scholarly journals Replication/Assembly Defective Avian Flavivirus With Internal Deletions in the Capsid Can Be Used as an Approach for Living Attenuated Vaccine

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu He ◽  
Xiaoli Wang ◽  
Jiaqi Guo ◽  
Li Mao ◽  
Senzhao Zhang ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Functional Requirements ◽  
Lethal Challenge ◽  
Live Vaccines ◽  
Large Deletions ◽  
C Terminus ◽  
Viral Virulence ◽  
Viral Morphogenesis ◽  
Shed Light

Avian Tembusu virus (TMUV) is a novel flavivirus causing severe egg drop and fatal encephalitis in avian in Asia. In the present study, we screened the structural and functional requirements of TMUV capsid protein (CP) for viral morphogenesis using reverse genetics methods in combination with replicon packaging assays. TMUV-CP showed dramatic functional and structural flexibility, and even though 44 residues were removed from the N-terminus, it was still capable of packaging replicon RNA; in addition, 33 residues were deleted from the C-terminus (containing nearly the entire α4-helix), and infectious particles were still produced, although α4-α4’ is supposedly vital for CP dimerization and nucleocapsid formation. We further analyzed two mutants (ΔC20-43 and ΔC64-96 viruses) with relatively large deletions that still replicated well in BHK-21 cells. Our data indicate that internal deletions within CP impaired viral replication or assembly, resulting in attenuated virus proliferation in cells and attenuated virulence in duck embryos, and these deletion mutations are quite stable in cell culture. An in vivo assay indicated that both ΔC20-43 virus and ΔC64-96 virus were highly attenuated in ducklings but still immunogenic. Single-dose immunization with ΔC20-43 virus or ΔC64-96 virus could protect ducklings from a lethal challenge with good antigen clearance. Together, our data shed light on replication/assembly defective TMUV with internal deletions in CP and provide an effective approach to attenuate viral virulence in live vaccines without changing the antigen composition.

scholarly journals Characterization of the Glycoprotein Stable Signal Peptide in Mediating Pichinde Virus Replication and Virulence

2016 ◽  
Vol 90 (22) ◽  
pp. 10390-10397 ◽  
Author(s):  
Junjie Shao ◽  
Xiaoying Liu ◽  
Hinh Ly ◽  
Yuying Liang
Keyword(s):  
Cell Culture ◽  
Membrane Fusion ◽  
Viral Entry ◽  
Reverse Genetics ◽  
Cell Entry ◽  
Pichinde Virus ◽  
Viral Virulence ◽  
Stable Signal

ABSTRACTArenaviruses can cause lethal hemorrhagic fevers in humans with few preventative and therapeutic measures. The arenaviral glycoprotein stable signal peptide (SSP) is unique among signal peptides in that it is an integral component of the mature glycoprotein complex (GPC) and plays important roles not only in GPC expression and processing but also in the membrane fusion process during viral entry. Using the Pichinde virus (PICV) reverse genetics system, we analyzed the effects of alanine substitutions at many conserved residues within the SSP on viral replication in cell culture and in a guinea pig infection model. Our data showed that the K33A, F49A, and C57A mutations abolished GPC-mediated cell entry and therefore could not allow for the generation of viable recombinant viruses, demonstrating that these residues are essential for the PICV life cycle. The G2A mutation caused a marked reduction of cell entry at the membrane fusion step, and while this mutant virus was viable, it was significantly attenuatedin vitroandin vivo. The N20A mutation also reduced membrane fusion activity and viral virulence in guinea pigs, but it did not significantly affect cell entry or viral growth in cell culture. Two other mutations (N37A and R55A) did not affect membrane fusion or viral growthin vitrobut significantly reduced viral virulencein vivo. Taken together, our data suggest that the GPC SSP plays an essential role in mediating viral entry and also contributes to viral virulencein vivo.IMPORTANCESeveral arenaviruses, such as Lassa fever virus, can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, and no FDA-approved vaccines or therapies are currently available. Viral entry into cells is mediated by arenavirus GPC that consists of an SSP, the receptor-binding GP1, and transmembrane GP2 protein subunits. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we have shown for the first time in the context of virus infections of cell culture and of guinea pigs that the SSP plays an essential role in mediating the membrane fusion step as well as in other yet-to-be-determined processes during viral infection. Our study provides important insights into the biological roles of GPC SSP and implicates it as a good target for the development of antivirals against deadly human arenavirus pathogens.

scholarly journals Lipidation of a bacterial effector is critical for bacterial evasion of host-defense

2020 ◽  
Author(s):  
Natalia Cattelan ◽  
Hongjiao Yu ◽  
Kornelia Przybyszewska ◽  
Rosa Angela Colamarino ◽  
Massimiliano Baldassarre ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Subcellular Fractionation ◽  
Effector Protein ◽  
Host Bacterium ◽  
Post Translational Modification ◽  
C Terminus ◽  
Mouse Macrophages ◽  
Caax Motif ◽  
Shed Light

AbstractThe Rab32 antimicrobial pathway has been shown to restrict Salmonella Typhi, in mouse macrophages. The broad-host pathogen Salmonella Typhimurium however has evolved a strategy to evade the Rab32 antimicrobial pathway, via its effector protein GtgE. GtgE is a cysteine protease that specifically mediates the cleavage and inactivation of Rab32. Here we show that GtgE association and targeting to membranes is critical for its efficient proteolytic activity. The C-terminus of GtgE contains a CaaX motif, which can be post-translationally modified by the host’s prenylation machinery. Using a combination of confocal microscopy and subcellular fractionation we show that a cysteine in the CaaX motif is crucial for GtgE membrane targeting and, more importantly, GtgE localization to the Salmonella-containing vacuole. We also demonstrated that prenylation of CaaX is important for an effective and fast Rab32 cleavage, which in turn helps Salmonella to successfully survive in macrophages and establish an in vivo infection in mice. Our findings shed light on the importance of a host mediated post-translational modification that targets GtgE to the membranes where it can efficiently cleave and inactivate Rab32, leading to a better Salmonella survival in macrophages.Author summarySalmonella species includes a large group of bacteria that cause disease in different hosts. While some serovars are host generalists, others are restricted to humans. This is the case of Salmonella Typhi, responsible for Typhoid fever, a disease that affects millions globally. We have previously discovered an antimicrobial activity in macrophages that is controlled by Rab32. While the broad-host bacterium Salmonella Typhimurium effectively counteracts this mechanism through the delivery of two effectors, GtgE and SopD2, Salmonella Typhi does not express those effectors and cannot survive in mouse macrophages. In this article, we demonstrate how Salmonella Typhimurium exploits a host machinery to modify GtgE. We show that this host mediated modification is important for GtgE intracellular localization and effective Rab32 targeting, resulting in both a better intracellular survival and infection in vivo.

scholarly journals The Cell Wall-Anchored Streptomyces reticuli Avicel-Binding Protein (AbpS) and Its Gene

1998 ◽  
Vol 180 (7) ◽  
pp. 1647-1654 ◽  
Author(s):  
Stefan Walter ◽  
Egbert Wellmann ◽  
Hildgund Schrempf
Keyword(s):  
Reverse Genetics ◽  
Binding Protein ◽  
Fluorescein Isothiocyanate ◽  
Proteinase K ◽  
Logarithmic Phase ◽  
C Terminus ◽  
Late Logarithmic Phase ◽  
In Vivo Labelling ◽  
Α Helix

ABSTRACT Streptomyces reticuli produces a 35-kDa cellulose-binding protein (AbpS) which interacts strongly with crystalline forms of cellulose (Avicel, bacterial microcrystalline cellulose, and tunicin cellulose); other polysaccharides are recognized on weakly (chitin and Valonia cellulose) or not at all (xylan, starch, and agar). The protein could be purified to homogeneity due to its affinity to Avicel. After we sequenced internal peptides, the corresponding gene was identified by reverse genetics. In vivo labelling experiments with fluorescein isothiocyanate (FITC), FITC-labelled secondary antibodies, or proteinase K treatment revealed that the anchored AbpS protrudes from the surfaces of the hyphae. When we investigated the hydrophobicity of the deduced AbpS, one putative transmembrane segment was predicted at the C terminus. By analysis of the secondary structure, a large centrally located α-helix which has weak homology to the tropomyosin protein family was found. Physiological studies showed that AbpS is synthesized during the late logarithmic phase, independently of the carbon source.

scholarly journals Development of Live-Attenuated Arenavirus Vaccines Based on Codon Deoptimization

2015 ◽  
Vol 89 (7) ◽  
pp. 3523-3533 ◽  
Author(s):  
Benson Yee Hin Cheng ◽  
Emilio Ortiz-Riaño ◽  
Aitor Nogales ◽  
Juan Carlos de la Torre ◽  
Luis Martínez-Sobrido
Keyword(s):  
Mammalian Cells ◽  
Reverse Genetics ◽  
Cultured Cells ◽  
Hemorrhagic Fever ◽  
Wild Type ◽  
Lethal Challenge ◽  
Live Attenuated Vaccine ◽  
Codon Deoptimization

ABSTRACTArenaviruses have a significant impact on public health and pose a credible biodefense threat, but the development of safe and effective arenavirus vaccines has remained elusive, and currently, no Food and Drug Administration (FDA)-licensed arenavirus vaccines are available. Here, we explored the use of a codon deoptimization (CD)-based approach as a novel strategy to develop live-attenuated arenavirus vaccines. We recoded the nucleoprotein (NP) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) with the least frequently used codons in mammalian cells, which caused lower LCMV NP expression levels in transfected cells that correlated with decreased NP activity in cell-based functional assays. We used reverse-genetics approaches to rescue a battery of recombinant LCMVs (rLCMVs) encoding CD NPs (rLCMV/NPCD) that showed attenuated growth kineticsin vitro. Moreover, experiments using the well-characterized mouse model of LCMV infection revealed that rLCMV/NPCD1and rLCMV/NPCD2were highly attenuatedin vivobut, upon a single immunization, conferred complete protection against a subsequent lethal challenge with wild-type (WT) recombinant LCMV (rLCMV/WT). Both rLCMV/NPCD1and rLCMV/NPCD2were genetically and phenotypically stable during serial passages in FDA vaccine-approved Vero cells. These results provide proof of concept of the safety, efficacy, and stability of a CD-based approach for developing live-attenuated vaccine candidates against human-pathogenic arenaviruses.IMPORTANCESeveral arenaviruses cause severe hemorrhagic fever in humans and pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections, while antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and is associated with side effects. Here, we describe the generation of recombinant versions of the prototypic arenavirus LCMV encoding codon-deoptimized viral nucleoproteins (rLCMV/NPCD). We identified rLCMV/NPCD1and rLCMV/NPCD2to be highly attenuatedin vivobut able to confer protection against a subsequent lethal challenge with wild-type LCMV. These viruses displayed an attenuated phenotype during serial amplification passages in cultured cells. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated arenavirus vaccines.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Role of Artesunate in Potentiation of β-lactam Against Methicillin Resistant Staphylococcus aureus (MRSA) Isolated from Bovine Mastitis and its Histopathology Impact In-Vivo Study

2020 ◽  
Keyword(s):  
Clavulanic Acid ◽  
Resistance Mechanism ◽  
Bovine Mastitis ◽  
Lethal Challenge ◽  
Experimental Procedure ◽  
Appropriate Treatment ◽  
Amoxicillin Clavulanic Acid ◽  
Mrsa Infection

This article discusses the augmenting influence of Artesunate (ART) in combination with β-lactams (amoxicillin/clavulanic acid) antibiotic in sepsis mice models infected by a lethal challenge dose of live coagulase positive enterotoxigenic (Sec) MRSA that was isolated from a case of chronic bovine mastitis. The main goal is to find an appropriate treatment to overcome resistance mechanism of MRSA towards β-lactams antibiotic. Fifty healthy adult Swiss mice divided into 5 equal groups were used in the experimental procedure. The infected group that treated with both ART and β-lactams (amoxicillin/clavulanic acid) antibiotic revealed complete inhibition of MRSA count with complete normal macroscopic and histopathological features. We suggest that ART can potentiate the antibacterial action of β-lactams (amoxicillin/Clavulanic) acid against MRSA infection. The combination of ART and antibiotic can overcome MRSA resistance mechanism and so could be considered a novel candidate to overcome mastitis and/or sepsis caused by MRSA.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

scholarly journals TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
April Bell ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Cell ◽  
Central Nervous System Tumor ◽  
Post Translational Modifications ◽  
Reporter Mouse ◽  
C Terminus ◽  
Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

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