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Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we image tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing membrane modification and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virus particles bud. Naturally occurring furin site variants determine whether virus particles mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation.

The nuclear pore complex (NPC) is composed of multiple nucleoporins (NUPs) and enables the exchange of RNA and proteins between the nucleus and cytoplasm. NUP98 is one of the major components of the NPC, being involved in the RNA export pathway by interacting with several transport factors. Previous studies have suggested both proviral and antiviral functions of NUP98 in viral infection, yet little is known about its function in orthoflavivirus infection. In this study we show that NUP98 is a proviral cellular protein that is recruited to the cytoplasm during orthoflavivirus infection. We observe that NUP98 is found specifically in the vicinity of the replication vesicles during infections with tick-borne encephalitis virus, Japanese encephalitis virus, and yellow fever virus. Furthermore, using surface plasmon resonance, cross-link immunoprecipitation, and cross-link immunoprecipitation-sequencing we observe that the C-Terminal domain of NUP98 directly interacts with a conserved site of the viral RNA (vRNA) in the E coding region promoting viral replication. We identified a peptide that binds to NUP98 that is antivirally active against several orthoflaviviruses by outcompeting the binding between NUP98 and vRNA, making NUP98 an attractive target for antiviral development.

Orthoflaviviruses are RNA viruses that cause serious diseases in humans, with currently no antivirals available. Targeting host factors is emerging as an attractive antiviral approach. However, as a first step, there is a need to understand which host proteins are hijacked and for what purpose. Here, using a combination of fluorescence microscopy, knock-down, crosslinking immunoprecipitation sequencing, mass spectrometry, and in vitro and biophysical assays, we identify nucleoporin-153 (NUP153) as a proviral factor during orthoflavivirus infection. We show that NUP153 is recruited to the virus amplification site on the endoplasmic reticulum to impact the structural to nonstructural viral protein ratios. We find that NUP153 interacts with both the viral proteins NS3 and NS5, and a highly conserved G-rich motif on the viral RNA. These interactions specifically promote the production of viral structural proteins, leading to an efficient virion assembly, virus release and spread to new cells. We propose that NUP153 acts as a key regulator in viral protein ratios, a mechanism that appears conserved among orthoflaviviruses.

Deep mutational scanning (DMS) has emerged as a powerful approach for evaluating the effects of mutations on binding or function. Here, we developed a DMS by phage display protocol to define the specificity determinants of short linear motifs (SLiMs) binding to peptide-binding domains. We first designed a benchmarking DMS library to evaluate the performance of the approach on well-known ligands for 11 different peptide-binding domains, including the talin-1 PTB domain, the G3BP1 NTF2 domain, and the MDM2 SWIB domain. Comparison with a set of reference motifs from the eukaryotic linear motif (ELM) database confirmed that the DMS by phage display analysis correctly identifies known motif binding determinants and provides novel insights into specificity determinants, including defining a non-canonical talin-1 PTB binding motif with a putative extended conformation. A second DMS library was designed, aiming to provide information on the binding determinants for 19 SLiM-based interactions between human and SARS-CoV-2 proteins. The analysis confirmed the affinity determining residues of viral peptides binding to host proteins and refined the consensus motifs in human peptides binding to five domains from SARS-CoV-2 proteins, including the non-structural protein (NSP) 9. The DMS analysis further pinpointed mutations that increased the affinity of ligands for NSP3 and NSP9. An affinity-improved cell-permeable NSP9-binding peptide was found to exert stronger antiviral effects than the wild-type peptide. Our study demonstrates that DMS by phage display can efficiently be multiplexed and applied to refine binding determinants and shows how the results can guide peptide-engineering efforts.

Tick-borne encephalitis virus (TBEV) is a neurotropic flavivirus that causes thousands of human infections annually. Viral tropism in the brain is determined by the presence of necessary receptors, entry factors, and the ability of the virus to overcome host defenses. The viral structural proteins, pre-membrane (prM), and envelope (E) play an important role in receptor binding, membrane fusion, particle maturation, and antibody neutralization. To understand how these proteins influence virus distribution and tropism in the brain, we generated a chimeric virus harboring the prM and ectodomain of E from TBEV in the background of the low-pathogenic Langat virus (LGTV). We solved the atomic structures of both the chimeric virus and LGTV to compare them to the known TBEV structure. We show that this chimeric virus remains low-pathogenic, while being structurally and antigenically similar to TBEV. Using 3D optical whole brain imaging combined with immunohistochemistry, we found that both LGTV and the chimeric virus primarily infect the cerebral cortex, with no significant differences in their localization or tropism. In contrast, TBEV shows high infection of the cerebellum and a strong preference toward Purkinje cells, indicating that factors other than the prM and E proteins are important for determining TBEV tropism in the brain. Together, this provides new insights into the roles of the structural and non-structural proteins of tick-borne flaviviruses. IMPORTANCE: Although an effective vaccine exists, there is no treatment for those infected by the tick-borne encephalitis virus (TBEV). This study aimed to better understand how the virus's surface proteins influence viral tropism and pathogenicity. We created a chimeric virus with prM and E proteins of TBEV in the genetic background of the low-pathogenic Langat virus (LGTV). The chimeric virus remained low pathogenic, similar to LGTV. Both viruses infected similar brain regions, while TBEV showed a strong preference for the cerebellum and Purkinje cells. This means that other parts of the virus, such as non-structural proteins or NCR, likely decide how the virus behaves in the brain. This study also presents the first cryogenic electron microscopy structure of LGTV, the first whole-brain imaging of TBEV infection in mouse brain, and a new model system to study surface proteins in tick-borne flaviviruses-laying groundwork for future studies on viral tropism, antibody cross-reactivity, and virus-receptor interaction.

Flavivirus assembly is driven by the envelope glycoproteins pre-membrane (prM) and envelope (E) in the neutral pH environment of the endoplasmic reticulum. Newly budded, spiky particles are exported through the Golgi apparatus, where mildly acidic pH induces a major surface rearrangement. The glycoproteins reorganize into (prM/E)\₂ complexes at the surface of smooth particles, with prM trapped at the E dimer interface, thereby exposing a furin cleavage site (FCS) for proteolytic maturation into infectious virions. Here, we show that in the absence of furin, immature tick-borne flavivirus particles—tick-borne encephalitis virus, Langat virus, and Louping ill virus—remain fully infectious and pathogenic in female BALB/c mice, in contrast to mosquito-borne flaviviruses such as Usutu, West Nile, and Zika viruses. We further show that the FCS in tick-borne viruses remains exposed at neutral pH, allowing furin at the surface of target cells to activate viral fusogenicity, while mosquito-borne counterparts require acidic re-exposure. Mutations increasing the dynamic behavior of the E dimer mimic the mosquito-borne phenotype, with retracted FCS at neutral pH and loss of infectivity. Our multidisciplinary approach—combining virological assays, targeted mutagenesis, structural modeling, and molecular dynamics simulations—highlights the role of E dimer dynamics in regulating flavivirus maturation and infectivity.

Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), a severe and sometimes life-threatening disease characterized by viral invasion of the central nervous system with symptoms of neuroinflammation1,2. As with other orthoflaviviruses—enveloped, arthropod-borne RNA viruses—host factors required for TBEV entry remain poorly defined. Here we used a genome-scale CRISPR–Cas9-based screen to identify LRP8, an apolipoprotein E and reelin receptor with high expression in the brain, as a TBEV receptor. LRP8 downregulation reduced TBEV infection in human cells, and its overexpression enhanced infection. LRP8 bound directly to the TBEV E glycoprotein and mediated viral attachment and internalization into cells. An LRP8-based soluble decoy blocked infection of human cell lines and neuronal cells and protected mice from lethal TBEV challenge. LRP8’s role as a TBEV receptor has implications for TBEV neuropathogenesis and the development of antiviral countermeasures.

A recent study byLindman and colleagueshighlights a cell type-specific function of receptor-interacting kinase 3 (RIPK3) in astrocytes during neurotropic flavivirus infection. Despite a proinflammatory transcriptional profile, RIPK3 in astrocytes can attenuate neuroinflammation and reduce leucocyte infiltration through upregulation of Serpin clade A member 3N (SerpinA3N), protecting mice from excessive neuroinflammation and increasing overall survival.

Flavivirus infection involves extensive remodeling of the endoplasmic reticulum (ER), which is key to both the replication of the viral RNA genome as well as the assembly and release of new virions. However, little is known about how viral proteins and host factors cooperatively facilitate such a vast transformation of the ER, and how this influences the different steps of the viral life cycle. In this study, we screened for host proteins that were enriched in close proximity to the tick-borne encephalitis virus (TBEV) protein NS4B and found that the top candidates were coupled to trafficking between ER exit sites (ERES) and the Golgi. We characterized the role of ACBD3, one of the identified proteins, and showed that it promotes TBEV infection. Depletion of ACBD3 inhibited virus replication and resulted in abnormal transformation of the ER, leading to reduced virion release. ACBD3's proviral mechanism did not involve the recruitment of PI4PK as previously described for enteroviruses. Instead, productive TBEV infection required the full-length ACBD3, which localizes to ER-Golgi contact sites together with NS4B. We propose that NS4B and ACBD3 promote replication by coordinating the transformation of the ER, which is required for RNA replication and particle release. The transformation involves direct coupling to the Golgi which facilitates efficient virion transport.

IMPORTANCE: Flaviviruses like tick-borne encephalitis have significant effects on human health. During flavivirus infection, the viral particles enter the host cells and transform the endoplasmic reticulum (ER), which is a membranous organelle and the main site of cellular protein synthesis. Although this is critical for successful infection, the details of the process are unknown. Here, we found that the viral protein NS4B and the host protein ACBD facilitate this transformation by ensuring that the ER is coupled to the Golgi apparatus, the organelle responsible for transporting material out of the cell. TBEV uses ACBD3 to guarantee that the connection sites between the transformed ER and the Golgi remain functional so that RNA is replicated and the produced viral particles are exported from the cell and can infect further cells. Our work sheds light both on the basic biology of flavivirus infection, and virus-induced remodeling of membranous organelles.

Introduction: Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited.

Methods: T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images.

Results: Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR.

Discussion: The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging.