Bluetongue Virus Nonstructural Protein NS3/NS3a Is Not Essential for Virus Replication
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{"title"=>"Bluetongue virus nonstructural protein NS3/NS3a is not essential for virus replication", "type"=>"journal", "authors"=>[{"first_name"=>"René G P", "last_name"=>"Van Gennip", "scopus_author_id"=>"6602101853"}, {"first_name"=>"Sandra G P", "last_name"=>"Van De Water", "scopus_author_id"=>"25625722400"}, {"first_name"=>"Piet A.", "last_name"=>"Van Rijn", "scopus_author_id"=>"7003818933"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"24465709", "sgr"=>"84906229475", "doi"=>"10.1371/journal.pone.0085788", "scopus"=>"2-s2.0-84906229475", "pui"=>"372852965", "issn"=>"19326203"}, "id"=>"a7828ef0-fe51-35cd-a945-0c670436779b", "abstract"=>"Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUG(Met)→GCC(Ala) mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.", "link"=>"http://www.mendeley.com/research/bluetongue-virus-nonstructural-protein-ns3ns3a-not-essential-virus-replication", "reader_count"=>11, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>1, "Student > Ph. D. Student"=>4, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>1, "Student > Ph. D. Student"=>4, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>7, "Medicine and Dentistry"=>1, "Immunology and Microbiology"=>1, "Earth and Planetary Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>7}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}}, "reader_count_by_country"=>{"United Kingdom"=>1, "Spain"=>1}, "group_count"=>1}

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  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1351763/Figure_1.tif"], "description"=>"<p>(A) Schematic representation of BTV NS3. Numbers indicate amino acid position according to the NS3 amino acid sequence The two cytoplasmic domains (IC), the two transmembrane domains (TM; yellow), the extracellular domain (EC; orange), the calpactin S100A10/p11 binding site (blue), the late domain motifs (LD; green), the VP2 binding domain (VP2 BD: red), and the conserved N-linked glycosylation site at position 150 (NLG) are indicated. (B) NS3 is an integral membrane protein that interacts to cellular release factors calpactin S100A10/p11 and Tsg101, whereas the C-terminal domain binds VP2 on the outside of the virus particle. (C) The putative amino acid sequence in the regions of the <i>Sty</i>I, <i>Bsi</i>WI sites and StyI-filled or BsiWI-filled are shown in single letter code. Changed amino acids by a 4-basepairs insertion are underlined. (D) Single and double AUG→GCC mutations are shown. Dots and dashes indicate no change and absence of expression, respectively.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Transmembrane proteins", "genetics", "gene expression", "Protein translation", "microbiology", "Virology", "Viral classification", "RNA viruses", "Viral replication", "Viral vaccines", "Veterinary diseases", "Veterinary virology", "btv", "ns3", "putative", "amino", "sequences", "mutant"], "article_id"=>904450, "categories"=>["Biological Sciences", "Medicine"], "users"=>["René G. P. van Gennip", "Sandra G. P. van de Water", "Piet A. van Rijn"], "doi"=>["http://dx.doi.org/10.1371/journal.pone.0085788.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Schematic_representation_of_BTV_NS3_and_putative_amino_acid_sequences_of_mutant_viruses_/904450", "title"=>"Schematic representation of BTV NS3 and putative amino acid sequences of mutant viruses.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-20 03:29:06"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1351765/Figure_2.tif"], "description"=>"<p>(A) anti-VP7 immunostained cells after 2 or 13 days post transfection with BTV1 Seg-1-9 and mutated Seg-10 StyI-filled resulting in revertant virus StyI-rev1 showing CPE at 13 dpt. (B) sequence analysis of Seg-10 amplicons from StyI-rev1 passages at different time points after transfection. In single letter code, putative translation of all three frames is shown of which the middle is the ORF of NS3 with the insertion of Alanine (A) in StyI-rev1. Here the analysis is shown by use of a sequence primer located downstream of the <i>Sty</i>I site. Note the mixed sequence at 8 dpt upstream of the filled site due the introduction of the point deletion at the <i>Sty</i>I site in a subpopulation of the fragments. (C) Comparison of the amino acid sequences of NS3 of wtBTV1/8(S10), the 4-basepairs insertions, and the rescued revertant viruses Sty-rev1, 2 and BsiWI-rev1. Amino acids changes in the regions of the 4-basepairs insertion of the revertant viruses Sty-rev1, 2 and BsiWI-rev1 are in bold.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Transmembrane proteins", "genetics", "gene expression", "Protein translation", "microbiology", "Virology", "Viral classification", "RNA viruses", "Viral replication", "Viral vaccines", "Veterinary diseases", "Veterinary virology", "revertant", "viruses", "sty-rev"], "article_id"=>904452, "categories"=>["Biological Sciences", "Medicine"], "users"=>["René G. P. van Gennip", "Sandra G. P. van de Water", "Piet A. van Rijn"], "doi"=>["http://dx.doi.org/10.1371/journal.pone.0085788.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Analysis_of_revertant_viruses_Sty_rev_and_BsiWI_rev_/904452", "title"=>"Analysis of revertant viruses Sty-rev and BsiWI-rev.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-20 03:29:06"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1351766/Figure_3.tif"], "description"=>"<p>(A) Sequence analysis of Seg-10 amplicons from AUG mutant viruses shown in DNASTAR Lasergene Seqman assembly software. The mutated codons are indicated in rectangles (B) Plaque morphology of BSR cells infected with wtBTV1/8(S10), mutAUG1, mutAUG2 and mutAUG1+2 virus grown under 1% methylcellulose overlay medium are shown. At 2 dpi cells were fixed and immunostained with anti-VP7 Mab.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Transmembrane proteins", "genetics", "gene expression", "Protein translation", "microbiology", "Virology", "Viral classification", "RNA viruses", "Viral replication", "Viral vaccines", "Veterinary diseases", "Veterinary virology", "plaque", "phenotype", "aug", "mutant"], "article_id"=>904453, "categories"=>["Biological Sciences", "Medicine"], "users"=>["René G. P. van Gennip", "Sandra G. P. van de Water", "Piet A. van Rijn"], "doi"=>["http://dx.doi.org/10.1371/journal.pone.0085788.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Sequence_analysis_and_plaque_phenotype_of_AUG_mutant_viruses_/904453", "title"=>"Sequence analysis and plaque phenotype of AUG mutant viruses.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-20 03:29:06"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1351768/Figure_4.tif"], "description"=>"<p>Westernblot analysis of expression of NS3 and VP5 in cell lysates from BSR cells infected with wtBTV1/8(S10) (lane 1), mutAUG1 (lane 2), mutAUG2 (lane 3), mutAUG1+2 (lane 4) and mock-infected cells (lane 5) using antibodies against BTV NS3 or VP5.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Transmembrane proteins", "genetics", "gene expression", "Protein translation", "microbiology", "Virology", "Viral classification", "RNA viruses", "Viral replication", "Viral vaccines", "Veterinary diseases", "Veterinary virology", "viral", "aug", "mutant"], "article_id"=>904455, "categories"=>["Biological Sciences", "Medicine"], "users"=>["René G. P. van Gennip", "Sandra G. P. van de Water", "Piet A. van Rijn"], "doi"=>["http://dx.doi.org/10.1371/journal.pone.0085788.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Detection_of_viral_proteins_of_AUG_mutant_viruses_/904455", "title"=>"Detection of viral proteins of AUG mutant viruses.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-20 03:29:06"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1351769/Figure_5.tif"], "description"=>"<p>Virus growth and virus release of AUG mutant viruses grown on BSR cells and KC cells infected in duplicate with wtBTV1/8 (S10), mutAUG1, mutAUG2 and mutAUG1+2 viruses and harvested at indicated time points post infection. Virus titers were determined in supernatant and in cells by endpoint dilution, and the mean values are expressed as <sup>10</sup>logTCID<sub>50</sub>/ml. (A) released virus from BSR cells (upper left panel) or (B) BSR cell-associated virus (upper right panel). (C) released virus from KC cells (lower left panel) or (D) KC cell-associated virus (lower right panel).</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Transmembrane proteins", "genetics", "gene expression", "Protein translation", "microbiology", "Virology", "Viral classification", "RNA viruses", "Viral replication", "Viral vaccines", "Veterinary diseases", "Veterinary virology", "kinetics", "aug", "mutant", "viruses", "mammalian"], "article_id"=>904456, "categories"=>["Biological Sciences", "Medicine"], "users"=>["René G. P. van Gennip", "Sandra G. P. van de Water", "Piet A. van Rijn"], "doi"=>["http://dx.doi.org/10.1371/journal.pone.0085788.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Replication_kinetics_of_AUG_mutant_viruses_on_mammalian_and_insect_cells_/904456", "title"=>"Replication kinetics of AUG mutant viruses on mammalian and insect cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-20 03:29:06"}

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Relative Metric

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