increased virus replication. Notably, C19orf66 had an inhibitory effect on frameshift production of JEV NS1'. The inhibition was more significant when C19orf66 and JEV NS1-NS2A were co-expressed in the 293T cells. Both C19orf66-209 and C19orf66-Zincmut did not significantly change the NS1' to NS1 ratio and had weaker antiviral effects than C19orf66. Similarly, C19orf66-209 and C19orf66-Zincmut had no significant effect on the expression of the JEV NS3 protein, whose expression was down-regulated by C19orf66 via the lysosome-dependent pathway. These findings suggest that C19orf66 may possess at least two different mechanisms of antagonizing JEV infection. This study identified C19orf66 as a novel interferon-stimulated gene product that can inhibit JEV replication by targeting -1 PRF and the NS3 protein. The study provides baseline information for the future development of broad-spectrum antiviral agents against JEV.