SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.

Download Full-text

A Possible Role of Allatostatin C in Inhibiting Ecdysone Biosynthesis Revealed in the Mud Crab Scylla paramamosain

Frontiers in Marine Science ◽

10.3389/fmars.2021.740251 ◽

2021 ◽

Vol 8 ◽

Author(s):

An Liu ◽

Wenyuan Shi ◽

Dongdong Lin ◽

Haihui Ye

Keyword(s):

Scylla Paramamosain ◽

Dependent Manner ◽

Mud Crab ◽

Methyl Farnesoate ◽

Independent Manner ◽

Wide Range ◽

Negative Effect

C-type allatostatins (C-type ASTs) are a family of structurally related neuropeptides found in a wide range of insects and crustaceans. To date, the C-type allatostatin receptor in crustaceans has not been deorphaned, and little is known about its physiological functions. In this study, we aimed to functionally define a C-type ASTs receptor in the mud crab, Scylla paramamosian. We showed that C-type ASTs receptor can be activated by ScypaAST-C peptide in a dose-independent manner and by ScypaAST-CCC peptide in a dose-dependent manner with an IC50 value of 6.683 nM. Subsequently, in vivo and in vitro experiments were performed to investigate the potential roles of ScypaAST-C and ScypaAST-CCC peptides in the regulation of ecdysone (20E) and methyl farnesoate (MF) biosynthesis. The results indicated that ScypaAST-C inhibited biosynthesis of 20E in the Y-organ, whereas ScypaAST-CCC had no effect on the production of 20E. In addition, qRT-PCR showed that both ScypaAST-C and ScypaAST-CCC significantly decreased the level of expression of the MF biosynthetic enzyme gene in the mandibular organ, suggesting that the two neuropeptides have a negative effect on the MF biosynthesis in mandibular organs. In conclusion, this study provided new insight into the physiological roles of AST-C in inhibiting ecdysone biosynthesis. Furthermore, it was revealed that AST-C family peptides might inhibit MF biosynthesis in crustaceans.

Download Full-text

Relative contribution of non-structural protein 1 in dengue pathogenesis

10.1101/2020.02.01.929885 ◽

2020 ◽

Author(s):

Pei Xuan Lee ◽

Donald Heng Rong Ting ◽

Clement Peng Hee Boey ◽

Eunice Tze Xin Tan ◽

Janice Zuo Hui Chia ◽

...

Keyword(s):

Critical Role ◽

Health Concern ◽

Structural Protein ◽

Promising Candidate ◽

Relative Contribution ◽

Structural Region ◽

Strain Dependent ◽

Pro Inflammatory Cytokines

AbstractDengue is a major public health concern in the tropical and sub-tropical world with no effective treatment. The controversial live attenuated virus vaccine Dengvaxia has boosted the pursuit of sub-unit vaccine approaches, and the non-structural protein 1 (NS1) has recently emerged as a promising candidate. However, we found that NS1 immunization or passive transfer of NS1 antibodies failed to confer protection in symptomatic dengue mouse models using two non mouse-adapted DENV2 strains from the Cosmopolitan genotype that currently circulates in South-East Asia. Furthermore, exogenous administration of purified NS1 did not worsen in vivo vascular leakage in sub-lethally infected mice, thereby supporting that NS1 does not play a critical role in the pathogenesis of these DENV2 strains. Virus chimerization approaches indicated that the prME structural region, but not NS1, plays a critical role in driving in vivo fitness and virulence of the virus, through induction of key pro-inflammatory cytokines. This work highlights that the pathogenic role of NS1 is DENV strain-dependent, which warrants re-evaluation of NS1 as a universal dengue vaccine candidate.

Download Full-text

A Critical Role for BMP Signaling in Adult Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v120.21.1194.1194 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1194-1194

Author(s):

Ulrika Blank ◽

Sarah Warsi ◽

Silja Andradottir ◽

Emma Rörby ◽

Stefan Karlsson

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Critical Role ◽

Bmp Signaling ◽

Type Ii ◽

Post Transplantation ◽

Regenerative Capacity ◽

Hematopoietic Stem ◽

Donor Population

Abstract Abstract 1194 The Bone Morphogenetic Proteins (BMPs), which belong to the TGF-beta superfamily of ligands, figure prominently during development and are involved in a wide variety of biological processes throughout life. BMP ligands signal via Type I and Type II receptors, both of which are required at the cell surface for propagation of the signal intra-cellularly. Upon receptor activation, both the Smad1/5/8 pathway and the Tak1 MAPK circuitry can be activated, ultimately leading to transcriptional regulation of target genes (Blank et al., Development 2009). Although the BMP pathway plays a role during embryonic development of hematopoiesis, its role in adult hematopoiesis has remained elusive. Previous studies of the Smad1/5/8 pathway have indicated that this pathway is not involved in regulation of adult hematopoietic stem cells (HSCs) in vivo. However, previously published findings demonstrate that the BMP Type II receptor (BmprII) is highly expressed in HSCs, suggesting that BMPs may still play a role in adult HSC regulation via Smad-independent mechanisms. To fully elucidate the role of BMP signaling in hematopoietic cells, we utilized a conditional knockout mouse model targeted to the BmprII gene by Vav-Cre-mediated deletion. Steady state hematopoiesis was essentially normal in BmprII knockouts, but the more primitive LSK population in the bone marrow (BM) was significantly reduced in knockouts compared to littermate controls at 16 weeks of age (0.107% of BM vs. 0.133%, p≤0.05, n=8–10). This reduction in primitive cells translated functionally into a reduced colony forming capacity in vitro (86 colonies/90 000 cells plated vs. 112/90 000 cells plated for controls, p≤0.05, n=8–10). Additionally, when hematopoietic cells were challenged in vivo by transplanting 0.2×10e6 knockout or littermate control whole BM cells in a competitive fashion with 0×10e6 wild type whole BM cells into lethally irradiated recipient mice, the regenerative capacity of BmprII knockout cells was significantly reduced both short term in peripheral blood, at 4 weeks post transplantation (36.5% vs. 48.6% donor-derived cells, p≤0.05, n=7 donors per genotype), and long term in the BM at 16 weeks post transplantation (40.9% vs. 63.4% donor-derived cells, p≤0.05, n=7 donors per genotype). Furthermore, we found a reduction in the myeloid compartment in the BM of BmprII donor recipients at 16 weeks post transplantation (40.3% vs. 64.5% Gr1+/Mac1+ cells of the donor population, p≤0.05, n=7 donors per genotype) coupled with an increase in B-lymphoid cells (46.7% vs. 26.3% B220+ cells of the donor population, p≤0.05, n=7 donors per genotype). To quantify more primitive cells, LSK SLAM FACS analysis was performed, revealing a significant decrease in the numbers of LSK cells (3508 cells vs. 12022 cells per femur, p≤0.05, n=7 donors per genotype), as well as LSK SLAM cells (542 vs. 3023 cells per femur, p≤0.05) derived from BmprII donors. Our studies indicate that the BMP circuitry plays a critical role in HSC regulation and that inactivation of this pathway at the receptor level results in a reduced regenerative capacity in vivo. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

An MHV macrodomain mutant predicted to lack ADP-ribose binding activity is severely attenuated, indicating multiple roles for the macrodomain in coronavirus replication

10.1101/2021.03.30.437796 ◽

2021 ◽

Author(s):

Lynden S Voth ◽

Joseph J O'Connor ◽

Catherine M Kerr ◽

Ethan Doerger ◽

Nancy Schwarting ◽

...

Keyword(s):

Hepatitis Virus ◽

Single Point ◽

Binding Activity ◽

Multiple Roles ◽

Structural Protein ◽

Transcript Accumulation ◽

Murine Hepatitis Virus ◽

Point Mutant

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes ADP-ribose covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutant of a highly conserved asparagine-to-alanine mutation, which is known to largely eliminate Mac1 ADP-ribosylhydrolase activity. To determine if Mac1 ADP-ribose binding separately contributes to CoV replication, we compared the replication of a murine hepatitis virus (MHV) Mac1 mutant predicted to dramatically reduce ADP-ribose binding, D1329A, to the previously mentioned asparagine mutant, N1347A. D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. However, D1329A was significantly more attenuated than N1347A in all cell lines tested that were susceptible to MHV infection. In addition, D1329A retained some ability to block IFN-β transcript accumulation compared to N1347A, indicating that these two mutants impacted distinct Mac1 functions. Mac1 mutants predicted to eliminate both binding and hydrolysis activities were unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple roles in promoting the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics.

Download Full-text

ESL-1 Is a Major Physiological Leukocyte Ligand for E-Selectin That Cooperates with PSGL-1 and CD44, and Together Mediate All Binding Activity to Endothelial Selectins In Vivo.

Blood ◽

10.1182/blood.v108.11.1787.1787 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1787-1787

Author(s):

Andres Hidalgo ◽

Anna J. Peired ◽

Paul S. Frenette

Keyword(s):

Affinity Purification ◽

Critical Role ◽

Binding Activity ◽

Wild Type ◽

Selectin Ligands ◽

Tnf Α ◽

Polarized Cells ◽

Selectin Ligand ◽

Soluble E Selectin

Abstract Endothelial selectins mediate leukocyte (WBC) rolling on activated endothelium. ESL-1 was isolated by affinity purification more than 10 years ago but whether it is a physiological ligand for E-selectin remains unclear. Recent studies suggest that PSGL-1 and CD44 are physiological E-selectin ligands on neutrophils (PMNs)(J. Exp. Med. 2005 201:1183). To analyze the contribution of ESL-1, PSGL-1 and CD44, we knocked down ESL-1 expression using lentiviral transfer of short hairpin (sh) RNA into wild-type (WT), PSGL-1- or/and CD44-deficient bone marrow lineage-negative (Lin-) cells. Sorted GFP+ cells from mice transplanted with Lin- cells transduced with the shESL-1 exhibited >90% reduction in total ESL-1 levels by Q-PCR and Western blot analyses, and complete absence of ESL-1 on the PMN surface. shESL-1 transduced Lin- cells engrafted recipients mice with the same efficiency as control vector. Downregulation of ESL-1 slightly affected (~33%) the binding of soluble E-selectin to PMNs, but did not alter WBC rolling numbers in TNF-α-stimulated cremasteric venules. Absence of ESL-1 or CD44, but not PSGL-1, resulted in significant increases in mean rolling velocities (4.7 ± 0.4 μm/s for WT; 8.3 ± 0.4 μm/s for shESL-1*; 7.2 ± 0.2 μm/s for CD44−/−* and 5.4 ± 0.4 μm/s for PSGL-1−/−; *p<0.001). Moreover, WBCs lacking ESL-1 exhibited a characteristic “skipping” rolling motion, suggesting that ESL-1 plays a critical role in stabilizing WBC rolling. Remarkably, ESL-1 downregulation in the absence of PSGL-1 led to dramatic reductions in WBC binding to soluble E-selectin (~99%), and rolling (~93%) on TNF-α-stimulated venules. To determine the contribution of these 3 glycoproteins in WBC recruitment, mice transplanted with WT, PSGL-1- and/or CD44-deficient Lin- cells that were transduced with the shESL-1 or control vectors, were injected i.p. with thioglycollate and the number of peritoneal GFP+ PMNs determined. Only the absence of all 3 glycoproteins compromised PMN recruitment to the levels observed in P- and E-selectin double-deficient mice (82% reduction compared to WT; p<0.001), suggesting that ESL-1, PSGL-1 and CD44 comprise all endothelial selectin ligand activity on PMNs in vivo. To assess the role of E-selectin ligands in WBC activation, we monitored in vivo L-selectin clustering on the surface of over 4,000 rolling WBCs using high-speed multichannel fluorescence videomicroscopy. The majority of wild-type rolling WBCs exhibited polarization of L-selectin 150 to 230 min after TNF-α. L-selectin clustering was greatly reduced in E-selectin−/− mice (58 ± 4% polarized cells in WT vs 23.5 ± 1% in E-selectin−/− mice; p<0.001). To determine which E-selectin ligand(s) mediated L-selectin clustering in vivo, we evaluated L-selectin distribution on rolling WBCs deficient in ESL-1, CD44 or PSGL-1. Absence of CD44, but not ESL-1 or PSGL-1, reduced L-selectin redistribution to levels found in E-selectin−/− mice (20 ± 2% polarized cells), indicating that CD44 is the signalling E-selectin ligand required for L-selectin clustering. In addition, E-selectin-mediated CD44 signalling was p38-dependent, as assessed in WT mice treated with the p38 inhibitor SB203580 (77% reduction; p<0.001). Our results indicate that ESL-1, PSGL-1 and CD44 comprise all selectin ligand binding activity on WBCs, and suggest specialized contributions for each ligand where PSGL-1 mediates the initial tethering, ESL-1 is critical for the conversion into steady slow rolling, and CD44 is required for E-selectin-mediated signals contributing to WBC activation.

Download Full-text

Embryonic Lethality, Decreased Erythropoiesis, and Defective Octamer-Dependent Promoter Activation in Oct-1-Deficient Mice

Molecular and Cellular Biology ◽

10.1128/mcb.24.3.1022-1032.2004 ◽

2004 ◽

Vol 24 (3) ◽

pp. 1022-1032 ◽

Cited By ~ 53

Author(s):

Victoria E. H. Wang ◽

Tara Schmidt ◽

Jianzhu Chen ◽

Phillip A. Sharp ◽

Dean Tantin

Keyword(s):

Dna Binding ◽

Gene Dosage ◽

Critical Role ◽

Low Frequency ◽

Binding Activity ◽

Gene Dosage Effect ◽

Erythroid Precursor ◽

Octamer Motif

ABSTRACT Oct-1 is a sequence-specific DNA binding transcription factor that is believed to regulate a large group of tissue-specific and ubiquitous genes. Both Oct-1 and the related but tissue-restricted Oct-2 protein bind to a DNA sequence termed the octamer motif (5′-ATGCAAAT-3′) with equal affinity in vitro. To address the role of Oct-1 in vivo, an Oct-1-deficient mouse strain was generated by gene targeting. Oct-1-deficient embryos died during gestation, frequently appeared anemic, and suffered from a lack of Ter-119-positive erythroid precursor cells. This defect was cell intrinsic. Fibroblasts derived from these embryos displayed a dramatic decrease in Oct-1 DNA binding activity and a lack of octamer-dependent promoter activity in transient transfection assays. Interestingly, several endogenous genes thought to be regulated by Oct-1 showed no change in expression. When crossed to Oct-2 +/− animals, transheterozygotes were recovered at a very low frequency. These findings suggest a critical role for Oct-1 during development and a stringent gene dosage effect with Oct-2 in mediating postnatal survival.

Download Full-text

A Determinant of Sindbis Virus Neurovirulence Enables Efficient Disruption of Jak/STAT Signaling

Journal of Virology ◽

10.1128/jvi.00577-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 11429-11439 ◽

Cited By ~ 35

Author(s):

Jason D. Simmons ◽

Amy C. Wollish ◽

Mark T. Heise

Keyword(s):

Cellular Response ◽

Sindbis Virus ◽

Critical Role ◽

Single Amino Acid ◽

Type I ◽

Type Ii ◽

Virulence Potential ◽

Stat Signaling ◽

Infected Cells

ABSTRACT Previous studies with Venezuelan equine encephalitis virus and Sindbis virus (SINV) indicate that alphaviruses are capable of suppressing the cellular response to type I and type II interferons (IFNs) by disrupting Jak/STAT signaling; however, the relevance of this signaling inhibition toward pathogenesis has not been investigated. The relative abilities of neurovirulent and nonneurovirulent SINV strains to downregulate Jak/STAT signaling were compared to determine whether the ability to inhibit IFN signaling correlates with virulence potential. The adult mouse neurovirulent strain AR86 was found to rapidly and robustly inhibit tyrosine phosphorylation of STAT1 and STAT2 in response to IFN-γ and/or IFN-β. In contrast, the closely related SINV strains Girdwood and TR339, which do not cause detectable disease in adult mice, were relatively inefficient inhibitors of STAT1/2 activation. Decreased STAT activation in AR86-infected cells was associated with decreased activation of the IFN receptor-associated tyrosine kinases Tyk2, Jak1, and Jak2. To identify the viral factor(s) involved, we infected cells with several panels of AR86/Girdwood chimeric viruses. Surprisingly, we found that a single amino acid determinant, threonine at nsP1 position 538, which is required for AR86 virulence, was also required for efficient disruption of STAT1 activation, and this determinant fully restored STAT1 inhibition when it was introduced into the avirulent Girdwood background. These data indicate that a key virulence determinant plays a critical role in downregulating the response to type I and type II IFNs, which suggests that the ability of alphaviruses to inhibit Jak/STAT signaling relates to their in vivo virulence potential.

Download Full-text

Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5221 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5221-5231 ◽

Cited By ~ 295

Author(s):

K Gradin ◽

J McGuire ◽

R H Wenger ◽

I Kvietikova ◽

M L fhitelaw ◽

...

Keyword(s):

Dna Binding ◽

Critical Role ◽

Hypoxia Inducible Factor ◽

Binding Activity ◽

Receptor Function ◽

Expression Levels ◽

Dioxin Receptor ◽

Alpha Function

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.

Download Full-text

Ecdysone receptor in the mud crab Scylla paramamosain: a possible role in promoting ovarian development

Journal of Endocrinology ◽

10.1530/joe-14-0526 ◽

2015 ◽

Vol 224 (3) ◽

pp. 273-287 ◽

Cited By ~ 33

Author(s):

Jie Gong ◽

Haihui Ye ◽

Yinjie Xie ◽

Yanan Yang ◽

Huiyang Huang ◽

...

Keyword(s):

Ovarian Development ◽

Stage Iv ◽

Mature Stage ◽

Scylla Paramamosain ◽

Ecdysone Receptor ◽

Mud Crab ◽

Follicular Cells ◽

Vitellogenic Stage

In arthropods, it is known that ecdysteroids regulate molting, limb regeneration, and reproduction through activation of the ecdysone receptor (EcR). However, the ecdysteroid signaling pathway for promotion of ovarian development in crustaceans is still unclear. In this study, three cDNA isoforms of EcR were cloned from the mud crab Scylla paramamosain. qRT-PCR revealed that the SpEcR mRNA was abundant in the eyestalk, ovary and epidermis. During ovarian development, the SpEcR transcripts increased from stage I (undeveloped stage) and reached a peak at stage IV (late vitellogenic stage) before dropping to a lower level at stage V (mature stage). Meanwhile, levels of 20-hydroxyecdysone (20E) in the hemolymph, detected by HPLC-MS, displayed a similar pattern of increase with ovarian development. Results from in situ hybridization indicated that SpEcR mRNA was present in the follicular cells during vitellogenesis. Results from in vivo experiments revealed that 20E at 0.2 μg/g body weight significantly stimulated the expression of SpEcR and vitellogenin (SpVg) in female crabs during the early vitellogenic stage but not during the previtellogenic stage. This was confirmed by results from in vitro experiments which indicated that SpEcR and SpVg expression levels were significantly upregulated in early vitellogenic ovarian explants incubated with 5.0 μM 20E at 3 and 6 h but not in previtellogenic ovarian explants. Finally, results from in vitro gene silencing experiments indicated that the expression of SpEcR and SpVg in the ovary was significantly inhibited by SpEcR dsRNA. All these results together indicated that in S. paramamosain, 20E, and SpEcR, located in the follicular cells, play important roles in the promotion of ovarian development via regulating the expression of SpVg.

Download Full-text