SARS-CoV-2 envelope protein-derived extracellular vesicles act as potential media for viral spillover.

Extracellular vesicles (EVs) are shown to be a novel viral transmission model capable of increasing a virus's tropism. According to our earlier research, cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or transfected with envelope protein plasmids generate a novel type of EVs that are micrometer-sized and able to encase virus particles. Here, we showed the capacity of these EVs to invade various animals both in vitro and in vivo independent of the angiotensin-converting enzyme 2 receptor. First, via macropinocytosis, intact EVs produced from Vero E6 (monkey) cells were able to enter cells from a variety of animals, including cats, dogs, bats, hamsters, and minks, and vice versa. Second, when given to zebrafish with cutaneous wounds, the EVs showed favorable stability in aqueous environments and entered the fish. Moreover, infection of wild-type (WT) mice with heterogeneous EVs carrying SARS-CoV-2 particles led to a strong cytokine response and a notable amount of lung damage. Conversely, free viral particles did not infect WT mice. These results highlight the variety of processes behind viral transmission and cross-species evolution by indicating that EVs may be possible vehicles for SARS-CoV-2 spillover and raising risk concerns over EVs' potential for viral gene transfer.

Discovery of SARS-CoV-2-E channel inhibitors as antiviral candidates.

Lack of efficiency has been a major problem shared by all currently developed anti-SARS-CoV-2 therapies. Our previous study shows that SARS-CoV-2 structural envelope (2-E) protein forms a type of cation channel, and heterogeneously expression of 2-E channels causes host cell death. In this study we developed a cell-based high throughput screening (HTS) assay and used it to discover inhibitors against 2-E channels. Among 4376 compounds tested, 34 hits with cell protection activity were found. Followed by an anti-viral analysis, 15 compounds which could inhibit SARS-CoV-2 replication were identified. In electrophysiological experiments, three representatives showing inhibitory effect on 2-E channels were chosen for further characterization. Among them, proanthocyanidins directly bound to 2-E channel with binding affinity (KD) of 22.14 µM in surface plasmon resonance assay. Molecular modeling and docking analysis revealed that proanthocyanidins inserted into the pore of 2-E N-terminal vestibule acting as a channel blocker. Consistently, mutations of Glu 8 and Asn 15, two residues lining the proposed binding pocket, abolished the inhibitory effects of proanthocyanidins. The natural product proanthocyanidins are widely used as cosmetic, suggesting a potential of proanthocyanidins as disinfectant for external use. This study further demonstrates that 2-E channel is an effective antiviral drug target and provides a potential antiviral candidate against SARS-CoV-2.

A multi-targeting drug design strategy for identifying potent anti-SARS-CoV-2 inhibitors.

The COVID-19, caused by SARS-CoV-2, is threatening public health, and there is no effective treatment. In this study, we have implemented a multi-targeted anti-viral drug design strategy to discover highly potent SARS-CoV-2 inhibitors, which simultaneously act on the host ribosome, viral RNA as well as RNA-dependent RNA polymerases, and nucleocapsid protein of the virus, to impair viral translation, frameshifting, replication, and assembly. Driven by this strategy, three alkaloids, including lycorine, emetine, and cephaeline, were discovered to inhibit SARS-CoV-2 with EC50 values of low nanomolar levels potently. The findings in this work demonstrate the feasibility of this multi-targeting drug design strategy and provide a rationale for designing more potent anti-virus drugs.

Discovery of potential small molecular SARS-CoV-2 entry blockers targeting the spike protein.

An epidemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. SARS-CoV-2 relies on its spike protein to invade host cells by interacting with the human receptor protein Angiotensin-Converting Enzymes 2 (ACE2). Therefore, designing an antibody or small-molecular entry blockers is of great significance for virus prevention and treatment. This study identified five potential small molecular anti-virus blockers via targeting SARS-CoV-2 spike protein by combining in silico technologies with in vitro experimental methods. The five molecules were natural products that binding to the RBD domain of SARS-CoV-2 was qualitatively and quantitively validated by both native Mass Spectrometry (MS) and Surface Plasmon Resonance (SPR). Anti-viral activity assays showed that the optimal molecule, H69C2, had a strong binding affinity (dissociation constant KD) of 0.0947 µM and anti-virus IC50 of 85.75 µM.

Longitudinal Profile of Laboratory Parameters and Their Application in the Prediction for Fatal Outcome Among Patients Infected With SARS-CoV-2: A Retrospective Cohort Study.

BACKGROUND: Patients with coronavirus disease 2019 (COVID-19) experience a wide clinical spectrum, with over 2% developing fatal outcome. The prognostic factors for fatal outcome remain sparsely investigated. METHODS: A retrospective cohort study was performed in a cohort of patients with confirmed COVID-19 in one designated hospital in Wuhan, China, from 17 January-5 March 2020. The laboratory parameters and a panel of cytokines were consecutively evaluated until patients' discharge or death. The laboratory features that could be used to predict fatal outcome were identified. RESULTS: Consecutively collected data on 55 laboratory parameters and cytokines from 642 patients with COVID-19 were profiled along the entire disease course, based on which 3 clinical stages (acute stage, days 1-9; critical stage, days 10-15; and convalescence stage, day 15 to observation end) were determined. Laboratory findings based on 75 deceased and 357 discharged patients revealed that, at the acute stage, fatality could be predicted by older age and abnormal lactate dehydrogenase (LDH), urea, lymphocyte count, and procalcitonin (PCT) level. At the critical stage, the fatal outcome could be predicted by age and abnormal PCT, LDH, cholinesterase, lymphocyte count, and monocyte percentage. Interleukin 6 (IL-6) was remarkably elevated, with fatal cases having a more robust production than discharged cases across the whole observation period. LDH, PCT, lymphocytes, and IL-6 were considered highly important prognostic factors for COVID-19-related death. CONCLUSIONS: The identification of predictors that were routinely tested might allow early identification of patients at high risk of death for early aggressive intervention.

Comprehensive Interactome Analysis Reveals that STT3B Is Required for N-Glycosylation of Lassa Virus Glycoprotein.

Lassa virus (LASV) is the causative agent of a fatal hemorrhagic fever in humans. The glycoprotein (GP) of LASV mediates viral entry into host cells, and correct processing and modification of GP by host factors is a prerequisite for virus replication. Here, using an affinity purification-coupled mass spectrometry (AP-MS) strategy, 591 host proteins were identified as interactors of LASV GP. Gene ontology analysis was performed to functionally annotate these proteins, and the oligosaccharyltransferase (OST) complex was highly enriched. Functional studies conducted by using CRISPR-Cas9-mediated knockouts showed that STT3A and STT3B, the two catalytically active isoforms of the OST complex, are essential for the propagation of the recombinant arenavirus rLCMV/LASV glycoprotein precursor, mainly via affecting virus infectivity. Knockout of STT3B, but not STT3A, caused hypoglycosylation of LASV GP, indicating a preferential requirement of LASV for the STT3B-OST isoform. Furthermore, double knockout of magnesium transporter 1 (MAGT1) and tumor suppressor candidate 3 (TUSC3), two specific subunits of STT3B-OST, also caused hypoglycosylation of LASV GP and affected virus propagation. Site-directed mutagenesis analysis revealed that the oxidoreductase CXXC active-site motif of MAGT1 or TUSC3 is essential for the glycosylation of LASV GP. NGI-1, a small-molecule OST inhibitor, can effectively reduce virus infectivity without affecting cell viability. The STT3B-dependent N-glycosylation of GP is conserved among other arenaviruses, including both the Old World and New World groups. Our study provided a systematic view of LASV GP-host interactions and revealed the preferential requirement of STT3B for LASV GP N-glycosylation.IMPORTANCE Glycoproteins play vital roles in the arenavirus life cycle by facilitating virus entry and participating in the virus budding process. N-glycosylation of GPs is responsible for their proper functioning; however, little is known about the host factors on which the virus depends for this process. In this study, a comprehensive LASV GP interactome was characterized, and further study revealed that STT3B-dependent N-glycosylation was preferentially required by arenavirus GPs and critical for virus infectivity. The two specific thioredoxin subunits of STT3B-OST MAGT1 and TUSC3 were found to be essential for the N-glycosylation of viral GP. NGI-1, a small-molecule inhibitor of OST, also showed a robust inhibitory effect on arenavirus. Our study provides new insights into LASV GP-host interactions and extends the potential targets for the development of novel therapeutics against Lassa fever in the future.

Quantitative Proteomic Analysis Reveals Unfolded-Protein Response Involved in Severe Fever with Thrombocytopenia Syndrome Virus Infection.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging, highly pathogenic, infectious disease caused by infection with a newly discovered tick-borne phlebovirus, SFTS virus (SFTSV). Limited information on the molecular mechanism of SFTSV infection and pathogenesis impedes the development of effective vaccines and drugs for SFTS prevention and treatment. In this study, an isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis of SFTSV-infected HEK 293 cells was performed to explore dynamic host cellular protein responses toward SFTSV infection. A total of 433 of 5,606 host proteins involved in different biological processes were differentially regulated by SFTSV infection. The proteomic results highlighted a potential role of endoplasmic reticular stress-triggered unfolded-protein response (UPR) in SFTSV infection. Further functional studies confirmed that all three major branches of the UPR, including the PKR-like endoplasmic reticulum kinase (PERK), the activating transcription factor-6 (ATF6), and the inositol-requiring protein-1 (IRE1)/X-box-binding protein 1 (XBP1) pathways, were activated by SFTSV. However, only the former two pathways play a crucial role in SFTSV infection. Furthermore, expression of SFTSV glycoprotein (GP) alone was sufficient to stimulate the UPR, whereas suppression of PERK and ATF6 notably decreased GP expression. Interestingly, two other newly discovered phleboviruses, Heartland virus and Guertu virus, also stimulated the UPR, suggesting a common mechanism shared by these genetically related phleboviruses. This study provides a global view to our knowledge on how host cells respond to SFTSV infection and highlights that host cell UPR plays an important role in phlebovirus infection.IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe fever with thrombocytopenia syndrome in humans, with a mortality rate reaching up to 30% in some outbreaks. There are currently no U.S. Food and Drug Administration-approved vaccines or specific antivirals available against SFTSV. To comprehensively understand the molecular interactions occurring between SFTSV and the host cell, we exploit quantitative proteomic approach to investigate the dynamic host cellular responses to SFTSV infection. The results highlight multiple biological processes being regulated by SFTSV infection. Among these, we focused on exploration of the mechanism of how SFTSV infection stimulates the host cell's unfolded-protein response (UPR) and identified the UPR as a common feature shared by SFTSV-related new emerging phleboviruses. This study, for the first time to our knowledge, provides a global map for host cellular responses to SFTSV infection and highlighted potential host targets for further research.

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

A Comparative Quantitative Proteomic Analysis of HCMV-Infected Cells Highlights pUL138 as a Multifunctional Protein.

Human cytomegalovirus (HCMV) is a widespread virus that can establish life-long latent infection in large populations. The establishment of latent infection prevents HCMV from being cleared by host cells, and HCMV reactivation from latency can cause severe disease and death in people with immature or compromised immune systems. To establish persistent and latent infection in healthy individuals, HCMV encodes a large array of proteins that can modulate different components and pathways of host cells. It has been reported that pUL138 encoded by the UL133-UL138 polycistronic locus promotes latent infection in primary CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. In this study, recombinant HCMV HanUL138del was constructed by deleting the UL138 locus of Han, a clinical HCMV strain. Then, a comparative quantitative proteomic analysis of Han- and HanUL138del-infected MRC5 cells was performed to study the effect of pUL138 on host cells in the context of HCMV infection. Our results indicated that, during the early phase of HCMV infection, the innate immune response was differentially activated, while during the late phase of HCMV infection, multiple host proteins were differentially expressed between Han- and HanUL138del-infected cells, and these proteins are involved in the oxidation-reduction process, ER to Golgi vesicle-mediated transport, and extracellular matrix organization. Among these proteins, STEAP3, BORCS7, FAM172A, RELL1, and WDR48 were further demonstrated to affect HCMV infection. Our study provides a systematic view of the effect of pUL138 on the host cell proteome and highlights the proposition that multiple biological processes or host factors may be involved in the overall role of the UL133-UL138 polycistronic locus in HCMV persistence.

A Subcellular Quantitative Proteomic Analysis of Herpes Simplex Virus Type 1-Infected HEK 293T Cells.

Herpes simplex virus type 1 (HSV-1) is widespread double-stranded DNA (dsDNA) virus that establishes life-long latency and causes diverse severe symptoms. The mechanisms of HSV-1 infection and HSV-1's interactions with various host cells have been studied and reviewed extensively. Type I interferons were secreted by host cells upon HSV infection and play a vital role in controlling virus proliferation. A few studies, however, have focused on HSV-1 infection without the presence of interferon (IFN) signaling. In this study, HEK 293T cells with low toll-like receptor (TLR) and stimulator of interferon genes protein (STING) expression were infected with HSV-1 and subjected to a quantitative proteomic analysis. By using a subcellular fractionation strategy and high-performance mass spectrometry, a total of 6607 host proteins were quantified, of which 498 proteins were differentially regulated. A bioinformatics analysis indicated that multiple signaling pathways might be involved in HSV-1 infection. A further functional study indicated the role of Interferon-induced transmembrane protein 3 (IFITM3), Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2), and Tripartite motif-containing protein 27 (TRIM27) in inhibiting viral DNA replication and proliferation. Our data provide a global view of host responses to HSV-1 infection in HEK 293T cells and identify the proteins involved in the HSV-1 infection process.

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-ß) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.

Global quantitative proteomic analysis profiles host protein expression in response to Sendai virus infection.

Sendai virus (SeV) is an enveloped nonsegmented negative-strand RNA virus that belongs to the genus Respirovirus of the Paramyxoviridae family. As a model pathogen, SeV has been extensively studied to define the basic biochemical and molecular biologic properties of the paramyxoviruses. In addition, SeV-infected host cells were widely employed to uncover the mechanism of innate immune response. To identify proteins involved in the SeV infection process or the SeV-induced innate immune response process, system-wide evaluations of SeV-host interactions have been performed. cDNA microarray, siRNA screening and phosphoproteomic analysis suggested that multiple signaling pathways are involved in SeV infection process. Here, to study SeV-host interaction, a global quantitative proteomic analysis was performed on SeV-infected HEK 293T cells. A total of 4699 host proteins were quantified, with 742 proteins being differentially regulated. Bioinformatics analysis indicated that regulated proteins were mainly involved in "interferon type I (IFN-I) signaling pathway" and "defense response to virus," suggesting that these processes play roles in SeV infection. Further RNAi-based functional studies indicated that the regulated proteins, tripartite motif (TRIM24) and TRIM27, affect SeV-induced IFN-I production. Our data provided a comprehensive view of host cell response to SeV and identified host proteins involved in the SeV infection process or the SeV-induced innate immune response process.

Activation of the RLR/MAVS Signaling Pathway by the L Protein of Mopeia Virus.

The family Arenaviridae includes several important human pathogens that can cause severe hemorrhagic fever and greatly threaten public health. As a major component of the innate immune system, the RLR/MAVS signaling pathway is involved in recognizing viral components and initiating antiviral activity. It has been reported that arenavirus infection can suppress the innate immune response, and NP and Z proteins of pathogenic arenaviruses can disrupt RLR/MAVS signaling, thus inhibiting production of type I interferon (IFN-I). However, recent studies have shown elevated IFN-I levels in certain arenavirus-infected cells. The mechanism by which arenavirus infection induces IFN-I responses remains unclear. In this study, we determined that the L polymerase (Lp) of Mopeia virus (MOPV), an Old World (OW) arenavirus, can activate the RLR/MAVS pathway and thus induce the production of IFN-I. This activation is associated with the RNA-dependent RNA polymerase activity of Lp. This study provides a foundation for further studies of interactions between arenaviruses and the innate immune system and for the elucidation of arenavirus pathogenesis. IMPORTANCE: Distinct innate immune responses are observed when hosts are infected with different arenaviruses. It has been widely accepted that NP and certain Z proteins of arenaviruses inhibit the RLR/MAVS signaling pathway. The viral components responsible for the activation of the RLR/MAVS signaling pathway remain to be determined. In the current study, we demonstrate for the first time that the Lp of MOPV, an OW arenavirus, can activate the RLR/MAVS signaling pathway and thus induce the production of IFN-I. Based on our results, we proposed that dynamic interactions exist among Lp-produced RNA, NP, and the RLR/MAVS signaling pathway, and the outcome of these interactions may determine the final IFN-I response pattern: elevated or reduced. Our study provides a possible explanation for how IFN-I can become activated during arenavirus infection and may help us gain insights into the interactions that form between different arenavirus components and the innate immune system.

Subcellular quantitative proteomic analysis reveals host proteins involved in human cytomegalovirus infection.

Viral replication requires host cell macromolecules and energy, although host cells can alter their protein expression to restrict viral replication. To study the host cell response to human cytomegalovirus (HCMV) infection, a stable isotope labeling by amino acids in cell culture (SILAC)-based subcellular quantitative proteomic study of HCMV-infected human embryo lung fibroblast (HEL) cells was performed, and a total of 247 host proteins were identified as differentially regulated by HCMV. Western blotting and immunofluorescence confocal microscopy were performed to validate the data sets. Gene Ontology analysis indicated that cellular processes involving the metabolism, localization and immune system were regulated as a result of HCMV infection. Functional analysis of selected regulated proteins revealed that knockdown of HNRPD, PHB2 and UB2V2 can increase HCMV replication, while knockdown of A4 and KSRP resulted in decreased HCMV replication. Our study may improve our understanding of the dynamic interactions between HCMV and its host and provide multiple potential targets for anti-HCMV agent research.

Global quantitative proteomic analysis of human glioma cells profiled host protein expression in response to enterovirus type 71 infection.

Enterovirus 71 (EV71) is one of the leading causes of hand, foot and mouth disease with neurological complications in some cases. To study the pathogenesis of EV71 infection, large-scale analyses of EV71 infected cells have been performed. However, most of these studies employed rhabdomyosarcoma (RD) cells or used transcriptomic strategy. Here, we performed SILAC-based quantitative proteomic analysis of EV71-infected U251 cells, a human glioma cell line. A total of 3125 host proteins were quantified, in which 451 were differentially regulated as a result of EV71 infection at 8 or 20 hpi or both. Gene Ontology analysis indicates the regulated proteins were enriched in "metabolic process", "biological regulation" and "cellular process", implying that these biological processes were affected by EV71 infection. Furthermore, functional study indicated that TRAF2 and TRAF6 among the up-regulated proteins could inhibit the replication of EV71 at the early phase post infection, and the anti-EV71 function of both proteins was independent of interferon ß. Our study not only provided an overview of cellular response to EV71 infection in a human glioma cell line, but also found that TRAF2 and TRAF6 might be potential targets to inhibit the replication of EV71. All MS data have been deposited in the ProteomeXchange with identifier PXD002454 (http://proteomecentral.proteomexchange.org/dataset/PXD002454).

Analysis of EV71 infection progression using triple-SILAC-based proteomics approach.

Enterovirus 71 (EV71), a member of Picornaviridae, causes severe neurological and systemic illness in children. To better understand the virus-host cell interactions, we performed a triple-SILAC-based quantitative proteomics study monitoring host cell proteome changes after EV71 infection. Based on the quantitative data for more than 4100 proteins, ∼17% of the proteins were found as significantly changed (p<0.01) at either 8 or 20 hours post infection. Five biological processes and seven protein classes showed significant differences. Functional screening of nine regulated proteins discovered the regulatory role of CHCH2, a mitochondrial protein known as a transcriptional activator for cytochrome c oxidase, in EV71 replication. Further studies showed that CHCH2 served as a negative regulator of innate immune responses. All MS data have been deposited in the ProteomeXchange with identifier PXD002483 (http://proteomecentral.proteomexchange.org/dataset/PXD002483).

Identification of host proteins involved in Japanese encephalitis virus infection by quantitative proteomics analysis.

Japanese encephalitis virus (JEV) enters host cells via receptor-mediated endocytosis and replicates in the cytoplasm of infected cells. To study virus-host cell interactions, we performed a SILAC-based quantitative proteomics study of JEV-infected HeLa cells using a subcellular fractionation strategy. We identified 158 host proteins as differentially regulated by JEV (defined as exhibiting a greater than 1.5-fold change in protein abundance upon JEV infection). The mass spectrometry quantitation data for selected proteins were validated by Western blot and immunofluorescence confocal microscopy. Bioinformatics analyses were used to generate JEV-regulated host response networks consisting of regulated proteins, which included 35 proteins that were newly added based on the results of this study. The JEV infection-induced host response was found to be coordinated primarily through the immune response process, the ubiquitin-proteasome system (UPS), the intracellular membrane system, and lipid metabolism-related proteins. Protein functional studies of selected host proteins using RNA interference-based techniques were carried out in HeLa cells infected with an attenuated or a highly virulent strain of JEV. We demonstrated that the knockdown of interferon-induced transmembrane protein 3 (IFITM3), Ran-binding protein 2 (RANBP2), sterile alpha motif domain-containing protein 9 (SAMD9) and vesicle-associated membrane protein 8 (VAMP8) significantly increased JEV replication. The results presented here not only promote a better understanding of the host response to JEV infection but also highlight multiple potential targets for the development of antiviral agents.

Regulation of the WNT-CTNNB1 signaling pathway by severe fever with thrombocytopenia syndrome virus in a cap-snatching manner.

The segmented negative-strand RNA viruses (sNSVs) include highly pathogenic human and animal viruses such as Lassa virus (LASV), severe fever with thrombocytopenia syndrome virus (SFTSV), and influenza A virus (IAV). One of the conserved mechanisms at the stage of genome transcription of sNSVs is the cap-snatching process, providing druggable targets for the development of antivirals. SFTSV is an emerging tick-borne sNSV that causes severe hemorrhagic fever with a high fatality rate of 12%-50%. Here, we determined the correlation between death outcome and downregulation of the WNT-CTNNB1 signaling pathway through transcriptomic analysis of blood samples collected from SFTS patients. We further demonstrated that SFTSV affected this pathway by downregulating the mRNA levels of a series of pathway-related genes, including CTNNB1. Loss-of-function mutations or inhibitors targeting SFTSV cap-snatching activity effectively alleviated the inhibition of the WNT-CTNNB1 signaling pathway. Exogenous activation of the WNT-CTNNB1 signaling pathway enhanced SFTSV replication, while inhibition of this pathway reduced SFTSV replication. Treatment with a WNT-CTNNB1 signaling pathway inhibitor attenuated viral replication and decreased fatality in mice. Notably, downregulation of the WNT-CTNNB1 signaling pathway was also observed for other sNSVs, including LASV and IAV. These results suggested that RNAs related to the WNT-CTNNB1 signaling pathway might be utilized as a primer "pool" in a cap-snatching manner for viral transcription, which provides effective targets for the development of broad-spectrum antivirals against sNSVs.IMPORTANCEOne of the conserved mechanisms at the stage of genome transcription of segmented negative-strand RNA viruses (sNSVs) is the cap-snatching process, which is vital for sNSVs transcription and provides drugable targets for the development of antivirals. However, the specificity of RNAs snatched by sNSV is still unclear. By transcriptomics analysis of whole blood samples from SFTS patients, we found WNT-CTNNB1 signaling pathway was regulated according to the course of the disease. We then demonstrated that L protein of severe fever with thrombocytopenia syndrome virus (SFTSV) could interact with mRNAs of WNT-CTNNB1 signaling pathway-related gene, thus affecting WNT-CTNNB1 signaling pathway through its cap-snatching activity. Activation of WNT-CTNNB1 signaling pathway enhanced SFTSV replication, while inhibition of this pathway decreased SFTSV replication in vitro and in vivo. These findings suggest that WNT-associated genes may be the substrate for SFTSV "cap-snatching", and indicate a conserved sNSVs replication mechanism involving WNT-CTNNB1 signaling.