Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity

ABSTRACTAlphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicityin vitroandin vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination inAedes aegyptimosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis.IMPORTANCEChikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about the role the receptor binding protein (E2) plays on disease outcome in an infected host. To study this, our laboratory generated chimeric CHIKV containing corresponding regions of the Semliki Forest virus (SFV) E2 (domains A, B, and C) substituted into the CHIKV genome. Our results demonstrate that each domain of E2 likely plays a critical, but dissimilar role in the viral life cycle. Our experiments show that manipulation of E2 domains can be useful for studies on viral pathogenesis and potentially the production of vaccines and/or antivirals.

Download Full-text

Nuclear receptor-binding protein 1: a novel tumour suppressor and pseudokinase

Biochemical Society Transactions ◽

10.1042/bst20130069 ◽

2013 ◽

Vol 41 (4) ◽

pp. 1055-1060 ◽

Cited By ~ 9

Author(s):

Jason S. Kerr ◽

Catherine H. Wilson

Keyword(s):

Nuclear Receptor ◽

Receptor Binding ◽

Kinase Activity ◽

Binding Protein ◽

Kinase Domain ◽

Present Review ◽

Critical Components ◽

Receptor Binding Protein

Pseudokinases are a class of kinases which are structurally designated as lacking kinase activity. Despite the lack of kinase domain sequence conservation, there is increasing evidence that a number of pseudokinases retain kinase activity and/or have critical cellular functions, casting aside previous notions that pseudokinases simply exist as redundant kinases. Moreover, a number of recent studies have implicated pseudokinases as critical components in cancer formation and progression. The present review discusses the interactions and potential functions that nuclear receptor-binding protein 1, a pseudokinase recently described to have a tumour-suppressive role in cancer, may play in cellular homoeostasis and protein regulation. The recent findings highlighted in the present review emphasize the requirement to fully determine the function of pseudokinases in vitro and in vivo, the understanding of which may ultimately uncover new directions for drug discovery.

Download Full-text

Developing Innolysins Against Campylobacter jejuni Using a Novel Prophage Receptor-Binding Protein

Frontiers in Microbiology ◽

10.3389/fmicb.2021.619028 ◽

2021 ◽

Vol 12 ◽

Author(s):

Athina Zampara ◽

Martine C. Holst Sørensen ◽

Yilmaz Emre Gencay ◽

Dennis Grimon ◽

Sebastian Hougaard Kristiansen ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Receptor Binding ◽

Capsular Polysaccharide ◽

Binding Protein ◽

Log Reduction ◽

Cell Counts ◽

Phage Receptor ◽

Receptor Binding Protein

Campylobacter contaminated poultry remains the major cause of foodborne gastroenteritis worldwide, calling for novel antibacterials. We previously developed the concept of Innolysin composed of an endolysin fused to a phage receptor binding protein (RBP) and provided the proof-of-concept that Innolysins exert bactericidal activity against Escherichia coli. Here, we have expanded the Innolysin concept to target Campylobacter jejuni. As no C. jejuni phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of C. jejuni CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting C. jejuni (Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse C. jejuni strains after in vitro exposure for 45 min at 20°C, reaching up to 1.30 ± 0.21 log reduction in CAMSA2147 cell counts. Screening of a library of Innolysins Cj composed of distinct endolysins for growth inhibition, allowed us to select Innolysin Cj5 as an additional promising antibacterial candidate. Application of either Innolysin Cj1 or Innolysin Cj5 on chicken skin refrigerated to 5°C and contaminated with C. jejuni CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill C. jejuni in situ. The receptor of Innolysins Cj remains to be identified, however, the RBP component (H-fiber) recognizes a novel receptor compared to lytic phages binding to capsular polysaccharide or flagella. Identification of other unexplored Campylobacter phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against Campylobacter.

Download Full-text

Nuclear Receptor Binding Protein 2 Is Downregulated in Medulloblastoma, and Reduces Tumor Cell Survival upon Overexpression

Cancers ◽

10.3390/cancers12061483 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1483

Author(s):

Anqi Xiong ◽

Ananya Roy ◽

Argyris Spyrou ◽

Holger Weishaupt ◽

Voichita D. Marinescu ◽

...

Keyword(s):

Cell Lines ◽

Nuclear Receptor ◽

Receptor Binding ◽

Growth Regulation ◽

Binding Protein ◽

Histone Deacetylation ◽

Adult Brain ◽

Tissue Array ◽

Receptor Binding Protein

Pseudokinases, comprising 10% of the human kinome, are emerging as regulators of canonical kinases and their functions are starting to be defined. We previously identified the pseudokinase Nuclear Receptor Binding Protein 2 (NRBP2) in a screen for genes regulated during neural differentiation. During mouse brain development, NRBP2 is expressed in the cerebellum, and in the adult brain, mainly confined to specific neuronal populations. To study the role of NRBP2 in brain tumors, we stained a brain tumor tissue array for NRPB2, and find its expression to be low, or absent, in a majority of the tumors. This includes medulloblastoma (MB), a pediatric tumor of the cerebellum. Using database mining of published MB data sets, we also find that NRBP2 is expressed at a lower level in MB than in the normal cerebellum. Recent studies indicate that MB exhibits frequent epigenetic alternations and we therefore treated MB cell lines with drugs inhibiting DNA methylation or histone deacetylation, which leads to an upregulation of NRBP2 mRNA expression, showing that it is under epigenetic regulation in cultured MB cells. Furthermore, forced overexpression of NRBP2 in MB cell lines causes a dramatic decrease in cell numbers, increased cell death, impaired cell migration and inhibited cell invasion in vitro. Taken together, our data indicate that downregulation of NRBP2 may be a feature by which MB cells escape growth regulation.

Download Full-text

Conformational changes in Chikungunya virus E2 protein upon heparan sulfate receptor binding explain mechanism of E2–E1 dissociation during viral entry

Bioscience Reports ◽

10.1042/bsr20191077 ◽

2019 ◽

Vol 39 (6) ◽

Cited By ~ 5

Author(s):

Bibekananda Sahoo ◽

Tirumala Kumar Chowdary

Keyword(s):

Fusion Protein ◽

Heparan Sulfate ◽

Membrane Fusion ◽

Receptor Binding ◽

Chikungunya Virus ◽

Binding Protein ◽

Md Simulations ◽

Cell Entry ◽

Sequence Motif ◽

Receptor Binding Protein

AbstractReceptor binding is the first step in viral cell entry. In enveloped virus cell entry, viral and host membrane fusion follows receptor binding. Viral surface receptor-binding protein associates with membrane fusion protein and masks its structure, to prevent pre-mature fusion activity. Dissociation of receptor-binding protein from fusion protein is an essential step before membrane fusion. Mechanism of receptor binding leading to dissociation of receptor binding and fusion protein is poorly understood in alphaviruses. Chikungunya virus (CHIKV), an alphavirus, re-emerged as a global pathogen in recent past. CHIKV surface envelope proteins, E2 and E1, function as receptor binding and fusion protein, respectively. Site of heparan sulfate (HS) receptor binding on E2–E1 heterodimer and its effect on E2–E1 heterodimer conformation is not known. Using molecular docking, we mapped HS binding to a positively charged pocket on E2 that is structurally conserved in alphaviruses. Based on our results from docking and sequence analysis, we identified a novel HS-binding sequence motif in E2. Purified E2 binds to heparin and HS specifically through charge interactions. Binding affinity of E2 to HS is comparable with other known HS–protein interactions (Kd ∼ 1.8 μM). Mutation of charged residues in the predicted HS-binding motif of E2 to alanine resulted in reduction of HS binding. Molecular dynamics (MD) simulations on E2, after docking HS, predicted allosteric domain movements. Fluorescence spectroscopy, far-UV circular dichroism spectroscopy, fluorescence resonance energy transfer experiments on HS-bound E2 corroborate our findings from MD simulations. We propose a mechanism where receptor-binding results in allosteric domain movements in E2, explaining E2–E1 dissociation.

Download Full-text

A New Mutant Class, Made by Targeted Mutagenesis, of Phage PRD1 Reveals That Protein P5 Connects the Receptor Binding Protein to the Vertex

Journal of Virology ◽

10.1128/jvi.74.17.7781-7786.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 7781-7786 ◽

Cited By ~ 32

Author(s):

Jaana K. H. Bamford ◽

Dennis H. Bamford

Keyword(s):

Receptor Binding ◽

Spatial Model ◽

Binding Protein ◽

Dna Packaging ◽

Targeted Mutagenesis ◽

In Vitro Mutagenesis ◽

Particle Assembly ◽

Receptor Binding Protein

ABSTRACT Phage PRD1 and adenovirus share a number of structural and functional similarities, one of which is the vertex organization at the fivefold-symmetry positions. We developed an in vitro mutagenesis system for the linear PRD1 genome in order to make targeted mutations. The role of protein P5 in the vertex structure was examined by this method. Mutation in gene V revealed that protein P5 is essential. The absence of P5 did not compromise the particle assembly or DNA packaging but led to a deficient vertex structure where the receptor binding protein P2, in addition to protein P5, was missing. P5− particles also lost their DNA upon purification. Based on this and previously published information we propose a spatial model for the spike structure at the vertices. This resembles to the corresponding structure in adenovirus.

Download Full-text

A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

10.1101/2021.03.08.434494 ◽

2021 ◽

Author(s):

Andrea N.W. Lim ◽

Minmin Yen ◽

Kimberley D. Seed ◽

David W. Lazinski ◽

Andrew Camilli

Keyword(s):

Host Range ◽

Vibrio Cholerae ◽

Receptor Binding ◽

Binding Protein ◽

Mutant Phenotype ◽

Tail Fiber ◽

Missense Mutations ◽

Wild Type ◽

Stool Samples ◽

Receptor Binding Protein

AbstractICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants had missense mutations in putative tail fiber gene gp25 and putative adhesin gp23. Using site-specific mutagenesis we show that single or double mutations in gp25 are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild type V. cholerae cells. The strength of binding of host-range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first, gp25 mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication.ImportanceConcern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies a ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

Download Full-text