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• {"link"=>{"url"=>"http://www.citeulike.org/user/slowjimmy/article/10016605"}, "post_time"=>"2011-11-11 14:12:24", "tag"=>"mhv68", "linkout"=>[{"type"=>"DOI", "url"=>"http://dx.doi.org/10.1371/journal.pone.0011672"}, {"type"=>"Pubmed Central", "url"=>"http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908122/"}, {"type"=>"Pubmed", "url"=>"http://view.ncbi.nlm.nih.gov/pubmed/20657771"}, {"type"=>"Hubmed", "url"=>"http://www.hubmed.org/display.cgi?uids=20657771"}], "username"=>"slowjimmy", "article_id"=>"10016605"}

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{"title"=>"A gammaherpesvirus complement regulatory protein promotes initiation of infection by activation of protein kinase Akt/PKB", "type"=>"journal", "authors"=>[{"first_name"=>"Beatrix", "last_name"=>"Steer", "scopus_author_id"=>"16647094800"}, {"first_name"=>"Barbara", "last_name"=>"Adler", "scopus_author_id"=>"7102049703"}, {"first_name"=>"Stipan", "last_name"=>"Jonjic", "scopus_author_id"=>"7004268029"}, {"first_name"=>"James P.", "last_name"=>"Stewart", "scopus_author_id"=>"7404792586"}, {"first_name"=>"Heiko", "last_name"=>"Adler", "scopus_author_id"=>"7101733273"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"359319167", "pmid"=>"20657771", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0011672", "scopus"=>"2-s2.0-77955349674", "sgr"=>"77955349674"}, "id"=>"fef1357c-cbf3-3901-802e-372656f1f5e8", "abstract"=>"BACKGROUND: Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation.\\n\\nCONCLUSIONS/SIGNIFICANCE: In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein.", "link"=>"http://www.mendeley.com/research/gammaherpesvirus-complement-regulatory-protein-promotes-initiation-infection-activation-protein-kina", "reader_count"=>8, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>1, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>2, "Student > Master"=>1, "Student > Bachelor"=>1, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>1, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>2, "Student > Master"=>1, "Student > Bachelor"=>1, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Agricultural and Biological Sciences"=>5, "Medicine and Dentistry"=>2, "Psychology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}}, "reader_count_by_country"=>{"United Kingdom"=>1}, "group_count"=>0}

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• http://doi.org/10.1128/JVI.00841-14
• http://doi.org/10.1038/s41598-018-32350-0
• http://doi.org/10.1186/s12989-016-0181-1
• http://doi.org/10.1186/1743-422X-9-107
• http://doi.org/10.1007/s11262-012-0769-y
• http://doi.org/10.1111/brv.12034

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• 3439311

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Figshare

• {"files"=>["https://ndownloader.figshare.com/files/418185", "https://ndownloader.figshare.com/files/418227", "https://ndownloader.figshare.com/files/418253", "https://ndownloader.figshare.com/files/418276"], "description"=>"<div><h3>Background</h3><p>Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages.</p> <h3>Methodology/Principal Findings</h3><p>Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation.</p> <h3>Conclusions/Significance</h3><p>In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein.</p> </div>", "links"=>[], "tags"=>["gammaherpesvirus", "complement", "promotes", "initiation", "activation", "kinase"], "article_id"=>142574, "categories"=>["Cancer", "Cell Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.s001", "https://dx.doi.org/10.1371/journal.pone.0011672.s002", "https://dx.doi.org/10.1371/journal.pone.0011672.s003", "https://dx.doi.org/10.1371/journal.pone.0011672.s004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Gammaherpesvirus_Complement_Regulatory_Protein_Promotes_Initiation_of_Infection_by_Activation_of_Protein_Kinase_Akt_PKB/142574", "title"=>"A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2010-07-21 00:42:54"}
• {"files"=>["https://ndownloader.figshare.com/files/839399"], "description"=>"<p>Cells were infected at a multiplicity of infection of 10 for 1h at 37°C. Then, the inoculum was removed and fresh medium, without (panel A) or with (panel B) phosphonoacetic acid (PAA), was added. Cells were harvested immediately thereafter and after 6, 18 and 48 hours, respectively, and analyzed for gfp expression by FACS analysis. Data shown are means ± SD of three independent experiments. The asterisks indicate that the expression of gfp is significantly delayed after infection with the ORF4⁻Tet⁺ mutant, when compared to parental or revertant virus (p<0,02; Two-way ANOVA).</p>", "links"=>[], "tags"=>["gfp-expression", "nih3t3", "cells", "deletion", "mutant", "orf4"], "article_id"=>509851, "categories"=>["Virology", "Infectious Diseases", "Plant Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Kinetics_of_gfp_expression_after_infection_of_NIH3T3_cells_with_parental_virus_the_deletion_mutant_ORF4_8722_Tet_or_the_ORF4_revertant_/509851", "title"=>"Kinetics of gfp-expression after infection of NIH3T3 cells with parental virus, the deletion mutant ORF4⁻Tet⁺ or the ORF4 revertant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-07-21 02:44:11"}
• {"files"=>["https://ndownloader.figshare.com/files/839499"], "description"=>"<p>The expression of the mRNA of the MHV-68 immediate-early gene Rta (ORF50) was examined by quantitative RT-PCR. Cells were infected at a multiplicity of infection of 10 for 1h at 37°C. Then, the inoculum was removed and fresh medium with PAA was added. Total RNA was isolated from infected cells 18 hours after infection and reverse transcribed. The resulting cDNA was used as template for PCR amplification of L8 and MHV-68 ORF50, respectively. Data are presented as relative expression of ORF50 after normalization to the corresponding L8 levels using the delta-delta C_t method. 18 hours after infection, the expression of the ORF50 mRNA was significantly reduced (*, p = 0,002; **, p = 0,025; Student's t-test) in cells infected with the ORF4 mutants when compared with the parental or revertant virus. Data shown are means ± SD of two independent experiments, each determined in duplicates.</p>", "links"=>[], "tags"=>["kinetics", "mutants", "quantitative"], "article_id"=>509954, "categories"=>["Virology", "Infectious Diseases", "Plant Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Delayed_infection_kinetics_of_the_ORF4_8722_Tet_and_ORF4_STOP_mutants_as_determined_by_quantitative_RT_PCR_/509954", "title"=>"Delayed infection kinetics of the ORF4⁻Tet⁺ and ORF4.STOP mutants as determined by quantitative RT-PCR.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-07-21 02:45:54"}
• {"files"=>["https://ndownloader.figshare.com/files/839567"], "description"=>"<p>For immunofluorescence staining, NIH3T3 cells were seeded on coverslips in 12 well plates the day before staining. Cells were infected at a multiplicity of infection of 5–10 for 1h at 4°C to allow adsorption. Then, cells were washed three times and were either immediately (0h – upper panel) fixed with 4% paraformaldehyde or prewarmed medium was added and the cells were further incubated at 37°C. Three hours later (3h – median panel) or eight hours later (8h – lower panel), cells were fixed and permeabilized with 0.1% Triton-X100. Virus particles were visualized with polyclonal rabbit anti-MHV-68 antiserum followed by goat anti-rabbitCy3 antibody. Stained cells were mounted in ProLong® Gold antifade reagent with DAPI and analysed by using a Zeiss Axiovert 200M microscope. Cells were viewed with a 100× oil immersion objective. The top panel shows staining of uninfected cells as control. One representative experiment of 5 is shown.</p>", "links"=>[], "tags"=>["kinetics", "mutants", "fluorescence"], "article_id"=>510029, "categories"=>["Virology", "Infectious Diseases", "Plant Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Delayed_infection_kinetics_of_the_ORF4_8722_Tet_and_ORF4_STOP_mutants_as_determined_by_fluorescence_microscopy_/510029", "title"=>"Delayed infection kinetics of the ORF4⁻Tet⁺ and ORF4.STOP mutants as determined by fluorescence microscopy.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-07-21 00:00:29"}
• {"files"=>["https://ndownloader.figshare.com/files/839660"], "description"=>"<p>Cells were infected at a multiplicity of infection of 10 or left uninfected ( = time point 0). At the indicated time points after infection, the relative phosphorylation levels of the protein kinase Akt were determined by a cell-based protein phosphorylation ELISA. The data are presented as the amount of activated (phosphorylated) Akt protein (pAkt) relative to total Akt protein (tAkt) (panel A). Total Akt levels are additionally depicted in panel B. Data shown are means ± sem of three to seven independent determinations, each performed in triplicate. The asterisks indicate that the difference between ORF4⁻Tet⁺ and ORF4 revertant is statistically significant (p<0,05; Two-way ANOVA).</p>", "links"=>[], "tags"=>["phosphorylation", "kinase", "akt", "reduced", "deletion", "mutant"], "article_id"=>510112, "categories"=>["Virology", "Infectious Diseases", "Plant Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_relative_phosphorylation_level_of_the_protein_kinase_Akt_is_significantly_reduced_after_infection_with_the_deletion_mutant_ORF4_8722_Tet_/510112", "title"=>"The relative phosphorylation level of the protein kinase Akt is significantly reduced after infection with the deletion mutant ORF4⁻Tet⁺.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-07-21 00:01:52"}
• {"files"=>["https://ndownloader.figshare.com/files/839763"], "description"=>"<p>NIH3T3 cells were treated with the indicated doses of Triciribine (TCN), or as a control, with DMSO, starting 1 h prior to infection. Then, cells were infected at a multiplicity of infection of 5–10. After 1h at 37°C, the inoculum was removed and fresh medium, with TCN or DMSO, respectively, was added. Cells were harvested after 18 hours and analyzed for gfp expression by FACS analysis (panel A), or were analyzed after 1.5 hours by microscopy using a 100× oil immersion objective (panel B) as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011672#pone-0011672-g003\" target=\"_blank\">Figure 3</a>. Data shown in panel A are means ± SD of four independent experiments. The asterisk indicates a statistically significant difference of p = 0,005, and the two asterisks indicate a statistically significant difference of p = 0,0003 (Student's t-test). In panel B, one representative experiment of 2 is shown.</p>", "links"=>[], "tags"=>["akt", "activation", "cells", "infected", "leads", "phenotype", "resembling", "orf4", "mutant"], "article_id"=>510213, "categories"=>["Virology", "Infectious Diseases", "Plant Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Inhibition_of_Akt_activation_in_cells_infected_with_parental_virus_leads_to_a_phenotype_closely_resembling_the_phenotype_of_the_ORF4_mutant_virus_/510213", "title"=>"Inhibition of Akt activation in cells infected with parental virus leads to a phenotype closely resembling the phenotype of the ORF4 mutant virus.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-07-21 00:03:33"}
• {"files"=>["https://ndownloader.figshare.com/files/839841"], "description"=>"<p>A) The expression of total Akt (tAkt) and its phosphorylated form (pAkt) was analyzed in insulin-treated NIH3T3 cells by Western blotting. The cells were treated with 10 µg/ml insulin for various time periods (min) as indicated. pAkt (upper panel) was detected with anti-phospho-Akt antibody (#9271), and tAkt (lower panel) was detected with anti-total-Akt antibody (#9272), both from Cell Signaling Technology (Danvers, MA). B) NIH3T3 cells were treated with insulin starting 60 min prior to infection. Then, insulin was washed away, and cells were infected at a multiplicity of infection of 5–10. After 1h at 37°C, the inoculum was removed and fresh medium was added. Cells were harvested after 18 hours and analyzed for gfp expression by FACS analysis. Treatment of the cells with insulin resulted in a significant reversion of the phenotype of the ORF4⁻Tet⁺ mutant (p = 0,037; Student's t-test). Data shown are means ± SD of at least three independent experiments.</p>", "links"=>[], "tags"=>["akt", "insulin", "reverses", "phenotype", "mhv-68", "orf4", "deletion", "mutant"], "article_id"=>510290, "categories"=>["Virology", "Infectious Diseases", "Plant Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phosphorylation_of_Akt_by_insulin_reverses_the_phenotype_of_the_MHV_68_ORF4_deletion_mutant_ORF4_8722_Tet_/510290", "title"=>"Phosphorylation of Akt by insulin reverses the phenotype of the MHV-68 ORF4 deletion mutant ORF4⁻Tet⁺.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-07-21 00:04:50"}
• {"files"=>["https://ndownloader.figshare.com/files/839930"], "description"=>"<p>Cells were infected at a multiplicity of infection of 10 for 1h at 37°C. Then, the inoculum was removed and fresh medium, containing phosphonoacetic acid (PAA), was added. Cells were harvested 18 hours after infection and analyzed for gfp expression by FACS analysis. Data shown are means ± SD of three independent experiments. The asterisk indicates that the number of gfp-positive cells after infection with the ORF4⁻Tet⁺ mutant was significantly reduced when compared to parental virus or ORF4⁻Tet⁺ with KSHV-ORF4 (p<0,05; Student's t-test).</p>", "links"=>[], "tags"=>["kshv", "rescues", "phenotype", "mhv-68", "orf4", "deletion", "mutant"], "article_id"=>510379, "categories"=>["Virology", "Infectious Diseases", "Plant Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ORF4_of_KSHV_rescues_the_phenotype_of_the_MHV_68_ORF4_deletion_mutant_ORF4_8722_Tet_/510379", "title"=>"ORF4 of KSHV rescues the phenotype of the MHV-68 ORF4 deletion mutant ORF4⁻Tet⁺.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-07-21 00:06:19"}
• {"files"=>["https://ndownloader.figshare.com/files/840004"], "description"=>"<p>Both wildtype virus (wt; black) and ORF4 mutant virus (ΔORF4; grey) initially activate – in an ORF4-independent manner – the Akt pathway (a). After being endocytosed, the wt virus is able to maintain the activation of Akt in an ORF4-dependent manner which aids to leave the endosome rather quickly (b). In contrast, due to the lack of ORF4, ORF4 mutant virus is trapped much longer in the endosome and escapes with delay, most likely by additional, ORF4-independent mechanisms (c).</p>", "links"=>[], "tags"=>["Virology", "virology/host invasion and cell entry", "virology/viral replication and gene regulation", "virology/virulence factors and mechanisms"], "article_id"=>510456, "categories"=>["Virology", "Infectious Diseases", "Plant Biology"], "users"=>["Beatrix Steer", "Barbara Adler", "Stipan Jonjic", "James P. Stewart", "Heiko Adler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0011672.g008"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Current_model_Working_hypothesis_/510456", "title"=>"Current model/Working hypothesis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-07-21 00:07:36"}

PMC Usage Stats | Further Information

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• {"unique-ip"=>"8", "full-text"=>"11", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
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• {"unique-ip"=>"5", "full-text"=>"7", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"9"}
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Relative Metric

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F1000Prime