The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.

In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.

SFTSV nucleoprotein mediates DNA sensor cGAS degradation to suppress cGAS-dependent antiviral responses.

Cyclic GMP-AMP synthase (cGAS) is an important DNA pattern recognition receptor that senses double-stranded DNA derived from invading pathogens or self DNA in cytoplasm, leading to an antiviral interferon response. A tick-borne Bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), is an RNA virus that causes a severe emerging viral hemorrhagic fever in Asia with a high case fatality rate of up to 30%. However, it is unclear whether cGAS interacts with SFTSV infection. In this study, we found that SFTSV infection upregulated cGAS RNA transcription and protein expression, indicating that cGAS is an important innate immune response against SFTSV infection. The mechanism of cGAS recognizing SFTSV is by cGAS interacting with misplaced mitochondrial DNA in the cytoplasm. Depletion of mitochondrial DNA significantly inhibited cGAS activation under SFTSV infection. Strikingly, we found that SFTSV nucleoprotein (N) induced cGAS degradation in a dose-dependent manner. Mechanically, N interacted with the 161-382 domain of cGAS and linked the cGAS to LC3. The cGAS-N-LC3 trimer was targeted to N-induced autophagy, and the cGAS was degraded in autolysosome. Taken together, our study discovered a novel antagonistic mechanism of RNA viruses, SFTSV is able to suppress the cGAS-dependent antiviral innate immune responses through N-hijacking cGAS into N-induced autophagy. Our results indicated that SFTSV N is an important virulence factor of SFTSV in mediating host antiviral immune responses. IMPORTANCE: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne RNA virus that is widespread in East and Southeast Asian countries with a high fatality rate of up to 30%. Up to now, many cytoplasmic pattern recognition receptors, such as RIG-I, MDA5, and SAFA, have been reported to recognize SFTSV genomic RNA and trigger interferon-dependent antiviral responses. However, current knowledge is not clear whether SFTSV can be recognized by DNA sensor cyclic GMP-AMP synthase (cGAS). Our study demonstrated that cGAS could recognize SFTSV infection via ectopic mitochondrial DNA, and the activated cGAS-stimulator of interferon genes signaling pathway could significantly inhibit SFTSV replication. Importantly, we further uncovered a novel mechanism of SFTSV to inhibit innate immune responses by the degradation of cGAS. cGAS was degraded in N-induced autophagy. Collectively, this study illustrated a novel virulence factor of SFTSV to suppress innate immune responses through autophagy-dependent cGAS degradation.

Host factor MxA restricts Dabie bandavirus infection by targeting the viral NP protein to inhibit NP-RdRp interaction and ribonucleoprotein activity.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high case mortality rates, which is caused by Dabie bandavirus (DBV), a novel pathogen also termed as SFTS virus (SFTSV). Currently, no specific therapeutic drugs or vaccines are available for SFTS. Myxovirus resistance protein A (MxA) has been shown to inhibit multiple viral pathogens; however, the role of MxA in DBV infection is unknown. Here, we demonstrated that DBV stimulates MxA expression which, in turn, restricts DBV infection. Mechanistic target analysis revealed that MxA specifically interacts with the viral nucleocapsid protein (NP) in a manner independent of RNA. Minigenome reporter assay showed that in agreement with its targeting of NP, MxA inhibits DBV ribonucleoprotein (RNP) activity. In detail, MxA interacts with the NP N-terminal and disrupts the interaction of NP with the viral RNA-dependent RNA polymerase (RdRp) but not NP multimerization, the critical activities of NP for RNP formation and function. Furthermore, MxA N-terminal domain was identified as the functional domain inhibiting DBV infection, and, consistently, then was shown to interact with NP and obstruct the NP-RdRp interaction. Additionally, threonine 103 within the N-terminal domain is important for MxA inhibition to DBV, and its mutation (T103A) attenuates MxA binding to NP and obstruction of the NP-RdRp interaction. This study uncovers MxA inhibition of DBV with a series of functional and mechanistical analyses, providing insights into the virus-host interactions and probably helping inform the development of antiviral agents in the future.IMPORTANCEDBV/SFTSV is an emerging high-pathogenic virus. Since its first identification in China in 2009, cases of DBV infection have been reported in many other countries, posing a significant threat to public health. Uncovering the mechanisms of DBV-host interactions is necessary to understand the viral pathogenesis and host response and may advance the development of antiviral therapeutics. Here, we found that host factor MxA whose expression is induced by DBV restricts the virus infection. Mechanistically, MxA specifically interacts with the viral NP and blocks the NP-RdRp interaction, inhibiting the viral RNP activity. Further studies identified the key domain and amino acid residue required for MxA inhibition to DBV. Consistently, they were then shown to be important for MxA targeting of NP and obstruction of the NP-RdRp association. These findings unravel the restrictive role of MxA in DBV infection and the underlying mechanism, expanding our knowledge of the virus-host interactions.

The change of Siglec-9 expression in peripheral blood NK cells of SFTS patients can affect the function of NK cells.

OBJECTIVES: To investigate the changes and mechanism of Siglec-9 on NK cells in peripheral blood of patients with severe fever with thrombocytopenia syndrome (SFTS). METHODS: First, we used single-cell RNA sequencing to analyze the frequency of NK cells in Peripheral Blood Mononuclear Cells (PBMCs) of SFTS patients and healthy controls (HCs), as well as the differences in the genes on NK cells. Secondly, we analyzed the expression of Siglec-9 and other receptors on NK cells by flow cytometry. Thirdly, we analyzed the correlation between Siglec-9 on NK cells and DBV viral load in plasma. RESULTS: Compared with HCs, the frequency of NK cells in peripheral blood of SFTS patients was significantly decreased, and the activating receptors on NK cells were reduced. The expression of Siglec-9 on NK cells and the frequency of Siglec-9+NK cells decreased significantly in SFTS patients. The expression of Siglec-9 on CD16+CD56dim NK cells was negatively correlated with DBV viral load. In addition, Siglec-9+NK cells expressed higher levels of activating receptors and exhibited stronger effector functions than Siglec-9-NK cells. CONCLUSIONS: The decreased expression of Siglec-9 on NK cells predicts NK cell dysfunction in SFTS patients, suggesting that Siglec-9 may be a potential marker for functional NK cell subsets in SFTS patients.

Activation of the NLRP3 inflammasome and elevation of interleukin-1ß secretion in infection by sever fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging phlebovirus that causes a hemorrhagic fever known as the severe fever with thrombocytopenia syndrome (SFTS). Inflammasomes are a molecular platform that are assembled to process pro-caspase 1 and subsequently promote secretion of interleukin (IL)-1ß/IL-18 for proinflammatory responses induced upon infection. We hypothesize that inflammasome activation and pyroptosis induced in SFTS results in elevated levels of IL-1ß/IL-18 responsible for high fever and hemorrhage in the host, characteristic of SFTS. Here we report that IL-1ß secretion was elevated in SFTS patients and infected mice and IL-1ß levels appeared to be reversibly associated to disease severity and viral load in patients' blood. Increased caspase-1 activation, IL-1ß/IL-18 secretion, cell death, and processing of gasdermin D were detected, indicating that pyroptosis was induced in SFTSV-infected human peripheral blood monocytes (PBMCs). To characterize the mechanism of pyroptosis induction, we knocked down several NOD-like receptors (NLRs) with respective shRNAs in PBMCs and showed that the NLR family pyrin domain containing 3 (NLRP3) inflammasome was critical for processing pro-caspase-1 and pro-IL-1ß. Our data with specific inhibitors for NLRP3 and caspase-1 further showed that activation of the NLRP3 inflammasome was key to caspase-1 activation and IL-1ß secretion which may be inhibitory to viral replication in PBMCs infected with SFTSV. The findings in this study suggest that the activation of the NLPR3 inflammasome and pyroptosis, leading to IL-1ß/IL-18 secretion during the SFTSV infection, could play important roles in viral pathogenesis and host protection. Pyroptosis as part of innate immunity might be essential in proinflammatory responses and pathogenicty in humans infected with this novel phlebovirus.

Subcellular localization of nucleocapsid protein of SFTSV and its assembly into the ribonucleoprotein complex with L protein and viral RNA.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes novel zoonotic diseases in Asian countries including China, Japan, South Korea, and Vietnam. In phleboviruses, viral proteins play a critical role in viral particle formation inside the host cells. Viral glycoproteins (GPs) and RNA-dependent RNA polymerase (RdRp) are colocalized in the Golgi apparatus and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The nucleocapsid (N) protein was widely expressed in the cytoplasm, even in cells coexpressing GP. However, the role of SFTSV N protein remains unclear. The subcellular localization of SFTSV structural proteins was investigated using a confocal microscope. Subsequently, minigenome and immunoprecipitation assays were carried out. The N protein interacts with viral RNA (vRNA) and further shows translational activity with RdRp which is L protein and localized in the ERGIC and Golgi apparatus when co-expressed with GP. On the other hand, mutant N protein did not interact with vRNA either localized in the ERGIC or Golgi apparatus. The interaction between the N protein of SFTSV and vRNA is important for the localization of viral proteins and viral assembly. This study provides useful insights into the life cycle of SFTSV, which will lead to the detection of antiviral targets.

In Silico Structure-Based Design of Antiviral Peptides Targeting the Severe Fever with Thrombocytopenia Syndrome Virus Glycoprotein Gn.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus in Asia that causes severe disease. Despite its clinical importance, treatment options for SFTSV infection remains limited. The SFTSV glycoprotein Gn plays a major role in mediating virus entry into host cells and is therefore a potential antiviral target. In this study, we employed an in silico structure-based strategy to design novel cyclic antiviral peptides that target the SFTSV glycoprotein Gn. Among the cyclic peptides, HKU-P1 potently neutralizes the SFTSV virion. Combinatorial treatment with HKU-P1 and the broad-spectrum viral RNA-dependent RNA polymerase inhibitor favipiravir exhibited synergistic antiviral effects in vitro. The in silico peptide design platform in this study may facilitate the generation of novel antiviral peptides for other emerging viruses.

Nonstructural Protein NSs Hampers Cellular Antiviral Response through LSm14A during Severe Fever with Thrombocytopenia Syndrome Virus Infection.

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) plays multiple functions in the virus life cycle. Proteomic screening for host proteins interacting with NSs identified the cellular protein LSm14A. LSm14A, a member of the LSm family involved in RNA processing in the processing bodies, binds to viral RNA or synthetic homolog and mediates IFN regulatory factor 3 activation and IFN-ß induction. NSs interacted with and colocalized with LSm14A, and this interaction effectively inhibited downstream phosphorylation and dimerization of IFN regulatory factor 3, resulting in the suppression of antiviral signaling and IFN induction in several cell types of human origin. Knockdown of NSs resulted in the suppression of SFTSV replication in host cells. Viral RNA bound to LSm14A-NSs protein complex during the interaction. A newly discovered LRRD motif of NSs functioned to interact with LSm14A. Altogether, our data demonstrated a mechanism used by SFTSV to inhibit host innate immune response.

The Role of Non-Structural Protein NSs in the Pathogenesis of Severe Fever with Thrombocytopenia Syndrome.

Viral non-structural proteins, such as NSs of the newly emerging severe fever with thrombocytopenia syndrome virus, are well established virulence factors, mediating viral pathogenesis and disease progression through various mechanisms. NSs has been described as a potent interferon antagonist and NF-κB agonist, two divergent signaling pathways in many immune responses upon a viral encounter. In this review, we highlight the many mechanisms used by NSs on the host that promote viral replication and hyper-inflammation. Understanding these host-pathogen interactions is crucial for antiviral therapy development.

SFTSV Infection Induced Interleukin-1ß Secretion Through NLRP3 Inflammasome Activation.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus that causes hemorrhagic fever. Previous studies showed that SFTSV-infected patients exhibited elevated levels of pro-inflammatory cytokines like interleukin-1ß (IL-1ß), indicating that SFTSV infection may activate inflammasomes. However, the detailed mechanism remains poorly understood. Herein, we found that SFTSV could stimulate the IL-1ß secretion in the infected human peripheral blood mononuclear cells (PBMCs), human macrophages, and C57/BL6 mice. We demonstrate that the maturation and secretion of IL-1ß during SFTSV infection is mediated by the nucleotide and oligomerization domain, leucine-rich repeat-containing protein family, pyrin-containing domain 3 (NLRP3) inflammasome. This process is dependent on protease caspase-1, a component of the NLRP3 inflammasome complex. For the first time, our study discovered the role of NLRP3 in response to SFTSV infection. This finding may lead to the development of novel drugs to impede the pathogenesis of SFTSV infection.

Host restriction of emerging high-pathogenic bunyaviruses via MOV10 by targeting viral nucleoprotein and blocking ribonucleoprotein assembly.

Bunyavirus ribonucleoprotein (RNP) that is assembled by polymerized nucleoproteins (N) coating a viral RNA and associating with a viral polymerase can be both the RNA synthesis machinery and the structural core of virions. Bunyaviral N and RNP thus could be assailable targets for host antiviral defense; however, it remains unclear which and how host factors target N/RNP to restrict bunyaviral infection. By mass spectrometry and protein-interaction analyses, we here show that host protein MOV10 targets the N proteins encoded by a group of emerging high-pathogenic representatives of bunyaviruses including severe fever with thrombocytopenia syndrome virus (SFTSV), one of the most dangerous pathogens listed by World Health Organization, in RNA-independent manner. MOV10 that was further shown to be induced specifically by SFTSV and related bunyaviruses in turn inhibits the bunyaviral replication in infected cells in series of loss/gain-of-function assays. Moreover, animal infection experiments with MOV10 knockdown corroborated the role of MOV10 in restricting SFTSV infection and pathogenicity in vivo. Minigenome assays and additional functional and mechanistic investigations demonstrate that the anti-bunyavirus activity of MOV10 is likely achieved by direct impact on viral RNP machinery but independent of its helicase activity and the cellular interferon pathway. Indeed, by its N-terminus, MOV10 binds to a protruding N-arm domain of N consisting of only 34 amino acids but proving important for N function and blocks N polymerization, N-RNA binding, and N-polymerase interaction, disabling RNP assembly. This study not only advances the understanding of bunyaviral replication and host restriction mechanisms but also presents novel paradigms for both direct antiviral action of MOV10 and host targeting of viral RNP machinery.

Analysis of Severe Fever With Thrombocytopenia Syndrome in Critical Ill Patients in Central China.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonosis infected by virus (SFTSV) in central and eastern China, which is associated with high mortality. However, limited clinical data have been reported about this critical illness. PATIENTS AND METHODS: Retrospective cohort study in intensive care unit (ICU) patients with SFTSV infection admitted in 2014 to 2019. Diagnosis was confirmed using reverse transcription polymerase chain reaction on serum samples. RESULTS: One hundred sixteen patients with SFTSV infection were included (mean age 63 ±â€Š9 years, 59 [51.3%] males). Non-survivors (43.1%) were older, and had lower Glasgow Coma Score, higher Acute Physiology and Chronic Health Evaluation II, and sequential organ failure assessment score at ICU admission. In addition, non-survivors had more severe respiratory failure (PaO2/FiO2: 208 ±â€Š14 mm Hg vs. 297 ±â€Š15 mm Hg), more frequent shock (25[50%] vs. 7[10.6%]), and required more frequently mechanical ventilation (78% vs. 19.7%; P < 0.001) and vasopressor support (56% vs. 9.1%; P < 0.001). Non-survivors experienced more obvious monocyte loss. After adjustment for potential confounding factors, older age, elevated lactate level, and elevated creatinine level were the independent risk factors for death. CONCLUSION: We provided knowledge about the clinical characteristics of SFTS admitted in ICU. Older age, elevated lactate level, and elevated creatinine level may be useful for identifying patients with poor outcome and intensive medical intervention can be provided for patients as soon as possible to reduce mortality.