Siah2- and LRSAM1-mediated K63-linked ubiquitination of snakehead vesiculovirus nucleoprotein facilitates viral replication.

Nucleoprotein (N) is well known for its function in the encapsidation of the genomic RNAs of negative-strand RNA viruses, which leads to the formation of ribonucleoproteins that serve as templates for viral transcription and replication. However, the function of the N protein in other aspects during viral infection is far from clear. In this study, the N protein of snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was proved to be ubiquitinated mainly via K63-linked ubiquitination. We identified nine host E3 ubiquitin ligases that interacted with SHVV N, among which seven E3 ubiquitin ligases facilitated ubiquitination of the N protein. Further investigation revealed that only two E3 ubiquitin ligases, Siah E3 ubiquitin protein ligase 2 (Siah2) and leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1), mediated K63-linked ubiquitination of the N protein. SHVV infection upregulated the expression of Siah2 and LRSAM1, which maintained the stability of SHVV N. Besides, overexpression of Siah2 or LRSAM1 promoted SHVV replication, while knockdown of Siah2 or LRSAM1 inhibited SHVV replication. Deletion of the ligase domain of Siah2 or LRSAM1 did not affect their interactions with SHVV N but reduced the K63-linked ubiquitination of SHVV N and SHVV replication. In summary, Siah2 and LRSAM1 mediate K63-linked ubiquitination of SHVV N to facilitate SHVV replication, which provides novel insights into the role of the N proteins of negative-strand RNA viruses. IMPORTANCE: Ubiquitination of viral protein plays an important role in viral replication. However, the ubiquitination of the nucleoprotein (N) of negative-strand RNA viruses has rarely been investigated. This study aimed at investigating the ubiquitination of the N protein of a fish rhabdovirus SHVV (snakehead vesiculovirus), identifying the related host E3 ubiquitin ligases, and determining the role of SHVV N ubiquitination and host E3 ubiquitin ligases in viral replication. We found that SHVV N was ubiquitinated mainly via K63-linked ubiquitination, which was mediated by host E3 ubiquitin ligases Siah2 (Siah E3 ubiquitin protein ligase 2) and LRSAM1 (leucine-rich repeat and sterile alpha motif containing 1). The data suggested that Siah2 and LRSAM1 were hijacked by SHVV to ubiquitinate the N protein for viral replication, which exhibited novel anti-SHVV targets for drug design.

Grass carp reovirus VP56 and VP35 induce formation of viral inclusion bodies for replication.

Viral inclusion bodies (VIBs) are subcellular structures required for efficient viral replication. How type II grass carp reovirus (GCRV-II), the mainly prevalent strain, forms VIBs is unknown. In this study, we found that GCRV-II infection induced punctate VIBs in grass carp ovary (GCO) cells and that non-structural protein 38 (NS38) functioned as a participant in VIB formation. Furthermore, VP56 and VP35 induced VIBs and recruited other viral proteins via the N-terminal of VP56 and the middle domain of VP35. Additionally, we found that the newly synthesized viral RNAs co-localized with VP56 and VP35 in VIBs during infection. Taken together, VP56 and VP35 induce VIB formation and recruit other viral proteins and viral RNAs to the VIBs for viral replication, which helps identify new targets for developing anti-GCRV-II drugs to disrupt viral replication.

Snakehead vesiculovirus hijacks SH3RF1 for replication via mediating K63-linked ubiquitination at K264 of the phosphoprotein.

Snakehead vesiculovirus (SHVV) is a type of rhabdovirus that causes serious economic losses in snakehead fish culture in China. However, no specific antiviral drugs or vaccines are currently available for SHVV infection. In this study, 4D label-free ubiquitome analysis of SHVV-infected cells revealed dozens of ubiquitinated sites on the five SHVV proteins. We focused on investigating the ubiquitination of phosphoprotein (P), a viral polymerase co-factor involved in viral replication. SHVV-P was proved to be ubiquitinated via K63-linked ubiquitination at lysine 264 (K264). Overexpression of wild-type P, but not its K264R mutant, facilitated SHVV replication, indicating that K264 ubiquitination of the P protein is critical for SHVV replication. RNAi screening of 26 cellular E3 ubiquitin ligases identified five pro-viral factors for SHVV replication, including macrophage erythroblast attacher (MAEA), TNF receptor-associated factor 7 (TRAF7), and SH3 domain-containing ring finger protein 1 (SH3RF1), which interacted with and mediated ubiquitination of SHVV P. TRAF7 and SH3RF1, but not MAEA, mediated K63-linked ubiquitination of SHVV P, while only SH3RF1 mediated K264 ubiquitination of SHVV P. Besides, overexpression of SH3RF1 promoted SHVV replication and maintained the stability of SHVV P. In summary, SH3RF1 mediated K63-linked ubiquitination of SHVV P at K264 to facilitate SHVV replication, providing targets for developing anti-SHVV drugs and live-attenuated SHVV vaccines. Our study provides novel insights into the role of P protein in the replication of single-stranded, negative-sense RNA viruses.

DDX23 of black carp negatively regulates MAVS-mediated antiviral signaling in innate immune activation.

Mammalian DDX23 is involved in multiple biological processes, such as RNA processing and antiviral responses. However, the function of teleost DDX23 still remains unclear. In this paper, we have cloned the DDX23 homologue of black carp (Mylopharyngodon piceus) (bcDDX23) and elucidated its role in the antiviral innate immunity. The coding region of bcDDX23 comprises 2427 nucleotides and encodes 809 amino acids. The transcription of bcDDX23 was promoted by the stimulation of LPS, poly(I:C), and SVCV; and immunoblotting (IB) assay showed that bcDDX23 migrated aground 94.5 kDa. Immunofluorescence (IF) assay revealed that bcDDX23 was mainly distributed in the nucleus, and the amount of cytosolic bcDDX23 was significantly increased after SVCV infection. The reporter assay showed that bcDDX23 inhibited bcMAVS-mediated transcription of the IFN promoter. And the co-immunoprecipitation (co-IP) assays identified the interaction between bcDDX23 and bcMAVS. Furthermore, co-expressed bcDDX23 significantly inhibited bcMAVS-mediated antiviral ability against SVCV in EPC cells, and knockdown of bcDDX23 enhanced the resistance of host cells against SVCV. Overall, our results conclude that bcDDX23 targets bcMAVS and suppresses MAVS-mediated IFN signaling, which sheds light on the regulation of IFN signaling in teleost fish.

p38MAPK- and GSK3-Mediated Phosphorylation of Snakehead Vesiculovirus Phosphoprotein at Threonine 160 Facilitates Viral Replication.

Phosphoprotein (P), co-factor of the polymerase (large protein, L) of single-stranded negative-sense RNA viruses, is phosphorylated during viral infection and its phosphorylation has been reported to play important roles in viral replication. However, the function of P phosphorylation in viral replication is still far from clear. Snakehead vesiculovirus (SHVV) is a kind of fish rhabdovirus that has caused serious economic losses in snakehead fish culture in China without any effective preventive or therapeutical measures currently. In this study, 4D label-free phosphoproteomics sequencing of SHVV-infected cells identified five phosphorylated sites on SHVV P, among which threonine 160 (T160) was proved to be phosphorylated. Overexpression of wild-type P, but not P-T160A or P-T160E mutant, promoted SHVV replication, suggesting that the T160 phosphorylation on the P protein is critical for SHVV replication. Moreover, we found that T160A or T160E mutation on SHVV P had no effect on the interactions of P-nucleoprotein (N), P-P, or P-L. Further study revealed that p38 mitogen-activated protein kinase (p38MAPK) and glycogen synthase kinase 3 (GSK3) interacted with SHVV P and mediated the T160 phosphorylation. Besides, overexpression of p38MAPK or GSK3 facilitated, while knockdown or activity inhibition of p38MAPK or GSK3 suppressed, SHVV replication. Overall, p38MAPK- and GSK3-mediated phosphorylation of the P protein at T160 is required for SHVV replication, which provided targets for designing anti-SHVV drugs and developing live-attenuated SHVV vaccines. Our study helps understand the role of P phosphorylation in the replication of single-stranded negative-sense RNA viruses. IMPORTANCE Phosphorylation of viral proteins plays important roles in viral replication. Currently, the role of phosphorylation of phosphoprotein (P) in the replication of single-stranded negative-sense RNA viruses is far from clear. Identification of the phosphorylated sites on viral P protein and the related host kinases is helpful for developing live-attenuated vaccines and designing antiviral drugs. This study focused on identifying the phosphorylated sites on P protein of a fish rhabdovirus SHVV, determining the related host kinases, and revealing the effects of the phosphorylated sites and kinases on SHVV replication. We found that SHVV P was phosphorylated at T160, which was mediated by the kinases p38MAPK and GSK3 to promote SHVV replication. This study is the first time to study the role of P phosphorylation in fish rhabdovirus replication.

hnRNPA1 impedes snakehead vesiculovirus replication via competitively disrupting viral phosphoprotein-nucleoprotein interaction and degrading viral phosphoprotein.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) plays an important role in regulating the replication of many viruses. However, it remains elusive whether and how hnRNPA1 regulates fish virus replication. In this study, the effects of twelve hnRNPs on the replication of snakehead vesiculovirus (SHVV) were screened. Three hnRNPs, one of which was hnRNPA1, were identified as anti-SHVV factors. Further verification showed that knockdown of hnRNPA1 promoted, while overexpression of hnRNPA1 inhibited, SHVV replication. SHVV infection reduced the expression level of hnRNPA1 and induced the nucleocytoplasmic shuttling of hnRNPA1. Besides, we found that hnRNPA1 interacted with the viral phosphoprotein (P) via its glycine-rich domain, but not with the viral nucleoprotein (N) or large protein (L). The hnRNPA1-P interaction competitively disrupted the viral P-N interaction. Moreover, we found that overexpression of hnRNPA1 enhanced the polyubiquitination of the P protein and degraded it through proteasomal and lysosomal pathways. This study will help understanding the function of hnRNPA1 in the replication of single-stranded negative-sense RNA viruses and providing a novel antiviral target against fish rhabdoviruses.

Black carp RNF5 inhibits STING/IFN signaling through promoting K48-linked ubiquitination and degradation of STING.

Ubiquitination is one of the important post-translational modifications (PTMs) of proteins that plays a vital role in regulating substrate degradation to ensure cellular homeostasis. Ring finger protein 5 (RNF5) is an essential E3 ubiquitin ligase for inhibiting STING-mediated interferon (IFN) signaling in mammals. Nevertheless, the function of RNF5 in STING/IFN pathway remains obscure in teleost. Here, we reported that over-expression of black carp RNF5 (bcRNF5) inhibited STING-mediated transcription activity of bcIFNa, DrIFNφ1, NF-κB and ISRE promoters and antiviral activity against SVCV. Moreover, knockdown of bcRNF5 increased the expression of host genes, including bcIFNa, bcIFNb, bcILß, bcMX1 and bcViperin, and also enhanced the antiviral capability of host cells. Immunofluorescence (IF) and Co-immunoprecipitation (Co-IP) assay confirmed that bcRNF5 was mainly localized in the cytoplasm and interacted with bcSTING. The expression level of bcSTING protein was attenuated by co-expressed bcRNF5 and MG132 treatment rescued this attenuating effect, suggesting that bcRNF5-mediated bcSTING degradation was dependent on the proteasome pathway. Subsequent, Co-IP and immunoblot (IB) experiments identified that bcRNF5 triggered the K48-linked but not K63-linked ubiquitination of bcSTING. Altogether, above results conclude that RNF5 suppresses STING/IFN signaling by enhancing K48-linked ubiquitination and protease degradation of STING in black carp.

DDX3X Is Hijacked by Snakehead Vesiculovirus Phosphoprotein To Facilitate Virus Replication via Stabilization of the Phosphoprotein.

Asp-Glu-Ala-Asp (DEAD) box helicase 3 X-linked (DDX3X) plays important regulatory roles in the replication of many viruses. However, the role of DDX3X in rhabdovirus replication has seldomly been investigated. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was used to study the role of DDX3X in rhabdovirus replication. DDX3X was identified as an interacting partner of SHVV phosphoprotein (P). The expression level of DDX3X was increased at an early stage of SHVV infection and then decreased to a normal level at a later infection stage. Overexpression of DDX3X promoted, while knockdown of DDX3X using specific small interfering RNAs (siRNAs) suppressed, SHVV replication, indicating that DDX3X was a proviral factor for SHVV replication. The N-terminal and core domains of DDX3X (DDX3X-N and DDX3X-Core) were determined to be the regions responsible for its interaction with SHVV P. Overexpression of DDX3X-Core suppressed SHVV replication by competitively disrupting the interaction between full-length DDX3X and SHVV P, suggesting that full-length DDX3X-P interaction was required for SHVV replication. Mechanistically, DDX3X-mediated promotion of SHVV replication was due not to inhibition of interferon expression but to maintenance of the stability of SHVV P to avoid autophagy-lysosome-dependent degradation. Collectively, our data suggest that DDX3X is hijacked by SHVV P to ensure effective replication of SHVV, which suggests an important anti-SHVV target. This study will help elucidate the role of DDX3X in regulating the replication of rhabdoviruses. IMPORTANCE Growing evidence has suggested that DDX3X plays important roles in virus replication. In one respect, DDX3X inhibits the replication of viruses, including hepatitis B virus, influenza A virus, Newcastle disease virus, duck Tembusu virus, and red-spotted grouper nervous necrosis virus. In another respect, DDX3X is required for the replication of viruses, including hepatitis C virus, Japanese encephalitis virus, West Nile virus, murine norovirus, herpes simplex virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because DDX3X has rarely been investigated in rhabdovirus replication, this study aimed at investigating the role of DDX3X in rhabdovirus replication by using the fish rhabdovirus SHVV as a model. We found that DDX3X was required for SHVV replication, with the mechanism that DDX3X interacts with and maintains the stability of SHVV phosphoprotein. Our data provide novel insights into the role of DDX3X in virus replication and will facilitate the design of antiviral drugs against rhabdovirus infection.

Negatively regulation of MAVS-mediated antiviral innate immune response by E3 ligase RNF5 in black carp.

Mitochondrial antiviral signaling protein (MAVS) is as an adaptor in RIG-I like receptor (RLR) signaling, which plays the key role in interferon (IFN) production during host antiviral innate immune activation. MAVS is fine tuned to avoid excess IFN production, which have been extensively studied in human and mammals. However, the regulation of MAVS in teleost still remains obscure. In this manuscript, we cloned ring finger protein 5 (bcRNF5) of black carp (Mylopharyngodon piceus) and characterized this teleost E3 ubiquitin ligase as a negative regulator of MAVS. The coding region of bcRNF5 consists of 615 nucleotides which encode 205 amino acids, containing two trans-membrane domain (TM) and a ring-finger domain (RING). The transcription regulation of bcRNF5 varies in host cells in response to stimulations of LPS, poly (I:C), grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). bcRNF5 migrates around 22 KDa in immunoblot (IB) assay and distributes mainly in cytoplasm by immunofluorescent (IF) staining test. Moreover, bcRNF5 significantly inhibits bcMAVS-mediated IFN promoter transcription. In addition, both IF and co-immunoprecipitation assay showed that bcRNF5 interacts with bcMAVS. Furthermore, bcMAVS-mediated antiviral ability is distinctly impaired by bcRNF5. Taken together, these results conclude that bcRNF5, as a negative regulator of the MAVS-mediated IFN signaling, may play a key role in host protection upon virus infection in black carp.

MicroRNA miR-722 Inhibits Cyprinid Herpesvirus 3 Replication via Targeting the Viral Immune Evasion Protein ORF89, Which Negatively Regulates IFN by Degrading IRF3.

Cyprinid herpesvirus 3 (CyHV-3) has caused severe economic losses to carp culture, but its pathogenicity is far from clear. Our previous study has revealed that microRNA (miR)-722 was upregulated during CyHV-3 infection, indicating that miR-722 might play an important role in CyHV-3 replication. In this study, we found that overexpression of miR-722 inhibited CyHV-3 replication and promoted IFN expression. The putative target gene of miR-722 was searched over the CyHV-3 genome, and ORF89 was identified and validated as a target gene of miR-722. Overexpression of ORF89 markedly reduced the expression of IFN and IFN-stimulated genes. Mechanistically, ORF89 interacted with and degraded IFN regulatory factor 3 (IRF3), and inhibited the entry of IRF3 into the nucleus by suppressing the dimerization of IRF3. Moreover, ORF89-mediated suppression of IFN expression could be restored by adding miR-722. To our knowledge, our findings confirm a novel virus-host combat, in which CyHV-3 evades host antiviral immunity by its ORF89 protein, whereas host miR-722, upregulated on CyHV-3 infection, targets ORF89 to impede CyHV-3 replication.

Hsp90 Regulates GCRV-II Proliferation by Interacting with VP35 as Its Receptor and Chaperone.

The frequent outbreak of grass carp hemorrhagic disease caused by grass carp reovirus (GCRV), especially the mainly prevalent type II GCRV (GCRV-II), has seriously affected the grass carp culture in China. However, its pathogenic mechanism is still far from clear. In this study, the GCRV-II outer capsid protein VP35 was used as bait to capture interacting partners from Ctenopharyngon idellus kidney (CIK) cells, and heat shock protein 90 (Hsp90) was selected and confirmed interacting with VP35 through the C-terminal domain of Hsp90. Knockdown of Hsp90 or inhibition of Hsp90 activity suppressed GCRV-II proliferation, demonstrating that Hsp90 is an essential factor for GCRV-II proliferation. The confocal microscopy and flow cytometry showed that Hsp90 localized at both membrane and cytoplasm of CIK cells. The entry of GCRV-II into CIK cells was efficiently blocked by incubating the cells with Hsp90 antibody or by pretreating the virus with recombinant Hsp90 protein. Whereas overexpression of Hsp90 in CIK cells, grass carp ovary (GCO) cells, or 293T cells promoted GCRV-II entry, indicating that the membrane Hsp90 functions as a receptor of GCRV-II. Furthermore, Hsp90 interacted with clathrin and mediated GCRV-II entry into CIK cells through clathrin endocytosis pathway. In addition, we found that the cytoplasmic Hsp90 acted as a chaperone of VP35 because inhibition of Hsp90 activity enhanced VP35 polyubiquitination and degraded VP35 through the proteasome pathway. Collectively, our data suggest that Hsp90 functions both as a receptor for GCRV-II entry and a chaperone for the maturation of GCRV-II VP35, thus ensuring efficient proliferation of GCRV-II. IMPORTANCE Identification of viral receptors has always been the research hot spot in virus research field as receptor functions at the first stage of viral infection, which can be designed as efficient antiviral drug targets. GCRV-II, the causative agent of the grass carp epidemic hemorrhagic disease, has caused tremendous losses in grass carp culture in China. To date, the receptor of GCRV-II remains unknown. This study focused on identifying cellular receptor interacting with the GCRV-II outer capsid protein VP35, studying the effects of their interaction on GCRV-II proliferation, and revealing the underlying mechanisms. We demonstrated that Hsp90 acts both as a receptor of GCRV-II by interacting with VP35 and as a chaperone for the maturation of VP35, thus ensuring efficient proliferation of GCRV-II. Our data provide important insights into the role of Hsp90 in GCRV-II life cycle, which will help understand the mechanism of reovirus infection.

Construction of Attenuated Strains for Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) via Reverse Genetic System.

The nervous necrosis virus (NNV) mainly attacks the central nervous system of fish to cause viral nervous necrosis, which is an acute and serious prevalent disease in fish. Among different genotypes of NNV, red-spotted grouper nervous necrosis virus (RGNNV) is the most widely reported, with the highest number of susceptible species. To better understand the pathogenicity of RGNNV, we first developed a reverse genetic system for recombinant RGNNV rescue using B7GG and striped snakehead (SSN-1) cells. Furthermore, we constructed attenuated RGNNV strains rRGNNV-B2-M1 and rRGNNV-B2-M2 with the loss of B2 protein expression, which grew slower and induced less Mx1 expression than that of wild-type RGNNV. Moreover, rRGNNV-B2-M1 and rRGNNV-B2-M2 were less virulent than the wild-type RGNNV. Our study provides a potential tool for further research on the viral protein function, virulence pathogenesis, and vaccine development of RGNNV, which is also a template for the rescue of other fish viruses.

MicroRNA miR-155 inhibits cyprinid herpesvirus 3 replication via regulating AMPK-MAVS-IFN axis.

Since emerged in the late 1990s, cyprinid herpesvirus 3 (CyHV-3) has caused huge economic losses in common and koi carp culture worldwide. Accumulating evidences suggest that teleost fish microRNA (miRNA), a class of non-coding RNA of ∼22 nucleotides, can participate in many cellular processes, especially in host antiviral defenses. However, the roles of miRNAs in CyHV-3 infection are still unclear. Here, using high-throughput miRNA sequencing and quantitative real-time PCR (qRT-PCR) verification, we found that miR-155 was significantly upregulated in common carp brain (CCB) cells upon CyHV-3 infection. Overexpression of miR-155 effectively inhibited CyHV-3 replication in CCB cells and promoted type I interferon (IFN-I) expression. Further study revealed that miR-155 targeted the 3' untranslated region (UTR) of the mRNA of 5'AMP-activated protein kinase (AMPK), and that AMPK could interact with and degrade the mitochondrial antiviral signaling protein (MAVS), resulting in the reduction of interferon (IFN) expression. Collectively, our results show that miR-155, induced by CyHV-3 infection, exhibits anti-CyHV-3 activity via regulating AMPK-MAVS-IFN axis, which will help design anti-CyHV-3 drugs.

Hsp90 Is Required for Snakehead Vesiculovirus Replication via Stabilization of the Viral L Protein.

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction caused by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction, and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensures the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help us to understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. IMPORTANCE It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help us to understand the polymerase function of negative-stranded RNA viruses.

Leader RNA regulates snakehead vesiculovirus replication via interacting with viral nucleoprotein.

Leader RNA, a kind of virus-derived small noncoding RNA, has been proposed to play an important role in regulating virus replication, but the underlying mechanism remains elusive. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus causing high mortality to the cultured snakehead fish in China, was used to unveil the molecular function of leader RNA. High-throughput small RNA sequencing of SHVV-infected cells showed that SHVV produced two groups of leader RNAs (named legroup1 and legroup2) during infection. Overexpression and knockout experiments reveal that legroup1, but not legroup2, affects SHVV replication. Mechanistically, legroup1-mediated regulation of SHVV replication was associated with its interaction with the viral nucleoprotein (N). Moreover, the nucleotides 6-10 of legroup1 were identified as the critical region for its interaction with the N protein, and the amino acids 1-45 of N protein were proved to confer its interaction with the legroup1. Taken together, we identified two groups of SHVV leader RNAs and revealed a role in virus replication for one of the two types of leader RNAs. This study will help understand the role of leader RNA in regulating the replication of negative-stranded RNA viruses.

Immunogenicity study of OmpU subunit vaccine against Vibrio mimicus in yellow catfish, Pelteobagrus fulvidraco.

The outer membrane protein U (OmpU) is a conserved outer membrane protein in a variety of pathogenic Vibrio species and has been considered as a vital protective antigen for vaccine development. Vibrio mimicus (V. mimicus) is the pathogen causing ascites disease in aquatic animals. In this study, the prokaryotically expressed and purified His-tagged OmpU of V. mimicus (His-OmpU) was used as a subunit vaccine. The formalin inactivated V. mimicus, purified His tag (His-tag), and PBS were used as controls. The vaccinated yellow catfish were challenged with V. mimicus at 28 days post-vaccination, and the results showed that the His-OmpU and inactivated V. mimicus groups exhibited much higher survival rates than the His-tag and PBS groups. To fully understand the underlying mechanism, we detected the expression levels of several immune-related genes in the spleen of fish at 28 days post-vaccination and 24 h post-challenge. The results showed that most of the detected immune-related genes were significantly upregulated in His-OmpU and inactivated V. mimicus groups. In addition, we performed the serum bactericidal activity assay, and the results showed that the serum from His-OmpU and inactivated V. mimicus groups exhibited much stronger bactericidal activity against V. mimicus than those of His-tag and PBS groups. Finally, the serum agglutination antibody was detected, and the antibody could be detected in His-OmpU and inactivated V. mimicus groups with the antibody titers increasing along with the time post-vaccination, but not in His-tag or PBS group. Our data reveal that the recombinant OmpU elicits potent protective immune response and is an effective vaccine candidate against V. mimicus in yellow catfish.

Structural and Functional Characterization of the Phosphoprotein Central Domain of Spring Viremia of Carp Virus.

Spring viremia of carp virus (SVCV) is a highly pathogenic Vesiculovirus in the common carp. The phosphoprotein (P protein) of SVCV is a multifunctional protein that acts as a polymerase cofactor and an antagonist of cellular interferon (IFN) response. Here, we report the 1.5-Å-resolution crystal structure of the P protein central domain (PCD) of SVCV (SVCVPCD). The PCD monomer consists of two ß sheets, an α helix, and another two ß sheets. Two PCD monomers pack together through their hydrophobic surfaces to form a dimer. The mutations of residues on the hydrophobic surfaces of PCD disrupt the dimer formation to different degrees and affect the expression of host IFN consistently. Therefore, the oligomeric state formation of the P protein of SVCV is an important mechanism to negatively regulate host IFN response.IMPORTANCE SVCV can cause spring viremia of carp with up to 90% lethality, and it is the homologous virus of the notorious vesicular stomatitis virus (VSV). There are currently no drugs that effectively cure this disease. P proteins of negative-strand RNA viruses (NSVs) play an essential role in many steps during the replication cycle and an additional role in immunosuppression as a cofactor. All P proteins of NSVs are oligomeric, but the studies on the role of this oligomerization mainly focus on the process of virus transcription or replication, and there are few studies on the role of PCD in immunosuppression. Here, we present the crystal structure of SVCVPCD A new mechanism of immune evasion is clarified by exploring the relationship between SVCVPCD and host IFN response from a structural biology point of view. These findings may provide more accurate target sites for drug design against SVCV and provide new insights into the function of NSVPCD.

Bacillus velezensis LG37: transcriptome profiling and functional verification of GlnK and MnrA in ammonia assimilation.

BACKGROUND: In recent years, interest in Bacillus velezensis has increased significantly due to its role in many industrial water bioremediation processes. In this study, we isolated and assessed the transcriptome of Bacillus velezensis LG37 (from an aquaculture pond) under different nitrogen sources. Since Bacillus species exhibit heterogeneity, it is worth investigating the molecular mechanism of LG37 through ammonia nitrogen assimilation, where nitrogen in the form of molecular ammonia is considered toxic to aquatic organisms. RESULTS: Here, a total of 812 differentially expressed genes (DEGs) from the transcriptomic sequencing of LG37 grown in minimal medium supplemented with ammonia (treatment) or glutamine (control) were obtained, from which 56 had Fold Change ≥2. BLAST-NCBI and UniProt databases revealed 27 out of the 56 DEGs were potentially involved in NH4+ assimilation. Among them, 8 DEGs together with the two-component regulatory system GlnK/GlnL were randomly selected for validation by quantitative real-time RT-PCR, and the results showed that expression of all the 8 DEGs are consistent with the RNA-seq data. Moreover, the transcriptome and relative expression analysis were consistent with the transporter gene amtB and it is not involved in ammonia transport, even in the highest ammonia concentrations. Besides, CRISPR-Cas9 knockout and overexpression glnK mutants further evidenced the exclusion of amtB regulation, suggesting the involvement of alternative transporter. Additionally, in the transcriptomic data, a novel ammonium transporter mnrA was expressed significantly in increased ammonia concentrations. Subsequently, OEmnrA and ΔmnrA LG37 strains showed unique expression pattern of specific genes compared to that of wild-LG37 strain. CONCLUSION: Based on the transcriptome data, regulation of nitrogen related genes was determined in the newly isolated LG37 strain to analyse the key regulating factors during ammonia assimilation. Using genomics tools, the novel MnrA transporter of LG37 became apparent in ammonia transport instead of AmtB, which transports ammonium nitrogen in other Bacillus strains. Collectively, this study defines heterogeneity of B. velezensis LG37 through comprehensive transcriptome analysis and subsequently, by genome editing techniques, sheds light on the enigmatic mechanisms controlling the functional genes under different nitrogen sources also reveals the need for further research.

Inhibition of Cyclophilin A on the replication of red spotted grouper nervous necrosis virus associates with multiple pro-inflammatory factors.

Cyclophilin A (CypA) is a ubiquitously expressed cellular protein and involves in diverse pathological conditions, including infection and inflammation. CypA acts as a key factor in the replication of several viruses. However, little is known about the role of CypA in the replication of the red-spotted grouper nervous necrosis virus (RGNNV). In the present report, grouper CypA (GF-CypA) was cloned from the grouper fin cell line (GF-1) derived from orange-spotted grouper (Epinephelus coioides). Sequence analysis found that GF-CypA open reading frame (ORF) of 495 bp encodes a polypeptide of 164 amino acids residues with a molecular weight of 17.4 kDa. The deduced amino acid sequence shared highly conserved regions with CypA of other animal species, showing that GF-CypA is a new member of Cyclophilin A family. We observed that GF-CypA was up-regulated in the GF-1 cells infected with RGNNV. Additionally, overexpression of CypA could significantly inhibit the replication of RGNNV in GF-1 cells. By contrast, when the GF-CypA was knock-downed by siRNA in GF-1 cells, the replication of RGNNV was enhanced. Furthermore, the expressions of pro-inflammatory factors, such as TNF-2, TNF-α, IL-1b, and ISG-15, were increased in GF-CypA transfected GF-1 cells challenged with RGNNV, indicating that GF-CypA might be involved in the regulation of the host pro-inflammatory factors. Altogether, we conclude that GF-CypA plays a vital role in the inhibitory effect of RGNNV replication that might be modulating the cytokines secretion in GF-1 cells during RGNNV infection. These results will shed new light on the function of CypA in the replication of RGNNV and will pave a new way for the prevention of the infection of RGNNV in fish.

Structural dynamics of bacteriophage P22 infection initiation revealed by cryo-electron tomography.

For successful infection, bacteriophages must overcome multiple barriers to transport their genome and proteins across the bacterial cell envelope. We use cryo-electron tomography to study the infection initiation of phage P22 in Salmonella enterica serovar Typhimurium, revealing how a channel forms to allow genome translocation into the cytoplasm. Our results show free phages that initially attach obliquely to the cell through interactions between the O antigen and two of the six tailspikes; the tail needle also abuts the cell surface. The virion then orients perpendicularly and the needle penetrates the outer membrane. The needle is released and the internal head protein gp7* is ejected and assembles into an extracellular channel that extends from the gp10 baseplate to the cell surface. A second protein, gp20, is ejected and assembles into a structure that extends the extracellular channel across the outer membrane into the periplasm. Insertion of the third ejected protein, gp16, into the cytoplasmic membrane probably completes the overall trans-envelope channel into the cytoplasm. Construction of a trans-envelope channel is an essential step during infection of Gram-negative bacteria by all short-tailed phages, because such virions cannot directly deliver their genome into the cell cytoplasm.