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.

SFTSV Infection Induces BAK/BAX-Dependent Mitochondrial DNA Release to Trigger NLRP3 Inflammasome Activation.

Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) is an emerging tick-borne virus that carries a high fatality rate of 12%-50%. In-depth understanding of the SFTSV-induced pathogenesis mechanism is critical for developing effective anti-SFTS therapeutics. Here, we report transcriptomic analysis of blood samples from SFTS patients. We observe a strong correlation between inflammatory responses and disease progression and fatal outcome. Quantitative proteomic analysis of SFTSV infection confirms the induction of inflammation and further reveals virus-induced mitochondrial dysfunction. Mechanistically, SFTSV infection triggers BCL2 antagonist/killer 1 (BAK) upregulation and BAK/BCL2-associated X (BAX) activation, leading to mitochondrial DNA (mtDNA) oxidization and subsequent cytosolic release. The cytosolic mtDNA binds and triggers NLRP3 inflammasome activation. Notably, the BAK expression level correlates with SFTS disease progression and fatal outcome. These findings provide insights into the clinical features and molecular underpinnings of severe SFTS, which may aid in patient care and therapeutic design, and may also be conserved during infection by other highly pathogenic viruses.

Calcium channel blockers reduce severe fever with thrombocytopenia syndrome virus (SFTSV) related fatality.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne infectious disease caused by a novel phlebovirus (SFTS virus, SFTSV), was listed among the top 10 priority infectious diseases by the World Health Organization due to its high fatality of 12%-50% and possibility of pandemic transmission. Currently, effective anti-SFTSV intervention remains unavailable. Here, by screening a library of FDA-approved drugs, we found that benidipine hydrochloride, a calcium channel blocker (CCB), inhibited SFTSV replication in vitro. Benidipine hydrochloride was revealed to inhibit virus infection through impairing virus internalization and genome replication. Further experiments showed that a broad panel of CCBs, including nifedipine, inhibited SFTSV infection. The anti-SFTSV effect of these two CCBs was further analyzed in a humanized mouse model in which CCB treatment resulted in reduced viral load and decreased fatality rate. Importantly, by performing a retrospective clinical investigation on a large cohort of 2087 SFTS patients, we revealed that nifedipine administration enhanced virus clearance, improved clinical recovery, and remarkably reduced the case fatality rate by >5-fold. These findings are highly valuable for developing potential host-oriented therapeutics for SFTS and other lethal acute viral infections known to be inhibited by CCBs in vitro.

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.

Complete Genome of a Novel Pseudoalteromonas Phage PHq0.

We isolated and purified a novel virulent Pseudoalteromonas bacteriophage PHq0 and the host bacterium Pseudoalteromonas BQ0 from seawater collected in a coastal area of the Yellow Sea of China. (36°06'N, 120°32'E). Transmission electron microscopy revealed that the phage had an icosahedral head of 50 nm in diameter with a long tail of 100 nm. The one-step growth curve showed the latent period of about 15 min, a rise period of 15 min, and a burst size of about 363 virions. The genome of phage PHq0 was found to consist of a linear, double-stranded 33,399-bp DNA molecule with a GC content of 40.29 % and 56 putative open reading frames (ORFs). Among these genes, 23 conserved domains were detected by BLASTP, 17 were functionally known, leaving 39 unknown putative genes, BLASTP results show that 57.14 % of the 56 predicted ORFs were not found to have any matches of putative functions or conserved domains in the BLASTP database which should be classified as a new member of the Siphoviridae family. The phage PHq0 genome adds a new Siphoviridae-family phage genome for marine bacteriophages which will provide useful basic information for further molecular research on interaction mechanism between bacteriophages and their hosts.