Viral Proteins Research Articles

Structure of coxsackievirus cloverleaf RNA and 3C pro dimer establishes the RNA-binding mechanism of enterovirus protease 3C pro

In positive-strand RNA viruses, the genome serves as a template for both protein translation and negative-strand RNA synthesis. Enteroviruses use the cloverleaf RNA structure at the 5′ end of the genome to balance these two processes. Cloverleaf acts as a promoter for RNA synthesis and forms a complex with viral 3CD protein, the precursor to 3C pro protease, and 3D pol polymerase. The interaction between cloverleaf and 3CD is mediated by the 3C pro domain, yet how 3C pro promotes specific RNA-binding is not clear. We report the structure of coxsackievirus cloverleaf RNA-3C pro complex, wherein two 3C pro molecules interact with cloverleaf stem-loop D. 3C pro dimer mainly recognizes the shape of the dsRNA helix through symmetric interactions, suggesting that 3C pro is a previously undiscovered type of RNA binding protein. We show that 3CD protein also dimerizes on cloverleaf RNA and binds the RNA with higher affinity than 3C pro . The structure provides insight into the RNA-binding mechanism of 3C pro or 3CD with other cis-acting replication elements.

Correction: Saha et al. Unveiling the Antiviral Potential of Minocycline: Modulation of Nuclear Export of Viral Ribonuclear Proteins during Influenza Virus Infection. Viruses 2024, 16, 1317

In the original publication [...]

Quantum Chemical and Metabolomic Characterization of Inhibitory Activity of Procyanidins and Flavonol Glucosides from Graptopetalum paraguayense E. Walther against Nonstructural Proteins of SARS-CoV-2.

This study explores the antiviral potential of Graptopetalum paraguayense (GP) against SARS-CoV-2 nonstructural proteins (NSPs). GP, a plant known for its health benefits, has previously shown strong antiviral activity against HSV-1, and although the two viruses have different taxonomy, its effectiveness against the new coronavirus was also examined. LC-ESI-QTOF-MS/MS analysis identified fourteen polyphenolic metabolites in GP, including procyanidins and flavonol glucosides, compounds associated with anti-coronavirus properties. These compounds were tested using molecular docking and ONIOM(B3LYP/6-31+G(d,p):UFF) quantum chemical calculations to assess their binding affinity to eight SARS-CoV-2 NSPs critical for viral replication and transcription. The results revealed that GP compounds, such as procyanidin B2, kaempferol-3-O-glucoside, and epicatechin-3-O-galactoside, had strong binding affinities to NSP14, NSP5, and NSP15, respectively, suggesting their potential to inhibit viral replication. Compared to known NSP inhibitors from the RCSB Protein Data Bank, GP-derived compounds demonstrated comparable or superior interaction energies in several cases. This investigation highlights GP's therapeutic potential as a natural antiviral against SARS-CoV-2. The findings provide new insights into the role of plant-derived polyphenols in targeting viral proteins and contribute to further experimental and clinical studies to validate these compounds as candidates for anti-coronavirus therapies.

Deciphering Host–Virus Interactions and Advancing Therapeutics for Chronic Viral Infection

Chronic viral infections like HIV, HBV, and HCV establish persistent interactions with the host immune system, resulting in immune evasion and long-term immune dysfunction. These viruses use a range of strategies to limit host defenses, such as downregulating MHC class I, disrupting interferon signaling, altering apoptosis pathways, and suppressing cytotoxic T-cell activity. Key viral proteins, including HIV Nef, HBV X protein, and HCV NS5A, interfere with antigen presentation and JAK/STAT signaling, thereby reducing antiviral immune responses. Chronic infections induce immune exhaustion due to persistent antigen exposure, which leads to the expression of inhibitory receptors like PD-1 and CTLA-4 on T cells. Viral epigenetic changes, such as N6-methyladenosine modifications and histone deacetylation, enhance immune evasion by modulating gene expression in infected cells. Viruses further manipulate host cytokine networks by promoting an immunosuppressive environment through IL-10 and TGF-β secretion, which suppress inflammatory responses and inhibit T-cell activation. This review examines the molecular/cellular mechanisms that enable chronic viruses to escape host immunity, focusing on antigenic variation, cytokine disruption, and control of apoptotic pathways. It also addresses how host genetic factors, such as HLA polymorphisms, influence disease progression. Lastly, we discuss host-targeted therapies, including immune checkpoint inhibitors, cytokine treatments, and CRISPR.

On the role of VP3-PI3P interaction in birnavirus endosomal membrane targeting.

Birnaviruses are a group of double-stranded RNA (dsRNA) viruses infecting birds, fish, and insects. Early endosomes (EE) constitute the platform for viral replication. Here, we study the mechanism of birnaviral targeting of EE membranes. Using the Infectious Bursal Disease Virus (IBDV) as a model, we validate that the viral protein 3 (VP3) binds to phosphatidylinositol-3-phosphate (PI3P) present in EE membranes. We identify the domain of VP3 involved in PI3P-binding, named P2 and localized in the core of VP3, and establish the critical role of the arginine at position 200 (R200), conserved among all known birnaviruses. Mutating R200 abolishes viral replication. Moreover, we propose a two-stage modular mechanism for VP3 association with EE. Firstly, the carboxy-terminal region of VP3 adsorbs on the membrane, and then the VP3 core reinforces the membrane engagement by specifically binding PI3P through its P2 domain, additionally promoting PI3P accumulation.

Multiplicity of infection and culture medium on the SARS-CoV-2 virus like-particles production by baculovirus/insect system.

This work aimed to assess the SARS-CoV-2 structural proteins' expression and virus-like particles (VLP) production by Baculovirus/Insect cell platform using two levels of Multiplicity of Infection (MOI), and two culture media, one of them a serum-free medium and the other one chemically defined. Two SARS-CoV-2 VLP were obtained from Sf9 cells coinfection using in both cases, three monocistronic recombinant baculoviruses holding the genes of Nucleocapsid (N; MOI = 2 or 0.2), Membrane (M; MOI = 1 or 0.1), and Envelope (E; MOI = 1 or 0.1) viral proteins, and the fourth one was changed between a baculovirus bearing Spike protein (S; MOI = 3 or 0.3) or receptor-binding domain (RBD; MOI = 3 or 0.3) genes of SARS-CoV-2. Similar performance was verified for both culture media in SARS-CoV-2 VLP production bearing four structural virus proteins or RBD domain. The SARS-CoV-2 structural proteins' expression was comparable at different MOIs (tenfold) as well as SARS-CoV-2 VLP size (around 100nm). The increase in specific death rates over the coinfection phase was confirmed in relatively high MOI assays. This finding was related to an exponential virus titer profile for high MOIs over the entire infection phase, meanwhile, a viral peak was observed at low MOIs, confirming a secondary infection. The SARS-CoV-2 VLP improved production carrying immunogenic S protein was confirmed concerning others holding RBD. However, the protein composition of produced VLP should be studied further to assess the VLP homogeneity when different culture media and MOIs are used.

Repurposing Efavirenz, the HIV Antiretroviral Drug for Chikungunya Virus Infection.

Chikungunya virus (CHIKV) has frequently recurred in recent decades, causing outbreaks worldwide in tropical and subtropical regions. The re-emergence of CHIKV poses a substantial risk to human health, as no efficacious drugs are currently available to curb new outbreaks. Here, the anti-CHIKV activity of efavirenz was investigated by in vitro cell culture-based antiviral assays in different relevant cell lines. Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI) used for the treatment of acquired immunodeficiency syndrome (AIDS), and it has good oral bioavailability, long half-life, and affordable low cost. This study demonstrated dose-dependent robust anti-CHIKV activity of efavirenz at low micromolar concentration in two different cell lines with 50% effective concentration (EC50) of 1.1 to 1.3 μM. Interestingly, efavirenz also inhibited the replication of Sindbis virus (SINV) at a low micromolar range indicating potential broad anti-alphavirus activity. Time of addition assay, direct transfection of virus replicon RNA, and minus-sense-specific reverse transcription polymerase chain reaction (RT-PCR) elucidated that efavirenz hinders the viral replication at an early stage after the virus entry by inhibiting the viral RNA synthesis. Further, the binding affinity of efavirenz toward purified capsid protein (CP) was observed, suggesting that CP could be one of the antiviral targets for efavirenz in addition to viral or host proteins involved in viral RNA replication. Finally, the in vivo efficacy of efavirenz was assessed in a murine model and a decrease in CHIKV viral load was observed. In summary, the present study underscores the potential of repurposing efavirenz for antiviral therapy against CHIKV to curb future viral outbreaks.

Effects of cellular membranes and the precore protein on hepatitis B virus core particle assembly and DNA replication.

Hepatitis B virus (HBV) is an important human pathogen that chronically infects 254 million people in the world. This virus contains a core particle, which plays an important role in the transport and replication of the viral DNA genome. The major protein constituent of this particle is the viral core protein. In this report, we examined how the subcellular compartments and the related precore protein might affect the core particle structure and viral DNA replication. We found that the subcellular localizations could affect the core particle assembly, and membranes and the precore protein could regulate HBV DNA replication. We also found that the inhibition of autophagic degradation increased the precore protein level, suggesting a role of autophagy in the regulation of precore protein activities. These findings provided important information for further understanding the HBV life cycle, which will aid in the development of novel drugs for the treatment of HBV patients.

Carbene-catalyzed Phthalide Ether Functionalization for Discovering Chiral Phytovirucides that Specifically Targets Viral Nia Protein to Inhibit Proliferation

Carbene-catalyzed Phthalide Ether Functionalization for Discovering Chiral Phytovirucides that Specifically Targets Viral Nia Protein to Inhibit Proliferation

Cas9-Rep fusion tethers donor DNA in vivo and boosts the efficiency of HDR-mediated genome editing.

Genome editing based on the homology-directed repair (HDR) pathway enables scar-free and precise genetic manipulations. However, the low frequency of HDR hinders its application in plant genome editing. In this study, we engineered the fusion of Cas9 and a viral replication protein (Rep) as a molecular bridge to tether donor DNA in vivo, which enhances the efficiency of targeted gene insertion via the HDR pathway. This Rep-bridged knock-in (RBKI) method combines the advantages of rolling cycle replication of viral replicons and in vivo enrichment of donor DNA at the target site for HDR. Chromatin immunoprecipitation indicated that the Cas9-Rep fusion protein bound up to 66-fold more donor DNA than Cas9 did. We exemplified the RBKI method by inserting small- to middle-sized tags (33-519 bp) into 3 rice genes. Compared to Cas9, Cas9-Rep fusion increased the KI frequencies by 4-7.6-fold, and up to 72.2% of stable rice transformants carried in-frame knock-in events in the T0 generation. Whole-genome sequencing of 6 plants segregated from heterozygous KI lines indicated that the knock-in events were faithfully inherited by the progenies with neither off-target editing nor random insertions of the donor DNA fragment. Further analysis suggested that the RBKI method reduced the number of byproducts from nonhomologous end joining; however, HDR-mediated knock-in tended to accompany microhomology-mediated end joining events. Together, these findings show that the in vivo tethering of donor DNAs with Cas9-Rep is an effective strategy to increase the frequency of HDR-mediated genome editing.

Marek's disease virus protein kinase US3 inhibits DNA-sensing antiviral innate immunity via abrogating activation of NF-κB.

Marek's disease virus (MDV) is an avian alphaherpesvirus associated with Marek's disease, an immunosuppressive and lymphoproliferative disease in chickens. The DNA sensing pathway mediates innate immune defense against infection by many DNA-containing pathogens, while viruses have evolved multiple strategies to evade the host immune response to survive in host cells. This study found that ectopic expression of MDV protein kinase US3 inhibited beta interferon (IFN-β) and interleukin-6 (IL-6) production induced by interferon-stimulatory and viral DNA. US3 was further shown to abolish the nuclear factor κB (NF-κB) activation. The US3 kinase activity was indispensable for its inhibitory function, as the kinase-dead US3 mutant (US3K220A) did not inhibit NF-κB activation. Further studies showed that US3 interacted with the Rel homology domains of the NF-κB subunits p65 and p50, which phosphorylated these transcription factors and blocked their nuclear translocation. Finally, US3 deficiency promoted IFN-β and IL-6 production, resulting in reduced viral replication and lower MDV-specific lesion incidence during MDV infection in chickens. Altogether, these findings reveal a novel mechanism for MDV to evade host antiviral immunity.IMPORTANCEMarek's disease virus (MDV) is an oncogenic avian alphaherpesvirus that causes an economically important disease affecting the health and welfare of poultry worldwide. Whereas human herpesviruses have been shown to evolve various strategies to inhibit the DNA sensing signaling for the evasion of the host's innate immunity, little is known regarding the mechanism for MDV to regulate this pathway. In this study, MDV US3 protein kinase was demonstrated to inhibit the activation of NF-κB in the DNA sensing pathway via binding to the Rel homology domains of the NF-κB subunits p65 and p50, which hyperphosphorylated these transcription factors and abolished their nuclear translocation. This is an important finding toward a better understanding of the functions of avian alphaherpesviruses encoded US3 protein kinase.

RNA-targeted proteomics identifies YBX1 as critical for efficient HCMV mRNA translation

Viruses have evolved unique strategies to circumvent host control of protein synthesis and enable viral protein synthesis in the face of the host response. Defining the factors that regulate viral messenger RNA (mRNA) translation is thus critical to understand how viruses replicate and cause disease. To identify factors that might regulate viral mRNA translation, we developed a technique for identifying proteins associated with a native RNA expressed from its endogenous promoter and genomic locus. This approach uses a guide RNA to target dCas13b fused to a biotin ligase domain to a specific RNA, where it covalently labels proteins in close proximity. Using this approach, we identified multiple proteins associated with transcripts encoding the human cytomegalovirus (HCMV) IE1 and IE2 proteins and found that several associated proteins positively or negatively regulate HCMV replication. We confirmed that one such protein, the cellular Y-box binding protein 1 (YBX1), binds to HCMV immediate early mRNAs and is required for efficient viral protein expression and virus replication. Ablating YBX1 expression reduced the association of HCMV immediate early mRNAs with polysomes, demonstrating a role for YBX1 as a positive regulator of viral RNA translation. These results provide a powerful tool for unraveling RNA-protein interactions that can be used in a wide range of biological processes and reveal a role for YBX1 as a critical regulator of HCMV immediate early gene expression.

Pearl Bodies as a Potential Source of Secondary Transmission of Papaya Leaf Distortion Mosaic Virus in Carica papaya.

Pearl bodies are small, pearl-like structures found on the surfaces of many tropical and subtropical plant species, including Carica papaya L., that serve as a nutritious food source for arthropods, helping plants defend against herbivores. This study investigated the role of pearl bodies in viral accumulation and transmission, focusing on papaya plants infected with papaya leaf distortion mosaic virus (PLDMV), a destructive pathogen threatening papaya production. Viral analysis of pearl bodies from PLDMV-infected plants confirmed the presence of viral particles, genomic RNA, and coat protein, with quantitative real-time RT-PCR showing significant viral RNA accumulation. These findings suggest that pearl bodies act as reservoirs for PLDMV. Infectivity tests demonstrated that ruptured pearl bodies from infected plants effectively transmitted PLDMV to healthy plants via mechanical inoculation. The fragility of stalks and cell surfaces of pearl bodies facilitate their detachment or rupture during mechanical activities, potentially releasing virus-laden contents into the environment. This poses a significant risk of contamination for agricultural tools, equipment, and personnel, contributing to secondary transmission. This study emphasizes the importance of recognizing pearl bodies as an overlooked source of viral spread, highlighting the need for careful handling and targeted management strategies to reduce virus transmission risks in papaya cultivation. This is the first report of pearl bodies' involvement in virus transmission, which provides novel insights into virus epidemiology and disease management in papaya cultivation and other crops with pearl bodies.

Adenovirus E1B-55K interferes with cellular IκB kinase complex subunit proteins

Human adenovirus (HAdV) infections can cause high mortality rates in immunocompromised patients due to the activation of unhampered cytokine storms that are mainly induced by activation of pro-inflammatory cytokines. NF-κB is a transcription factor that is involved in numerous biological processes such as regulation of cell death and proliferation, as well as the activation of innate immune responses including the expression of pro-inflammatory cytokines, chemokines, and other immune response genes. The IKK complex plays a crucial role in the NF-κB pathway by phosphorylating and activating IκB proteins, which leads to the degradation of IκB and the subsequent release and nuclear translocation of NF-κB dimers to initiate gene transcription. The host NF-κB pathway, particularly the formation of the IKK complex, is a common target for viruses to regulate host immune responses or to utilize or inhibit its function for efficient viral replication. So far, investigations of the immune response to adenovirus infection mainly focused on transduction of adenoviral vectors or high-titer infections. Therefore, the molecular mechanism of HAdV- and HAdV gene product-mediated modulation of the NF-κB response in lytic infection is not well understood. Here, we show that HAdV-C5 infection counteracts cellular IκB kinase complex formation. Intriguingly, the IKK complex protein IKKα is targeted to the nucleus and localizes juxtaposed to viral replication centers. Furthermore, IKKα interacts with the early viral E1B-55K protein and facilitates viral replication. Together, our data provide evidence for a novel HAdV-C5 mechanism to escape host immune responses by utilizing NF-κB pathway-independent nuclear functions of IKKα to support efficient viral progeny production.

Insight into the Role of the Aryl Hydrocarbon Receptor in Bovine Coronavirus Infection by an Integrated Approach Combining In Vitro and In Silico Methods

It is well known that the host response to different human and animal coronaviruses infection is regulated by the aryl hydrocarbon receptor, a ligand-activated transcription factor. The present study investigates the expression of the aryl hydrocarbon receptor during bovine coronavirus infection, through in vitro and in silico investigations. The in vitro studies demonstrate that the aryl hydrocarbon receptor and as well as its targets, CYP1A1 and CYP1B1, were significantly activated by bovine coronavirus infection in bovine cells (MDBK). During infection, the pretreatment of cells with non-cytotoxic doses of CH223191, a selective inhibitor of the aryl hydrocarbon receptor, resulted in a significant reduction in virus yield and a downregulation in the viral spike protein expression. These findings occurred in the presence of the inhibition of aryl hydrocarbon receptor signaling. Our results reveal that the bovine coronavirus acts on viral replication, upregulating the aryl hydrocarbon receptor and its downstream target proteins, CYP1A1 and CYP1B1. In addition, following the in silico studies, the three-dimensional structural model of the bovine aryl hydrocarbon receptor in complex with the antagonist CH223191 indicates that the molecular mechanism, by which the PASB and TAD domains of the receptor interact with the inhibitor, is mainly driven by an extensive network of hydrophobic interactions, with a series of hydrogen bonds contributing to stabilizing the complex. Interestingly, bioinformatic analyses revealed that the PASB and TAD domains in the human and bovine aryl hydrocarbon receptor present high similarity at the primary sequence and three-dimensional structure levels. Taken together, these findings represent a fundamental step for the development of innovative drugs targeting AhR as a potential object for CoVs therapy.

Detection of IgG Antibodies Against COVID-19 N-Protein by Hybrid Graphene–Nanorod Sensor

The COVID-19 pandemic highlighted the global necessity to develop fast, affordable, and user-friendly diagnostic alternatives. Alongside recognized tests such as ELISA, nanotechnologies have since been explored for direct and indirect diagnosis of SARS-CoV-2, the etiological agent of COVID-19. Accordingly, in this work, we report a method to detect anti-SARS-CoV-2 antibodies based on graphene-based field-effect transistors (GFETs), using a nanostructured platform of graphene with added gold nanorods (GNRs) and a specific viral protein. To detect anti-N-protein IgG antibodies for COVID-19 in human sera, gold nanorods were functionalized with the nucleocapsid (N) protein of SARS-CoV-2, and subsequently deposited onto graphene devices. Our test results demonstrate that the sensor is highly sensitive and can detect antibody concentrations as low as 100 pg/mL. Using the sensor to test human sera that were previously diagnosed with ELISA showed a 90% accuracy rate compared to the ELISA results, with the test completed in under 15 min. Integrating graphene and nanorods eliminates the need for a blocker, simplifying sensor fabrication. This hybrid sensor holds robust potential to serve as a simple and efficient point-of-care platform.

Detection of Viral Proteins in Rabies Virus-Infected Mouse Brain Tissue

Detection of Viral Proteins in Rabies Virus-Infected Mouse Brain Tissue

Interconnectedness threat: unveiling the mechanisms behind human papillomavirus-induced cervical cancer

Cervical cancer is the fourth leading cause of cancer-related deaths among women worldwide, causing over 660,000 new cases and 350,000 deaths in 2022, with a disproportionately high burden in low-resource countries where access to treatment is limited. Human papillomavirus (HPV) is a common sexually transmitted infection that accounts for approximately 95% of cervical cancer cases. Persistent HPV infection can progress to cervical dysplasia, categorized into varying severities (CIN1, CIN2, and CIN3), which significantly increases cancer risk. The mechanism of HPV-induced malignancy involves the disruption of cellular apoptosis by integrating viral genetic material into cervical cells, particularly within the transformation zone. The viral proteins E6 and E7 play pivotal roles in cervical carcinogenesis by inhibiting tumor suppressor proteins, promoting uncontrolled cell proliferation, and evading immune responses, ultimately driving progression toward malignancy. Timely detection and intervention are essential for managing HPV-related cervical cancers. Preventative measures such as HPV vaccination have demonstrated substantial efficacy. Six vaccines targeting high-risk (HR) HPV strains are recommended before sexual activity or exposure. Despite these advancements, barriers, such as misinformation, logistical challenges, and limited healthcare infrastructure, persist, particularly in underserved regions. Advances in diagnostic and therapeutic technologies have offered new avenues for addressing these challenges. Next-generation sequencing and CRISPR gene editing are emerging as promising tools for HPV-related cancer treatment that enable precise and targeted interventions. Furthermore, artificial intelligence (AI) and imaging innovations have significantly enhanced diagnostic accuracy and personalized care. Pap smears and HPV DNA testing are indispensable tools for early detection. To tackle HPV-related cervical cancer globally, a multifaceted approach is required. Public health education, vaccination programs, research, and international collaboration are crucial. Public health campaigns should combat misinformation, strengthen vaccination programs, and focus on novel therapies, screening technologies, and next-generation sequencing.

A comprehensive analysis of the genomic and proteomic profiles of a megalocytivirus isolated from Larimichthys crocea

IntroductionThe prevalence of viral diseases has posed significant challenges to the sustainable development of large yellow croaker (Larimichthys crocea) aquaculture, with megalocytivirus being one of the primary viral pathogens affecting this species. There have been two proteomic and genomic studies regarding two members of the genus Megalocytivirus: the spotted knifejaw iridovirus (SKIV) and the infectious spleen and kidney necrosis virus (ISKNV). However, both studies were conducted more than 10 years ago. To further investigate the pathogenesis of megalocytivirus, we sequenced the genome of the viral strain FD201807 isolated from L. crocea, and conducted a proteomics analysis.MethodsViral DNA was sequenced using the Illumina HiSeq 2000 platform. Viral proteins from purified virions and supernatants of viral infected cells were subjected to LC-MS/MS analysis, and the expression of four viral proteins was further confirmed by Western blotting. The entire viral genome was subjected to phylogenetic and bioinformatic analyses.ResultsThe FD201807 genome comprises 112,214 bp of double-stranded DNA with a G + C content of 53.53%. It contains 130 potential open reading frames, with coding capacities ranging from 41 to 1,293 amino acids. Phylogenetic analysis of the whole-genome sequence indicated that the closest known megalocytivirus related to FD201807 is Pompano iridovirus, with a sequence identity of 98.98%. Label-free proteomics analysis identified 27 viral proteins in the viral-infected cell culture supernatants and 46 viral proteins in the purified virus of FD201807. Among these, 19 viral proteins were detected in both the viral-infected cell culture supernatants and the purified virus samples, while 8 viral proteins were exclusively identified in the viral-infected cell culture supernatants. Notably, there were two proteins derived from the cultured cell line MFF-1 (mandarin fish fry cell line-1), namely cytochrome c and ubiquitin-activating enzyme E1, present in both the purified virus samples and the culture supernatant of infected cells. These cellular proteins may be associated with virus-host protein interactions and/or host cell apoptosis.DiscussionWe present the most comprehensive proteomic analysis to date of the megalocytivirus isolated from L. crocea, and help identify highly expressed proteins that may serve as future targets for immunotherapy and biochemical analysis.

Low-temperature enhances production of severe fever with thrombocytopenia syndrome virus virus-like particles

Tick-borne severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease caused by the SFTS virus (SFTSV). Serological assays based on the nucleocapsid protein and partial glycoprotein of this virus have been used for detecting SFTSV infections in humans and animals. However, whether the complete SFTSV glycoprotein (Gn/Gc) can induce the assembly of virus-like particles (VLPs) which can be used for serological surveillance and vaccine production remains unclear. In this study, we successfully expressed and secreted SFTSV Gn/Gc antigens by using a single plasmid encoding the complete glycoprotein sequence of the dominant genotype B virus. HEK293T and COS-1 cells were transfected with the aforementioned plasmid; cultivating these cells at 32 °C, instead of 37 °C, led to 4.0- and 3.3-fold higher antigen recovery, respectively. The secreted Gn/Gc antigens at 32 °C retained epitopes resembling those of the virion; these epitopes were recognized by a SFTS human–derived monoclonal antibody. Sucrose density gradient centrifugation, followed by transmission electron microscopy, confirmed the formation of VLPs with a diameter of approximately 100 nm. Overall, our findings highlight the potential of SFTSV VLPs for serological surveillance and vaccine development.Key points• Cultivating transfected cells at 32 °C boosts SFTSV glycoprotein production.• Complete SFTSV glycoprotein expression facilitates virus-like particle assembly.• The assembly does not require any other viral proteins or RNA.