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The arenavirus Lassa virus (LASV) causes a severe haemorrhagic fever with high mortality in man. The cellular receptor for LASV is dystroglycan(DG). DG is a ubiquitous receptor for extracellular matrix (ECM) proteins, which cooper...
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The arenavirus Lassa virus (LASV) causes a severe haemorrhagic fever with high mortality in man. The cellular receptor for LASV is dystroglycan(DG). DG is a ubiquitous receptor for extracellular matrix (ECM) proteins, which cooperates with β1 integrins to control cell-matrix interactions. Here, we investigated whether LASV binding to DG triggers signal transduction, mimicking the natural ligands. Engagement of DG by LASV resulted in the recruitment of the adaptor protein Grb2 and the protein kinase MEK1 by the cytoplasmic domain of DG without activating the MEK/ERK pathway, indicating assembly of an inactive signalling complex. LASV binding to cells however affected the activation of the MEK/ERK pathway via α6β1 integrins. The virus-induced perturbation of α6β1 integrin signalling critically depended on high-affinity LASV binding to DG and DG's cytoplasmic domain, indicating that LASV-receptor binding perturbed signalling cross-talk between DG and b1 integrins.
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The Phytoreovirus rice dwarf virus (RDV) has a complex nucleocapsid architecture composed of multiple proteins and RNAs. However, specific RNA-protein and protein-protein interactions involved in virion packaging have not been ent...
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The Phytoreovirus rice dwarf virus (RDV) has a complex nucleocapsid architecture composed of multiple proteins and RNAs. However, specific RNA-protein and protein-protein interactions involved in virion packaging have not been entirely elucidated. Inorder to define mechanisms governing RDV particle assembly, interactions between individual components were analyzed both in vivo and in vitro. The P7 core protein binds specifically and with high affinity to all 12 genomic RDV dsRNAs. P1, a putative RNApolymerase, P5, a putative guanyl-transferase and P7 are encapsidated within the virion and also bind viral transcripts based upon in vitro binding assays. P1, P5, P7 and genomic dsRNAs were lacking in empty particles purified from infected tissues thatalso yielded fractions containing intact, infectious particles. In addition, P7 forms complexes with P1 and P3, a core capsid protein, in viral particles. These results indicate the possibility that core proteins and dsRNAs interact as one unit suggesting a mechanism for assortment of viral RNAs and subsequent packaging into core particles.
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The host range of the murine coronavirus (MHV) is limited to susceptible mice and murine cell lines by interactions of the spike glycoprotein (S) with its receptor, mCEACAM1a. We identified five residues in S (S33, L79, T82, Y162 ...
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The host range of the murine coronavirus (MHV) is limited to susceptible mice and murine cell lines by interactions of the spike glycoprotein (S) with its receptor, mCEACAM1a. We identified five residues in S (S33, L79, T82, Y162 and K183) that are conserved in the receptor-binding domain of MHV strains, but not in related coronaviruses. We used targeted RNA recombination to generate isogenic viruses that differ from MHV-A59 by amino acid substitutions in S. Viruses with S33R and K183R substitutions had wild type growth, while L79A/T82A viruses formed small plaques. Viruses with S33G, L79M/T82M or K183G substitutions could only be recovered from cells that over-expressed a mutant mCEACAM1a. Viruses with Y162H or Y162Q substitutions were never recovered, while Y162A viruses formed minute plaques. However, viruses with Y162F substitutions had wild type growth, suggesting that Y162 may comprise part of a hydrophobic domain that contacts the MHV-binding site of mCEACAM1a.
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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus and the family Bunyaviridae. It is a negative-strand RNA virus comprised of small (S), medium (M), and large (L) genome segments. The S se...
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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus and the family Bunyaviridae. It is a negative-strand RNA virus comprised of small (S), medium (M), and large (L) genome segments. The S segment encodes for nucleocapsid protein, the M segment codes for envelope glycoproteins (Gn and Gc), and the L segment codes for the RNA-dependent RNA polymerase. Currently, there are a limited number of methods for rapidly diagnosing CCHFV infections. We developed a real-time, reverse transcription-polymerase chain reaction assay for the rapid detection of CCHFV by using the TaqMan((R))-minor groove binding protein probe technology. The primers and probes were designed to amplify and detect a region in the S segment of CCHFV that is conserved across multiple strains. The limit of detection of the assay was 10 genome copies of RNA. This primer and probe set was specific to 18 strains of CCHFV tested and did not cross-react with either a DNA panel of 78 organisms or a panel of 28 diverse RNA viruses. This will rapidly and specifically detect CCHFV, and it has been used to detect CCHFV infection in samples from humans, animals, and ticks.
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The eradication of smallpox and the cessation of global vaccination led to the increased prevalence of human infections in Central Africa. Serologic and protein-based diagnostic assay for MPXV detection is difficult due to cross-r...
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The eradication of smallpox and the cessation of global vaccination led to the increased prevalence of human infections in Central Africa. Serologic and protein-based diagnostic assay for MPXV detection is difficult due to cross-reactive antibodies that do not differentiate between diverse orthopoxvirus (OPXV) species. A previously characterized monoclonal antibody (mAb 69-126-3-7) against MPXV [1] was retested for cross-reactivity with various OPXVs. The 14.5 kDa band protein that reacted with mAb 69-126-3 was identified to be MPXV A29 protein (homolog of vaccinia virus Copenhagen A27). Amino acid sequence analysis of the MPXV A29 with other OPXV homologs identified four amino acid changes. Peptides corresponding to these regions were designed and evaluated for binding to mAb 69-126-3 by ELISA and Biolayer Interferometry (BLI). Further refinement and truncations mapped the specificity of this antibody to a single amino acid difference in a 30-mer peptide compared to other OPXV homologs. This particular residue is proposed to be essential for heparin binding by VACV A27 protein. Despite this substitution, MPXV A29 bound to heparin with similar affinity to that of VACV A27 protein, suggesting flexibility of this motif for heparin binding. Although binding of mAb 69-126-3-7 to MPXV A29 prevented interaction with heparin, it did not have any effect on the infectivity of MPXV. Characterization of 69-126-3-7 mAb antibody allows for the possibility of the generation of a serological based species-specific detection of OPXVs despite high proteomic homology. Published by Elsevier Inc.
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We report the electron microscopic structure of an alpha-dystroglycan (alpha-DG) fragment (DGEKFc4) that contains binding sites for lymphocytic choriomeningitis virus (LCMV) and the extracellular matrix (ECM) molecule laminin. In ...
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We report the electron microscopic structure of an alpha-dystroglycan (alpha-DG) fragment (DGEKFc4) that contains binding sites for lymphocytic choriomeningitis virus (LCMV) and the extracellular matrix (ECM) molecule laminin. In electron microscopic images, DGEKFc4 appears as dumbbell-shaped rods with a length of 7.5 +/- 0.5 nM and width of 3 +/- 0.3 nM. The C-terminal human Fc allows binding of anti-human Fc antibody resulting in formation of immune complexes that preserve alpha-DG binding to virus. Electron microscopy shows the antibody binding to near one end of the dumbbell-shaped rods. Because arenaviruses like LCMV or Lassa fever virus (LFV) generate poor neutralizing antibodies during natural infection or vaccination, we assayed whether the alpha-DG receptoid bodies generated could be used as an efficient antibody mimic. However, the receptor body formed by either alpha-DG fragment alone or complexed to antibody to human Fc failed to efficiently neutralize virus.
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Quantum dots (QDots) are fluorescent semiconductor nanocrystals with a narrow emission spectrum, high quantum yield, and excellent photostability. These unique properties of QDots have been utilized to develop a fluorescent bindin...
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Quantum dots (QDots) are fluorescent semiconductor nanocrystals with a narrow emission spectrum, high quantum yield, and excellent photostability. These unique properties of QDots have been utilized to develop a fluorescent binding assay using biotinylated human T cell leukemia virus type 1 (biot-HTLV-1) conjugated with streptavidin-coated QDots that enabled both qualitative and quantitative analyses of viral binding. The specificity and linearity of the assay was demonstrated utilizing T cells, the primary HTLV-1-susceptible cell population. Furthermore, differential binding of HTLV-1 was analyzed in additional cell types of clinical relevance including primary CD4(+) and CD8(+) T cells, dendritic cells (DCs), monocytes, bone marrow progenitor cells, and epithelial cells. DCs exhibited maximum binding affinity when compared to other examined cell types except the Jurkat and SUP-T1 T cell lines. Finally, blocking antibodies directed against a putative HTLV-1 receptor on DCs; DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin), were utilized to study the inhibition of HTLV-1 binding to target cells. Overall, these results demonstrated that this novel high throughput assay can be utilized to study the binding of a biotinylated virus and has implications for screening of viral binding inhibitors as well as host membrane proteins that may serve as receptors for viral entry.
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Epidemics caused by viral infections pose a significant global threat. Cytoskeletal vimentin is a major intermediate filament (IF) protein, and is involved in numerous functions, including cell signaling, epithelial–mesenchymal t...
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Epidemics caused by viral infections pose a significant global threat. Cytoskeletal vimentin is a major intermediate filament (IF) protein, and is involved in numerous functions, including cell signaling, epithelial–mesenchymal transition, intracellular organization and cell migration. Vimentin has important roles for the life cycle of particular viruses; it can act as a co-receptor to enable effective virus invasion and guide efficient transport of the virus to the replication site. Furthermore, vimentin has been shown to rearrange into cage-like structures that facilitate virus replication, and to recruit viral components to the location of assembly and egress. Surprisingly, vimentin can also inhibit virus entry or egress, as well as participate in host-cell defense. Although vimentin can facilitate viral infection, how this function is regulated is still poorly understood. In particular, information is lacking on its interaction sites, regulation of expression, post-translational modifications and cooperation with other host factors. This Review recapitulates the different functions of vimentin in the virus life cycle and discusses how they influence host-cell tropism, virulence of the pathogens and the consequent pathological outcomes. These insights into vimentin–virus interactions emphasize the importance of cytoskeletal functions in viral cell biology and their potential for the identification of novel antiviral targets.
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A foot-and-mouth disease virus (FMDV) polymerase (3D) with amino acid replacements G118D, V239M and G373D (triple DMD mutant) was obtained from a molecular clone derived from a virus population treated with ribavirin, in the trans...
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A foot-and-mouth disease virus (FMDV) polymerase (3D) with amino acid replacements G118D, V239M and G373D (triple DMD mutant) was obtained from a molecular clone derived from a virus population treated with ribavirin, in the transition to error catastrophe (virus extinction through lethal mutagenesis). DMD 3D was expressed in Escherichia coli, purified, and its activity compared with that of wild-type enzyme and mutant enzymes with either replacement G118D, G118A or D338A (the latter affecting the catalytic motif YGDD), generated by site-directed mutagenesis. No differences among the enzymes were noted in their interaction with monoclonal antibodies specific for the FMDV polymerase. Mutant enzymes with G118D or G118A showed a 100-fold decrease in polymerization activity relative to wild-type 3D, using poly(A)/oligo(dT)(15) and poly(A)/VPg as template-primers, under several reaction conditions. As expected, the activity of 3D with D338A was undetectable (<0.01 times the value for wild-type 3D). DMD and the G118 mutants showed impaired binding to template-primer RNA whereas the D338A mutant showed a binding similar to wild-type 3D. Transfection of cells with FMDV RNA encoding DMD 3D resulted in selection of revertant viruses that maintained only substitutions V239M and G373D. Consistently, when infectious transcripts encoded 3D with either G118D, G118A or D338A, viruses with reversions to the wild-type sequence were isolated. The implication of G118 in template-primer binding is supported by the location of this residue in the template-binding groove of the FMDV polymerase. In addition to identifying an amino acid residue that is critical for the binding of polymerase to RNA, the results document the presence of defective genomes in the transition of virus to error catastrophe.
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Despite decades of antiviral drug research and development, viruses still remain a top global healthcare problem. Compared to eukaryotic cells, viruses are composed by a limited numbers of proteins that, nevertheless, set up multi...
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Despite decades of antiviral drug research and development, viruses still remain a top global healthcare problem. Compared to eukaryotic cells, viruses are composed by a limited numbers of proteins that, nevertheless, set up multiple interactions with cellular components, allowing the virus to take control of the infected cell. Each virus/host interaction can be considered as a therapeutical target for new antiviral drugs but, unfortunately, the systematic study of a so huge number of interactions is time-consuming and expensive, calling for models overcoming these drawbacks. Surface plasmon resonance (SPR) is a label-free optical technique to study biomolecular interactions in real time by detecting reflected light from a prism-gold film interface. Launched 20 years ago, SPR has become a nearly irreplaceable technology for the study of biomolecular interactions. Accordingly, SPR is increasingly used in the field of virology, spanning from the study of biological interactions to the identification of putative antiviral drugs. From the literature available, SPR emerges as an ideal link between conventional biological experimentation and system biology studies functional to the identification of highly connected viral or host proteins that act as nodal points in virus life cycle and thus considerable as therapeutical targets for the development of innovative antiviral strategies.
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