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  • hsv 1  (ATCC)
    99
    ATCC hsv 1
    Human MxB is a herpesvirus restriction factor. (a) U87MG cells expressing HA-tagged Mx proteins were infected (MOI of 0.5) with GFP-encoding HIV-1 VSV-G pseudotyped lentiviral vector, <t>HSV-1,</t> MCMV, MHV68, Ad5, MVA, VSV, or IAV (H7N7) and subjected to fluorescence-activated cell sorter (FACS) analysis 20 hpi or 6 hpi (only VSV) or 48 hpi (only HIV-1). The percentage of GFP + cells relative to nontransduced control cells (NT) is shown. For Western blot analysis, Mx was detected by anti-HA antibody. Actin served as a control. (b) A549 cells expressing untagged Mx proteins were infected (MOI of 50) with GFP-encoding HCMV. Data represent percentages of GFP + cells relative to cells transduced with empty vector (ctrl) and the relative values of the mean fluorescence intensities (MFI). For Western blot analysis, MxB was detected by anti-MxB antibody. Actin served as a control. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. ***,
    Hsv 1, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 654 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biogenex hsv 1
    Genome organization and construction of HSV vectors. Schematic representation of the genome organization of <t>HSV-1.</t> (A) In the BAC pHSV-1(17 + )Lox-Luc, a luciferase cassette under a hCMV promoter was inserted between UL55 and UL56. (B) In pHSV-1(17 + )Lox-Luc-Δγ 1 34.5-Zeo (designated HSV-Zeo) both copies of γ 1 34.5 have been deleted, with a ZeoR selection cassette remaining in place of the 5′ copy of γ 1 34.5. (C) For pHSV-1(17 + )Lox-Luc-Δγ 1 34.5-LIF (designated HSV-LIF), the ZeoR cassette was replaced with LIF under control of an EF1alpha promoter.
    Hsv 1, supplied by Biogenex, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher hsv 1
    Degradation of mdm2 during <t>HSV-1</t> infection. (A) HFFF-1 cells were infected with wild-type HSV-1 (MOI of 10 PFU per cell) and then harvested at the indicated times after infection and analyzed for ICP0, ICP4, USP7, p53, p53 phosphorylated on serine 15,
    Hsv 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc hsv 1
    Deep sequence coverage of <t>HSV-1</t> variants from each strain group. Preprocessed sequence reads were aligned to the new draft genomes to assess the coverage depth of each assembly. Coverage depth is plotted on a log 10 scale ( y axis) across the length of the HSV-1 genome ( x axis). Major regions of the HSV-1 genome are diagrammed below the x axis, including the long and short internal repeats (IRL and IRS). Genomes and coverage are shown in a trimmed format ( 12 ), where the terminal copies of IRL and IRS are not included. Coverage tracks are overlaid for (A) HSV-1 KOS variants, (B) HSV-1 F variants, and (C) HSV-1 H166 and H166 Syncytial . The total number of sequence reads obtained was different for each HSV-1 strain, which affects overall coverage depth ( Table 3 ). However, peaks and valleys of coverage depth fall in similar locations on the HSV-1 genome, with the internal repeats (IRL and IRS) showing the most variability in coverage.
    Hsv 1, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies anti hsv 1 antibody
    (A) Schematic representation of the genome structure of <t>HSV-1</t> and the recombinant viruses. The two covalently linked components of HSV DNA, L and S, each consist of unique sequences, U L and U S , respectively, flanked by inverted repeats which are designated ab and b'a'. Recombinant virus HSV-BAC contains the wild-type γ 1 34.5 gene. Recombinant virus KY0234 lacks the coding region of the γ 1 ). Recombinant virus MC0201 was constructed by the HSV-BAC system with the VP35 gene from Ebola virus subtype Zaire replacing both copies of the γ 1 34.5 gene. Recombinant viruses MC0309 and MC0301 are repair viruses in which the VP35 gene of MC0201 was replaced with the wild-type γ 1 34.5 gene and a γ 1 34.5 gene containing an R215L point mutation, respectively. Restriction endonuclease abbreviations: N, NcoI; Be, BstEII; St, StuI; E, EcoRV. (B) Autoradiographic images of viral DNAs. Vero cells were infected with the indicated viruses at 10 PFU per cell. At 18 h after infection, cells were harvested and viral DNAs were prepared for Southern blot analysis as described in Materials and Methods. The VP35 and γ 1 34.5 genes were then detected by hybridization to electrophoretically separated digestion products of viral DNA transferred to a nitrocellulose sheet with either a 32 P-labeled EcoRI-XhoI or NotI fragment from the VP35 or γ 1 34.5 gene, respectively. Fragments representing VP35 or γ 1 34.5 are indicated on the right. (C) Expression of the VP35 or γ 1 34.5 gene products. Vero cells were either mock infected or infected with the indicated viruses at 10 PFU per cell. At 18 h postinfection, lysates of cells were prepared and subjected to Western immunoblot analysis with either anti-VP35, anti-γ 1 34.5, or anti-β-actin antibody. The positions of the VP35, γ 1 34.5, and β-actin proteins are shown on the right.
    Anti Hsv 1 Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad hsv 1 scaffolding protein
    Growth of BAC-derived UL34-null, UL31 R229L mutant virus on wt and mutant UL34-expressing cells. (A and B) Digital images show electrophoretically separated PCR products that are either digested with restriction enzyme (lanes 3 and 5) or undigested (lanes 2 and 4). The sizes of the undigested and digested products are indicated on the right of the gel. Lambda BstEII digest size standards are shown in lane 1, and the sizes of standard molecular weight bands are indicated on the left of the gel. (A) PCR products from the UL31 locus in virus rescued from wild-type <t>HSV-1</t> BAC (lanes 2 and 3) or UL34-null/UL31 R229L mutant (A, lanes 4 and 5) are shown. (B) PCR products from the UL34 locus in the same viruses are shown. Digital micrographs of immunofluorescently stained plaques formed on Vero (C to E), wt UL34-expressing RepAC cells (F to H), or CL04-expressing CL04AI cells (I to K) by viruses rescued from wt HSV-1(F) BAC (C, F, and I), UL34-null/UL31 wt BAC (D, G, and J), or UL34-null/UL31 R229L mutant BAC (E, H, and K).
    Hsv 1 Scaffolding Protein, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Medizinische Hochschule Hannover bac hsv1
    Growth of BAC-derived UL34-null, UL31 R229L mutant virus on wt and mutant UL34-expressing cells. (A and B) Digital images show electrophoretically separated PCR products that are either digested with restriction enzyme (lanes 3 and 5) or undigested (lanes 2 and 4). The sizes of the undigested and digested products are indicated on the right of the gel. Lambda BstEII digest size standards are shown in lane 1, and the sizes of standard molecular weight bands are indicated on the left of the gel. (A) PCR products from the UL31 locus in virus rescued from wild-type <t>HSV-1</t> BAC (lanes 2 and 3) or UL34-null/UL31 R229L mutant (A, lanes 4 and 5) are shown. (B) PCR products from the UL34 locus in the same viruses are shown. Digital micrographs of immunofluorescently stained plaques formed on Vero (C to E), wt UL34-expressing RepAC cells (F to H), or CL04-expressing CL04AI cells (I to K) by viruses rescued from wt HSV-1(F) BAC (C, F, and I), UL34-null/UL31 wt BAC (D, G, and J), or UL34-null/UL31 R229L mutant BAC (E, H, and K).
    Bac Hsv1, supplied by Medizinische Hochschule Hannover, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Agilent technologies rabbit anti hsv 1 serum
    Cell-to-cell spread of HSV gE ET domain mutants in HaCaT and ARPE-19 cells. Human HaCaT keratinocytes or retinal epithelial ARPE-19 cells were infected with wild-type <t>HSV-1</t> strain F or F-gEβ (derived from F), F-BAC, or F-BACgE448, which lacks
    Rabbit Anti Hsv 1 Serum, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology monoclonal anti hsv 1 tk
    A, Representative images (phase contrast or fluorescence, 10X) of BoHV-4-A-hCMVie-TK <t>HSV-1</t> -IRES-dsRed virus reconstitution following pBAC-BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed-pA electroporation into BEK or BEK expressing cre recombinase. B, Time after electroporation (hours) employed by pBAC-BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed-pA and pBAC-BoHV-4 to get viral reconstitution and plaque formation, following electroporation into BEK expressing cre cells (The data presented are the means ± standard errors of 3 electroporation for each BAC). C, Replication kinetics of BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed compared with BoHV-4-A. The data presented are the means ± standard errors of triplicate measurements ( P > .05 for all time points as measured by Student's t test). D, Representative images (10X) of plaque morphology and relative plaque size of BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed and BoHV-4-A on Vero cells. E, The plaque sizes (μm 2 ) software program. Bars represent means ± standard errors of 50 plaques for each virus; P > .05.
    Monoclonal Anti Hsv 1 Tk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATCC hsv 1 gfp
    Human MxB is a herpesvirus restriction factor. (a) U87MG cells expressing HA-tagged Mx proteins were infected (MOI of 0.5) with <t>GFP-encoding</t> HIV-1 VSV-G pseudotyped lentiviral vector, <t>HSV-1,</t> MCMV, MHV68, Ad5, MVA, VSV, or IAV (H7N7) and subjected to fluorescence-activated cell sorter (FACS) analysis 20 hpi or 6 hpi (only VSV) or 48 hpi (only HIV-1). The percentage of GFP + cells relative to nontransduced control cells (NT) is shown. For Western blot analysis, Mx was detected by anti-HA antibody. Actin served as a control. (b) A549 cells expressing untagged Mx proteins were infected (MOI of 50) with GFP-encoding HCMV. Data represent percentages of GFP + cells relative to cells transduced with empty vector (ctrl) and the relative values of the mean fluorescence intensities (MFI). For Western blot analysis, MxB was detected by anti-MxB antibody. Actin served as a control. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. ***,
    Hsv 1 Gfp, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    MACHEREY NAGEL recombinant
    Human MxB is a herpesvirus restriction factor. (a) U87MG cells expressing HA-tagged Mx proteins were infected (MOI of 0.5) with <t>GFP-encoding</t> HIV-1 VSV-G pseudotyped lentiviral vector, <t>HSV-1,</t> MCMV, MHV68, Ad5, MVA, VSV, or IAV (H7N7) and subjected to fluorescence-activated cell sorter (FACS) analysis 20 hpi or 6 hpi (only VSV) or 48 hpi (only HIV-1). The percentage of GFP + cells relative to nontransduced control cells (NT) is shown. For Western blot analysis, Mx was detected by anti-HA antibody. Actin served as a control. (b) A549 cells expressing untagged Mx proteins were infected (MOI of 50) with GFP-encoding HCMV. Data represent percentages of GFP + cells relative to cells transduced with empty vector (ctrl) and the relative values of the mean fluorescence intensities (MFI). For Western blot analysis, MxB was detected by anti-MxB antibody. Actin served as a control. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. ***,
    Recombinant, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Human MxB is a herpesvirus restriction factor. (a) U87MG cells expressing HA-tagged Mx proteins were infected (MOI of 0.5) with GFP-encoding HIV-1 VSV-G pseudotyped lentiviral vector, HSV-1, MCMV, MHV68, Ad5, MVA, VSV, or IAV (H7N7) and subjected to fluorescence-activated cell sorter (FACS) analysis 20 hpi or 6 hpi (only VSV) or 48 hpi (only HIV-1). The percentage of GFP + cells relative to nontransduced control cells (NT) is shown. For Western blot analysis, Mx was detected by anti-HA antibody. Actin served as a control. (b) A549 cells expressing untagged Mx proteins were infected (MOI of 50) with GFP-encoding HCMV. Data represent percentages of GFP + cells relative to cells transduced with empty vector (ctrl) and the relative values of the mean fluorescence intensities (MFI). For Western blot analysis, MxB was detected by anti-MxB antibody. Actin served as a control. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. ***,

    Journal: Journal of Virology

    Article Title: Human MxB Protein Is a Pan-herpesvirus Restriction Factor

    doi: 10.1128/JVI.01056-18

    Figure Lengend Snippet: Human MxB is a herpesvirus restriction factor. (a) U87MG cells expressing HA-tagged Mx proteins were infected (MOI of 0.5) with GFP-encoding HIV-1 VSV-G pseudotyped lentiviral vector, HSV-1, MCMV, MHV68, Ad5, MVA, VSV, or IAV (H7N7) and subjected to fluorescence-activated cell sorter (FACS) analysis 20 hpi or 6 hpi (only VSV) or 48 hpi (only HIV-1). The percentage of GFP + cells relative to nontransduced control cells (NT) is shown. For Western blot analysis, Mx was detected by anti-HA antibody. Actin served as a control. (b) A549 cells expressing untagged Mx proteins were infected (MOI of 50) with GFP-encoding HCMV. Data represent percentages of GFP + cells relative to cells transduced with empty vector (ctrl) and the relative values of the mean fluorescence intensities (MFI). For Western blot analysis, MxB was detected by anti-MxB antibody. Actin served as a control. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. ***,

    Article Snippet: The following viruses were used: HSV-1 (McIntyre) (ATCC, VR-539D); HSV-1 (F) ( ); HSV-1 GFP (17+ ) ( ); HSV-2 (MS) (ATCC, VR-734); HCMV (TB40-SE-EGFP) ( ); MCMV Δ1Δ6 GFP (generated by inserting a GFP expression cassette into a ΔMCMV bacterial artificial chromosome [BAC] via its FLP recombination target [FRT] site [ ]); wild-type MCMV rescued from BAC pSM3fr-MCK-2fl clone 3.3 ( ); MHV68 GFP ( ); Ad5 GFP ( ); MVA GFP ( ); VSV GFP ( ); and IAV GFP (A/Seal/MA/1/80-SC35M, H7N7) ( ).

    Techniques: Expressing, Infection, Plasmid Preparation, Fluorescence, FACS, Western Blot, Transduction

    MxB reduces viral gene expression and amplification of the herpesvirus genome. (a) HSV-1 capsid protein (anti-VP5) expression in A549 cells was analyzed by Western blotting at 24 hpi (MOI of 0.1). Shown is a blot representative of two independent experiments. (b) Expression of immediate early genes of HSV-1 ( ICP0 ) and MCMV ( ie2 ) in infected U87MG or A549 cells was measured by RT-qPCR. C T values were normalized to actin and plotted relative to the Δ C T values determined for the infected control cell line. Error bars represent geometric means of results from three independent experiments with technical duplicates. (c and d) Viral genome equivalents relative to GAPDH as a host-specific probe of HSV-1-infected (c) or MCMV-infected (d) A549 cells (MOI of 1). Error bars represent the SEM of (c) three independent HSV-1 experiments with technical quadruplicates or (d) two independent MCMV experiments with technical duplicates. Statistical analysis was performed using an unpaired t test for each time point. Significance is depicted relative to the control cells. ***,

    Journal: Journal of Virology

    Article Title: Human MxB Protein Is a Pan-herpesvirus Restriction Factor

    doi: 10.1128/JVI.01056-18

    Figure Lengend Snippet: MxB reduces viral gene expression and amplification of the herpesvirus genome. (a) HSV-1 capsid protein (anti-VP5) expression in A549 cells was analyzed by Western blotting at 24 hpi (MOI of 0.1). Shown is a blot representative of two independent experiments. (b) Expression of immediate early genes of HSV-1 ( ICP0 ) and MCMV ( ie2 ) in infected U87MG or A549 cells was measured by RT-qPCR. C T values were normalized to actin and plotted relative to the Δ C T values determined for the infected control cell line. Error bars represent geometric means of results from three independent experiments with technical duplicates. (c and d) Viral genome equivalents relative to GAPDH as a host-specific probe of HSV-1-infected (c) or MCMV-infected (d) A549 cells (MOI of 1). Error bars represent the SEM of (c) three independent HSV-1 experiments with technical quadruplicates or (d) two independent MCMV experiments with technical duplicates. Statistical analysis was performed using an unpaired t test for each time point. Significance is depicted relative to the control cells. ***,

    Article Snippet: The following viruses were used: HSV-1 (McIntyre) (ATCC, VR-539D); HSV-1 (F) ( ); HSV-1 GFP (17+ ) ( ); HSV-2 (MS) (ATCC, VR-734); HCMV (TB40-SE-EGFP) ( ); MCMV Δ1Δ6 GFP (generated by inserting a GFP expression cassette into a ΔMCMV bacterial artificial chromosome [BAC] via its FLP recombination target [FRT] site [ ]); wild-type MCMV rescued from BAC pSM3fr-MCK-2fl clone 3.3 ( ); MHV68 GFP ( ); Ad5 GFP ( ); MVA GFP ( ); VSV GFP ( ); and IAV GFP (A/Seal/MA/1/80-SC35M, H7N7) ( ).

    Techniques: Expressing, Amplification, Western Blot, Infection, Quantitative RT-PCR

    Genome organization and construction of HSV vectors. Schematic representation of the genome organization of HSV-1. (A) In the BAC pHSV-1(17 + )Lox-Luc, a luciferase cassette under a hCMV promoter was inserted between UL55 and UL56. (B) In pHSV-1(17 + )Lox-Luc-Δγ 1 34.5-Zeo (designated HSV-Zeo) both copies of γ 1 34.5 have been deleted, with a ZeoR selection cassette remaining in place of the 5′ copy of γ 1 34.5. (C) For pHSV-1(17 + )Lox-Luc-Δγ 1 34.5-LIF (designated HSV-LIF), the ZeoR cassette was replaced with LIF under control of an EF1alpha promoter.

    Journal: PLoS ONE

    Article Title: A Herpes Simplex Virus-Derived Replicative Vector Expressing LIF Limits Experimental Demyelinating Disease and Modulates Autoimmunity

    doi: 10.1371/journal.pone.0064200

    Figure Lengend Snippet: Genome organization and construction of HSV vectors. Schematic representation of the genome organization of HSV-1. (A) In the BAC pHSV-1(17 + )Lox-Luc, a luciferase cassette under a hCMV promoter was inserted between UL55 and UL56. (B) In pHSV-1(17 + )Lox-Luc-Δγ 1 34.5-Zeo (designated HSV-Zeo) both copies of γ 1 34.5 have been deleted, with a ZeoR selection cassette remaining in place of the 5′ copy of γ 1 34.5. (C) For pHSV-1(17 + )Lox-Luc-Δγ 1 34.5-LIF (designated HSV-LIF), the ZeoR cassette was replaced with LIF under control of an EF1alpha promoter.

    Article Snippet: Discussion In this study we constructed replicative vectors derived from HSV-1 (17+ ), deleted of the neurovirulence gene γ1 34.5, but expressing firefly luciferase for in vitro and in vivo tracking, using BAC mutagenesis.

    Techniques: BAC Assay, Luciferase, Selection

    Spread of HSV vectors in the CNS. (A) The HSV vectors expressed luciferase under an hCMV promoter which enables the study of viral spread in the CNS with an IVIS luminometer. 3 mg Luciferin were injected intraperitoneally (i.p.) to anesthetized mice five minutes prior to analysis. The figure shows two representative mice from each treatment group (follow-up of the same mice for all time points). (B-E) Immunohistochemistry for HSV-1 in the brain of untreated EAE mice (B), UV-irradiated vector (C), HSV-Zeo treated mice (D) and HSV-LIF treated mice (E) on day 9 post induction (day 3 post infection). Viral antigens were detected adjacent to the ventricles, in the ependymal cells, and in the corpus callosum in the HSV-vector treated groups (D, E). The arrows in the close-up fields indicate HSV detection. Five mice per group per time point were analyzed for all experimental settings. The scale bars are shown in the figure.

    Journal: PLoS ONE

    Article Title: A Herpes Simplex Virus-Derived Replicative Vector Expressing LIF Limits Experimental Demyelinating Disease and Modulates Autoimmunity

    doi: 10.1371/journal.pone.0064200

    Figure Lengend Snippet: Spread of HSV vectors in the CNS. (A) The HSV vectors expressed luciferase under an hCMV promoter which enables the study of viral spread in the CNS with an IVIS luminometer. 3 mg Luciferin were injected intraperitoneally (i.p.) to anesthetized mice five minutes prior to analysis. The figure shows two representative mice from each treatment group (follow-up of the same mice for all time points). (B-E) Immunohistochemistry for HSV-1 in the brain of untreated EAE mice (B), UV-irradiated vector (C), HSV-Zeo treated mice (D) and HSV-LIF treated mice (E) on day 9 post induction (day 3 post infection). Viral antigens were detected adjacent to the ventricles, in the ependymal cells, and in the corpus callosum in the HSV-vector treated groups (D, E). The arrows in the close-up fields indicate HSV detection. Five mice per group per time point were analyzed for all experimental settings. The scale bars are shown in the figure.

    Article Snippet: Discussion In this study we constructed replicative vectors derived from HSV-1 (17+ ), deleted of the neurovirulence gene γ1 34.5, but expressing firefly luciferase for in vitro and in vivo tracking, using BAC mutagenesis.

    Techniques: Luciferase, Injection, Mouse Assay, Immunohistochemistry, Irradiation, Plasmid Preparation, Infection

    Degradation of mdm2 during HSV-1 infection. (A) HFFF-1 cells were infected with wild-type HSV-1 (MOI of 10 PFU per cell) and then harvested at the indicated times after infection and analyzed for ICP0, ICP4, USP7, p53, p53 phosphorylated on serine 15,

    Journal: Journal of Virology

    Article Title: Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

    doi: 10.1128/JVI.79.19.12342-12354.2005

    Figure Lengend Snippet: Degradation of mdm2 during HSV-1 infection. (A) HFFF-1 cells were infected with wild-type HSV-1 (MOI of 10 PFU per cell) and then harvested at the indicated times after infection and analyzed for ICP0, ICP4, USP7, p53, p53 phosphorylated on serine 15,

    Article Snippet: Baculovirus Ac.CMV.His-ICP0 was constructed by inserting the NcoI-HpaI ICP0 genomic coding region of HSV-1, linked to an N-terminal polyhistidine tag and the human cytomegalovirus promoter region from pCIneo, into the Bac-to-Bac transfer plasmid vector pFastBac-HTa (Life Technologies).

    Techniques: Infection

    Effect of multiplicity of HSV-1 infection on the degradation of USP7. HFFF-2 cells were infected with wild-type HSV-1 at the indicated MOI and then samples that had been harvested at 2, 4, and 6 h after infection were analyzed for USP7 and ICP0 by Western

    Journal: Journal of Virology

    Article Title: Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

    doi: 10.1128/JVI.79.19.12342-12354.2005

    Figure Lengend Snippet: Effect of multiplicity of HSV-1 infection on the degradation of USP7. HFFF-2 cells were infected with wild-type HSV-1 at the indicated MOI and then samples that had been harvested at 2, 4, and 6 h after infection were analyzed for USP7 and ICP0 by Western

    Article Snippet: Baculovirus Ac.CMV.His-ICP0 was constructed by inserting the NcoI-HpaI ICP0 genomic coding region of HSV-1, linked to an N-terminal polyhistidine tag and the human cytomegalovirus promoter region from pCIneo, into the Bac-to-Bac transfer plasmid vector pFastBac-HTa (Life Technologies).

    Techniques: Infection, Western Blot

    Gene expression phenotype of M1 mutant HSV-1 in U2OS and HFFF-2 cells. (A) U2OS cells were infected with wild-type or M1 HSV-1 at an MOI of 2 PFU per cell. At 4 h postinfection, cycloheximide was added to a final concentration of 100 μg per ml

    Journal: Journal of Virology

    Article Title: Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

    doi: 10.1128/JVI.79.19.12342-12354.2005

    Figure Lengend Snippet: Gene expression phenotype of M1 mutant HSV-1 in U2OS and HFFF-2 cells. (A) U2OS cells were infected with wild-type or M1 HSV-1 at an MOI of 2 PFU per cell. At 4 h postinfection, cycloheximide was added to a final concentration of 100 μg per ml

    Article Snippet: Baculovirus Ac.CMV.His-ICP0 was constructed by inserting the NcoI-HpaI ICP0 genomic coding region of HSV-1, linked to an N-terminal polyhistidine tag and the human cytomegalovirus promoter region from pCIneo, into the Bac-to-Bac transfer plasmid vector pFastBac-HTa (Life Technologies).

    Techniques: Expressing, Mutagenesis, Infection, Concentration Assay

    FACS analysis of low-multiplicity wild-type and M1 HSV-1 infections in HFFF-2 cells. Cells in 35-mm plates were infected in duplicate with the wild-type or M1 mutant virus at MOIs of 0.1 and 0.5 PFU/cell, respectively. The dose of M1 virus was greater

    Journal: Journal of Virology

    Article Title: Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

    doi: 10.1128/JVI.79.19.12342-12354.2005

    Figure Lengend Snippet: FACS analysis of low-multiplicity wild-type and M1 HSV-1 infections in HFFF-2 cells. Cells in 35-mm plates were infected in duplicate with the wild-type or M1 mutant virus at MOIs of 0.1 and 0.5 PFU/cell, respectively. The dose of M1 virus was greater

    Article Snippet: Baculovirus Ac.CMV.His-ICP0 was constructed by inserting the NcoI-HpaI ICP0 genomic coding region of HSV-1, linked to an N-terminal polyhistidine tag and the human cytomegalovirus promoter region from pCIneo, into the Bac-to-Bac transfer plasmid vector pFastBac-HTa (Life Technologies).

    Techniques: FACS, Infection, Mutagenesis

    Gene expression phenotype of M1R rescuant virus in HFFF-2 cells. (A) Cells were infected with wild-type or M1R HSV-1 viruses at an MOI of 2 PFU per cell. Samples were harvested at the indicated time points for Western blot analysis of ICP0, ICP4, and

    Journal: Journal of Virology

    Article Title: Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

    doi: 10.1128/JVI.79.19.12342-12354.2005

    Figure Lengend Snippet: Gene expression phenotype of M1R rescuant virus in HFFF-2 cells. (A) Cells were infected with wild-type or M1R HSV-1 viruses at an MOI of 2 PFU per cell. Samples were harvested at the indicated time points for Western blot analysis of ICP0, ICP4, and

    Article Snippet: Baculovirus Ac.CMV.His-ICP0 was constructed by inserting the NcoI-HpaI ICP0 genomic coding region of HSV-1, linked to an N-terminal polyhistidine tag and the human cytomegalovirus promoter region from pCIneo, into the Bac-to-Bac transfer plasmid vector pFastBac-HTa (Life Technologies).

    Techniques: Expressing, Infection, Western Blot

    Reductions in USP7 levels by siRNA treatment reduce HSV-1 gene expression. (A) HeLa cells were mock transfected or transfected with anti-GFP control and anti-USP7 siRNAs. The cells were reseeded into 24-well dishes 2 days later, and the following day

    Journal: Journal of Virology

    Article Title: Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

    doi: 10.1128/JVI.79.19.12342-12354.2005

    Figure Lengend Snippet: Reductions in USP7 levels by siRNA treatment reduce HSV-1 gene expression. (A) HeLa cells were mock transfected or transfected with anti-GFP control and anti-USP7 siRNAs. The cells were reseeded into 24-well dishes 2 days later, and the following day

    Article Snippet: Baculovirus Ac.CMV.His-ICP0 was constructed by inserting the NcoI-HpaI ICP0 genomic coding region of HSV-1, linked to an N-terminal polyhistidine tag and the human cytomegalovirus promoter region from pCIneo, into the Bac-to-Bac transfer plasmid vector pFastBac-HTa (Life Technologies).

    Techniques: Expressing, Transfection

    Downregulation of USP7 requires ICP0 and its RING finger- and USP7-interacting domains. (A) HFFF-2 cells were infected with wild-type HSV-1 or ICP0-null mutant dl 1403 (MOI of 20 PFU/cell) and then samples were harvested 2, 4, 6, and 8 h later. Mock-infected

    Journal: Journal of Virology

    Article Title: Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

    doi: 10.1128/JVI.79.19.12342-12354.2005

    Figure Lengend Snippet: Downregulation of USP7 requires ICP0 and its RING finger- and USP7-interacting domains. (A) HFFF-2 cells were infected with wild-type HSV-1 or ICP0-null mutant dl 1403 (MOI of 20 PFU/cell) and then samples were harvested 2, 4, 6, and 8 h later. Mock-infected

    Article Snippet: Baculovirus Ac.CMV.His-ICP0 was constructed by inserting the NcoI-HpaI ICP0 genomic coding region of HSV-1, linked to an N-terminal polyhistidine tag and the human cytomegalovirus promoter region from pCIneo, into the Bac-to-Bac transfer plasmid vector pFastBac-HTa (Life Technologies).

    Techniques: Infection, Mutagenesis

    Levels of USP7 are reduced in a proteasome-dependent manner during HSV-1 infection of several cell types. (A) Vero cells were infected with wild-type HSV-1 (MOI of 20 PFU/cell) and samples were taken at 2, 4, and 8 h after virus adsorption. Whole-cell

    Journal: Journal of Virology

    Article Title: Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

    doi: 10.1128/JVI.79.19.12342-12354.2005

    Figure Lengend Snippet: Levels of USP7 are reduced in a proteasome-dependent manner during HSV-1 infection of several cell types. (A) Vero cells were infected with wild-type HSV-1 (MOI of 20 PFU/cell) and samples were taken at 2, 4, and 8 h after virus adsorption. Whole-cell

    Article Snippet: Baculovirus Ac.CMV.His-ICP0 was constructed by inserting the NcoI-HpaI ICP0 genomic coding region of HSV-1, linked to an N-terminal polyhistidine tag and the human cytomegalovirus promoter region from pCIneo, into the Bac-to-Bac transfer plasmid vector pFastBac-HTa (Life Technologies).

    Techniques: Infection, Adsorption

    Deep sequence coverage of HSV-1 variants from each strain group. Preprocessed sequence reads were aligned to the new draft genomes to assess the coverage depth of each assembly. Coverage depth is plotted on a log 10 scale ( y axis) across the length of the HSV-1 genome ( x axis). Major regions of the HSV-1 genome are diagrammed below the x axis, including the long and short internal repeats (IRL and IRS). Genomes and coverage are shown in a trimmed format ( 12 ), where the terminal copies of IRL and IRS are not included. Coverage tracks are overlaid for (A) HSV-1 KOS variants, (B) HSV-1 F variants, and (C) HSV-1 H166 and H166 Syncytial . The total number of sequence reads obtained was different for each HSV-1 strain, which affects overall coverage depth ( Table 3 ). However, peaks and valleys of coverage depth fall in similar locations on the HSV-1 genome, with the internal repeats (IRL and IRS) showing the most variability in coverage.

    Journal: mBio

    Article Title: Rapid Genome Assembly and Comparison Decode Intrastrain Variation in Human Alphaherpesviruses

    doi: 10.1128/mBio.02213-14

    Figure Lengend Snippet: Deep sequence coverage of HSV-1 variants from each strain group. Preprocessed sequence reads were aligned to the new draft genomes to assess the coverage depth of each assembly. Coverage depth is plotted on a log 10 scale ( y axis) across the length of the HSV-1 genome ( x axis). Major regions of the HSV-1 genome are diagrammed below the x axis, including the long and short internal repeats (IRL and IRS). Genomes and coverage are shown in a trimmed format ( 12 ), where the terminal copies of IRL and IRS are not included. Coverage tracks are overlaid for (A) HSV-1 KOS variants, (B) HSV-1 F variants, and (C) HSV-1 H166 and H166 Syncytial . The total number of sequence reads obtained was different for each HSV-1 strain, which affects overall coverage depth ( Table 3 ). However, peaks and valleys of coverage depth fall in similar locations on the HSV-1 genome, with the internal repeats (IRL and IRS) showing the most variability in coverage.

    Article Snippet: The authors who first described H166 and H166Syncytial noted that it is unusual for HSV-1 to cause meningitis ( ).

    Techniques: Sequencing

    Distribution of plaque morphologies before and after plaque purification. The graph depicts the number of plaques with a given area for each variant of plaque morphology shown in Fig. 1 . (A) Distribution of the original HSV-1 KOS stock and the large, syncytial, and hypersyncytial variants. Bins include the number of plaques with areas up to the value shown on the x axis (bins of 1 mm for HSV-1 KOS subclones). (B) Distribution of the original HSV-1 F stock and the large, small, and syncytial variants. Bins of 0.5 mm were used for HSV-1 F subclones. (C) Size distribution of plaques from clinical HSV-1 strains H166 and H166 Syncytial , using bins of 1 mm. Solid black lines indicate parental virus stocks, green lines indicate variants with standard cytopathic effect (CPE), and purple lines indicate syncytial variants. All plaques were quantified at 72 hpi. Note that the x axis scale varies across panels.

    Journal: mBio

    Article Title: Rapid Genome Assembly and Comparison Decode Intrastrain Variation in Human Alphaherpesviruses

    doi: 10.1128/mBio.02213-14

    Figure Lengend Snippet: Distribution of plaque morphologies before and after plaque purification. The graph depicts the number of plaques with a given area for each variant of plaque morphology shown in Fig. 1 . (A) Distribution of the original HSV-1 KOS stock and the large, syncytial, and hypersyncytial variants. Bins include the number of plaques with areas up to the value shown on the x axis (bins of 1 mm for HSV-1 KOS subclones). (B) Distribution of the original HSV-1 F stock and the large, small, and syncytial variants. Bins of 0.5 mm were used for HSV-1 F subclones. (C) Size distribution of plaques from clinical HSV-1 strains H166 and H166 Syncytial , using bins of 1 mm. Solid black lines indicate parental virus stocks, green lines indicate variants with standard cytopathic effect (CPE), and purple lines indicate syncytial variants. All plaques were quantified at 72 hpi. Note that the x axis scale varies across panels.

    Article Snippet: The authors who first described H166 and H166Syncytial noted that it is unusual for HSV-1 to cause meningitis ( ).

    Techniques: Purification, Variant Assay

    Plaque morphologies in HSV-1 stocks, before and after plaque purification. Lab-passaged stocks of classical HSV-1 KOS and F strains contain multiple plaque morphologies. (A) With multiple rounds of limiting dilution, distinct plaque morphologies can be separated into populations that breed true and exhibit greatly reduced diversity (see Table 1 for details). (B) HSV-1 KOS can be separated into large, syncytial, and hypersyncytial variants. (C) HSV-1 F can be separated into large, syncytial, and small variants. (D) We used a previously described BAC-cloned HSV-1 F genome that has been modified to encode an mRFP fusion to the viral capsid ( 38 ). Recovery of this cloned genome into mammalian cells regenerates the plaque-morphology diversity observed in HSV-1 F stocks. Phase and fluorescence images reveal that in addition to diversity in plaque morphology, fluorescently tagged viral stocks exhibit diversity in fluorescence (additional images can be found in Fig. S1 in the supplemental material). (E) The low-passage-number clinical isolates HSV-1 H166 and H166 Syncytial are distinct strains isolated from the cerebrospinal fluid of the same patient during an episode of viral meningitis. The size and appearance of cytopathic and syncytial plaques mirror those seen in the lab-isolated variants of HSV-1 KOS and F. Viruses were plated at limiting dilutions on monolayers of Vero cells and then fixed and stained with methylene blue at 72 hpi. Images were exported from Nikon NIS-Elements software. Contrast was inverted using Adobe Photoshop to show plaques more clearly. Bars: 1 mm (A), 5 mm (B, C, and E), and 2 mm (D). Further quantifications of plaque size distribution and frequency are found in Fig. 2 and Tables 1 and 2 .

    Journal: mBio

    Article Title: Rapid Genome Assembly and Comparison Decode Intrastrain Variation in Human Alphaherpesviruses

    doi: 10.1128/mBio.02213-14

    Figure Lengend Snippet: Plaque morphologies in HSV-1 stocks, before and after plaque purification. Lab-passaged stocks of classical HSV-1 KOS and F strains contain multiple plaque morphologies. (A) With multiple rounds of limiting dilution, distinct plaque morphologies can be separated into populations that breed true and exhibit greatly reduced diversity (see Table 1 for details). (B) HSV-1 KOS can be separated into large, syncytial, and hypersyncytial variants. (C) HSV-1 F can be separated into large, syncytial, and small variants. (D) We used a previously described BAC-cloned HSV-1 F genome that has been modified to encode an mRFP fusion to the viral capsid ( 38 ). Recovery of this cloned genome into mammalian cells regenerates the plaque-morphology diversity observed in HSV-1 F stocks. Phase and fluorescence images reveal that in addition to diversity in plaque morphology, fluorescently tagged viral stocks exhibit diversity in fluorescence (additional images can be found in Fig. S1 in the supplemental material). (E) The low-passage-number clinical isolates HSV-1 H166 and H166 Syncytial are distinct strains isolated from the cerebrospinal fluid of the same patient during an episode of viral meningitis. The size and appearance of cytopathic and syncytial plaques mirror those seen in the lab-isolated variants of HSV-1 KOS and F. Viruses were plated at limiting dilutions on monolayers of Vero cells and then fixed and stained with methylene blue at 72 hpi. Images were exported from Nikon NIS-Elements software. Contrast was inverted using Adobe Photoshop to show plaques more clearly. Bars: 1 mm (A), 5 mm (B, C, and E), and 2 mm (D). Further quantifications of plaque size distribution and frequency are found in Fig. 2 and Tables 1 and 2 .

    Article Snippet: The authors who first described H166 and H166Syncytial noted that it is unusual for HSV-1 to cause meningitis ( ).

    Techniques: Purification, BAC Assay, Clone Assay, Modification, Fluorescence, Isolation, Staining, Software

    (A) Schematic representation of the genome structure of HSV-1 and the recombinant viruses. The two covalently linked components of HSV DNA, L and S, each consist of unique sequences, U L and U S , respectively, flanked by inverted repeats which are designated ab and b'a'. Recombinant virus HSV-BAC contains the wild-type γ 1 34.5 gene. Recombinant virus KY0234 lacks the coding region of the γ 1 ). Recombinant virus MC0201 was constructed by the HSV-BAC system with the VP35 gene from Ebola virus subtype Zaire replacing both copies of the γ 1 34.5 gene. Recombinant viruses MC0309 and MC0301 are repair viruses in which the VP35 gene of MC0201 was replaced with the wild-type γ 1 34.5 gene and a γ 1 34.5 gene containing an R215L point mutation, respectively. Restriction endonuclease abbreviations: N, NcoI; Be, BstEII; St, StuI; E, EcoRV. (B) Autoradiographic images of viral DNAs. Vero cells were infected with the indicated viruses at 10 PFU per cell. At 18 h after infection, cells were harvested and viral DNAs were prepared for Southern blot analysis as described in Materials and Methods. The VP35 and γ 1 34.5 genes were then detected by hybridization to electrophoretically separated digestion products of viral DNA transferred to a nitrocellulose sheet with either a 32 P-labeled EcoRI-XhoI or NotI fragment from the VP35 or γ 1 34.5 gene, respectively. Fragments representing VP35 or γ 1 34.5 are indicated on the right. (C) Expression of the VP35 or γ 1 34.5 gene products. Vero cells were either mock infected or infected with the indicated viruses at 10 PFU per cell. At 18 h postinfection, lysates of cells were prepared and subjected to Western immunoblot analysis with either anti-VP35, anti-γ 1 34.5, or anti-β-actin antibody. The positions of the VP35, γ 1 34.5, and β-actin proteins are shown on the right.

    Journal: Journal of Virology

    Article Title: The VP35 Protein of Ebola Virus Inhibits the Antiviral Effect Mediated by Double-Stranded RNA-Dependent Protein Kinase PKR ▿

    doi: 10.1128/JVI.01006-06

    Figure Lengend Snippet: (A) Schematic representation of the genome structure of HSV-1 and the recombinant viruses. The two covalently linked components of HSV DNA, L and S, each consist of unique sequences, U L and U S , respectively, flanked by inverted repeats which are designated ab and b'a'. Recombinant virus HSV-BAC contains the wild-type γ 1 34.5 gene. Recombinant virus KY0234 lacks the coding region of the γ 1 ). Recombinant virus MC0201 was constructed by the HSV-BAC system with the VP35 gene from Ebola virus subtype Zaire replacing both copies of the γ 1 34.5 gene. Recombinant viruses MC0309 and MC0301 are repair viruses in which the VP35 gene of MC0201 was replaced with the wild-type γ 1 34.5 gene and a γ 1 34.5 gene containing an R215L point mutation, respectively. Restriction endonuclease abbreviations: N, NcoI; Be, BstEII; St, StuI; E, EcoRV. (B) Autoradiographic images of viral DNAs. Vero cells were infected with the indicated viruses at 10 PFU per cell. At 18 h after infection, cells were harvested and viral DNAs were prepared for Southern blot analysis as described in Materials and Methods. The VP35 and γ 1 34.5 genes were then detected by hybridization to electrophoretically separated digestion products of viral DNA transferred to a nitrocellulose sheet with either a 32 P-labeled EcoRI-XhoI or NotI fragment from the VP35 or γ 1 34.5 gene, respectively. Fragments representing VP35 or γ 1 34.5 are indicated on the right. (C) Expression of the VP35 or γ 1 34.5 gene products. Vero cells were either mock infected or infected with the indicated viruses at 10 PFU per cell. At 18 h postinfection, lysates of cells were prepared and subjected to Western immunoblot analysis with either anti-VP35, anti-γ 1 34.5, or anti-β-actin antibody. The positions of the VP35, γ 1 34.5, and β-actin proteins are shown on the right.

    Article Snippet: Samples were then sonicated, boiled, subjected to electrophoresis on denaturing 12% polyacrylamide gels, transferred to nitrocellulose membranes, blocked with 5% nonfat milk, and reacted with anti-γ1 34.5 antibody, anti-VP35 antibody, anti-HSV-1 antibody (Dako Corporation), anti-phosphorylated eIF-2α antibody (Biosource, Inc.), anti-eIF-2α antibody (Cell Signaling Technology, Inc.), anti-phosphorylated PKR (Cell Signaling Technology, Inc.), anti-PKR antibody (Santa Cruz), or anti-β-actin antibody (Sigma).

    Techniques: Recombinant, BAC Assay, Construct, Mutagenesis, Infection, Southern Blot, Hybridization, Labeling, Expressing, Western Blot

    (A) Viral protein accumulation in the presence and absence of interferon. Monolayers of Vero cells were not treated (−) or were pretreated (+) with human leukocyte alpha interferon (IFN α) (1,000 U/ml; Sigma) for 20 h. Cells were then mock infected or infected with the indicated viruses at 1.0 PFU per cell. At 18 h postinfection, cells were harvested, washed with phosphate-buffered saline, resuspended in disruption buffer, electrophoretically separated on a denaturing 12% polyacrylamide gel, transferred to a nitrocellulose sheet, and probed with a rabbit polyclonal antibody against all HSV-1 antigens as suggested by the manufacturer (Dako Corporation). β-Actin was probed as a loading control. (B) Effect of the VP35 protein on eIF-2α phosphorylation. Vero cells, untreated or pretreated with alpha interferon, were infected with viruses (1 PFU/cell), and lysates of cells were prepared 18 h postinfection. Samples were subjected to immunoblot analysis using rabbit antibody against phospho-Ser51 eIF-2α (Biosource, Inc.) or eIF-2α (Cell Signaling Technology, Inc.). The positions of eIF-2α and phosphorylated eIF-2α (p-eIF2α) are indicated on the left. The ratio between the amounts of phosphorylated eIF-2α and total eIF-2α in each lane were quantitated by densitometry, and the numbers indicate the ratios after normalization to mock-infected cells.

    Journal: Journal of Virology

    Article Title: The VP35 Protein of Ebola Virus Inhibits the Antiviral Effect Mediated by Double-Stranded RNA-Dependent Protein Kinase PKR ▿

    doi: 10.1128/JVI.01006-06

    Figure Lengend Snippet: (A) Viral protein accumulation in the presence and absence of interferon. Monolayers of Vero cells were not treated (−) or were pretreated (+) with human leukocyte alpha interferon (IFN α) (1,000 U/ml; Sigma) for 20 h. Cells were then mock infected or infected with the indicated viruses at 1.0 PFU per cell. At 18 h postinfection, cells were harvested, washed with phosphate-buffered saline, resuspended in disruption buffer, electrophoretically separated on a denaturing 12% polyacrylamide gel, transferred to a nitrocellulose sheet, and probed with a rabbit polyclonal antibody against all HSV-1 antigens as suggested by the manufacturer (Dako Corporation). β-Actin was probed as a loading control. (B) Effect of the VP35 protein on eIF-2α phosphorylation. Vero cells, untreated or pretreated with alpha interferon, were infected with viruses (1 PFU/cell), and lysates of cells were prepared 18 h postinfection. Samples were subjected to immunoblot analysis using rabbit antibody against phospho-Ser51 eIF-2α (Biosource, Inc.) or eIF-2α (Cell Signaling Technology, Inc.). The positions of eIF-2α and phosphorylated eIF-2α (p-eIF2α) are indicated on the left. The ratio between the amounts of phosphorylated eIF-2α and total eIF-2α in each lane were quantitated by densitometry, and the numbers indicate the ratios after normalization to mock-infected cells.

    Article Snippet: Samples were then sonicated, boiled, subjected to electrophoresis on denaturing 12% polyacrylamide gels, transferred to nitrocellulose membranes, blocked with 5% nonfat milk, and reacted with anti-γ1 34.5 antibody, anti-VP35 antibody, anti-HSV-1 antibody (Dako Corporation), anti-phosphorylated eIF-2α antibody (Biosource, Inc.), anti-eIF-2α antibody (Cell Signaling Technology, Inc.), anti-phosphorylated PKR (Cell Signaling Technology, Inc.), anti-PKR antibody (Santa Cruz), or anti-β-actin antibody (Sigma).

    Techniques: Infection

    Growth of BAC-derived UL34-null, UL31 R229L mutant virus on wt and mutant UL34-expressing cells. (A and B) Digital images show electrophoretically separated PCR products that are either digested with restriction enzyme (lanes 3 and 5) or undigested (lanes 2 and 4). The sizes of the undigested and digested products are indicated on the right of the gel. Lambda BstEII digest size standards are shown in lane 1, and the sizes of standard molecular weight bands are indicated on the left of the gel. (A) PCR products from the UL31 locus in virus rescued from wild-type HSV-1 BAC (lanes 2 and 3) or UL34-null/UL31 R229L mutant (A, lanes 4 and 5) are shown. (B) PCR products from the UL34 locus in the same viruses are shown. Digital micrographs of immunofluorescently stained plaques formed on Vero (C to E), wt UL34-expressing RepAC cells (F to H), or CL04-expressing CL04AI cells (I to K) by viruses rescued from wt HSV-1(F) BAC (C, F, and I), UL34-null/UL31 wt BAC (D, G, and J), or UL34-null/UL31 R229L mutant BAC (E, H, and K).

    Journal: Journal of Virology

    Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿

    doi: 10.1128/JVI.01638-09

    Figure Lengend Snippet: Growth of BAC-derived UL34-null, UL31 R229L mutant virus on wt and mutant UL34-expressing cells. (A and B) Digital images show electrophoretically separated PCR products that are either digested with restriction enzyme (lanes 3 and 5) or undigested (lanes 2 and 4). The sizes of the undigested and digested products are indicated on the right of the gel. Lambda BstEII digest size standards are shown in lane 1, and the sizes of standard molecular weight bands are indicated on the left of the gel. (A) PCR products from the UL31 locus in virus rescued from wild-type HSV-1 BAC (lanes 2 and 3) or UL34-null/UL31 R229L mutant (A, lanes 4 and 5) are shown. (B) PCR products from the UL34 locus in the same viruses are shown. Digital micrographs of immunofluorescently stained plaques formed on Vero (C to E), wt UL34-expressing RepAC cells (F to H), or CL04-expressing CL04AI cells (I to K) by viruses rescued from wt HSV-1(F) BAC (C, F, and I), UL34-null/UL31 wt BAC (D, G, and J), or UL34-null/UL31 R229L mutant BAC (E, H, and K).

    Article Snippet: Nitrocellulose sheets bearing proteins of interest were blocked in 5% nonfat milk plus 0.2% Tween 20 for at least 2 h. The membranes were probed with a previously described chicken polyclonal antibody directed against pUL34 (1:1,000) ( ) and subjected to reaction with alkaline phosphatase-conjugated anti-chicken secondary antibody (Aves Laboratories), with mouse monoclonal antibody directed against the HSV-1 scaffolding protein (1:2,000) (Serotec) and reacted with alkaline phosphatase-conjugated anti-mouse secondary antibody (Sigma), or with anti-FLAG M2 mouse monoclonal antibody (Sigma) and reacted with alkaline-phosphatase-conjugated anti-mouse.

    Techniques: BAC Assay, Derivative Assay, Mutagenesis, Expressing, Polymerase Chain Reaction, Molecular Weight, Staining

    Cell-to-cell spread of HSV gE ET domain mutants in HaCaT and ARPE-19 cells. Human HaCaT keratinocytes or retinal epithelial ARPE-19 cells were infected with wild-type HSV-1 strain F or F-gEβ (derived from F), F-BAC, or F-BACgE448, which lacks

    Journal:

    Article Title: The Extracellular Domain of Herpes Simplex Virus gE Is Indispensable for Efficient Cell-to-Cell Spread: Evidence for gE/gI Receptors

    doi: 10.1128/JVI.79.18.11990-12001.2005

    Figure Lengend Snippet: Cell-to-cell spread of HSV gE ET domain mutants in HaCaT and ARPE-19 cells. Human HaCaT keratinocytes or retinal epithelial ARPE-19 cells were infected with wild-type HSV-1 strain F or F-gEβ (derived from F), F-BAC, or F-BACgE448, which lacks

    Article Snippet: The cells were washed, permeabilized with 0.2% Tween 20, and stained with rabbit anti-HSV-1 serum (Dako, Copenhagen, Denmark) followed by donkey anti-rabbit immunoglobulin G (IgG) antibodies conjugated with horseradish peroxidase (Amersham), and plaques were visualized by the addition of a peroxidase substrate, 3,3′-diaminobenzidine hydrochloride (Sigma).

    Techniques: Infection, Derivative Assay, BAC Assay

    IgG binding of gE ET domain mutants. HaCaT cells were infected with wild-type HSV-1 (F); F-gEβ; F-gEΔCT; or F-BAC, F-BACgE488, or gE ET domain mutants at 10 PFU/cell for 18 h. The cells were incubated with 51 Cr-labeled, IgG-coated sheep

    Journal:

    Article Title: The Extracellular Domain of Herpes Simplex Virus gE Is Indispensable for Efficient Cell-to-Cell Spread: Evidence for gE/gI Receptors

    doi: 10.1128/JVI.79.18.11990-12001.2005

    Figure Lengend Snippet: IgG binding of gE ET domain mutants. HaCaT cells were infected with wild-type HSV-1 (F); F-gEβ; F-gEΔCT; or F-BAC, F-BACgE488, or gE ET domain mutants at 10 PFU/cell for 18 h. The cells were incubated with 51 Cr-labeled, IgG-coated sheep

    Article Snippet: The cells were washed, permeabilized with 0.2% Tween 20, and stained with rabbit anti-HSV-1 serum (Dako, Copenhagen, Denmark) followed by donkey anti-rabbit immunoglobulin G (IgG) antibodies conjugated with horseradish peroxidase (Amersham), and plaques were visualized by the addition of a peroxidase substrate, 3,3′-diaminobenzidine hydrochloride (Sigma).

    Techniques: Binding Assay, Infection, BAC Assay, Incubation, Labeling

    Cartoon of HSV-1 gE and description of gE ET domain mutants. (A) HSV gE is a type 1 membrane glycoprotein with a 25-amino-acid signal sequence (SS), a 396-residue extracellular (ET) domain, a 25-amino-acid transmembrane (TM) domain, and a 106-residue

    Journal:

    Article Title: The Extracellular Domain of Herpes Simplex Virus gE Is Indispensable for Efficient Cell-to-Cell Spread: Evidence for gE/gI Receptors

    doi: 10.1128/JVI.79.18.11990-12001.2005

    Figure Lengend Snippet: Cartoon of HSV-1 gE and description of gE ET domain mutants. (A) HSV gE is a type 1 membrane glycoprotein with a 25-amino-acid signal sequence (SS), a 396-residue extracellular (ET) domain, a 25-amino-acid transmembrane (TM) domain, and a 106-residue

    Article Snippet: The cells were washed, permeabilized with 0.2% Tween 20, and stained with rabbit anti-HSV-1 serum (Dako, Copenhagen, Denmark) followed by donkey anti-rabbit immunoglobulin G (IgG) antibodies conjugated with horseradish peroxidase (Amersham), and plaques were visualized by the addition of a peroxidase substrate, 3,3′-diaminobenzidine hydrochloride (Sigma).

    Techniques: Sequencing

    Immunoprecipitation of radiolabeled gE/gI from cells infected with HSV gE ET domain mutants. R-970 cells were infected with wild-type HSV-1 (F), F-gEβ, and various gE mutants. The cells were labeled with [ 35 S]methionine-cysteine from 6 to 9 h

    Journal:

    Article Title: The Extracellular Domain of Herpes Simplex Virus gE Is Indispensable for Efficient Cell-to-Cell Spread: Evidence for gE/gI Receptors

    doi: 10.1128/JVI.79.18.11990-12001.2005

    Figure Lengend Snippet: Immunoprecipitation of radiolabeled gE/gI from cells infected with HSV gE ET domain mutants. R-970 cells were infected with wild-type HSV-1 (F), F-gEβ, and various gE mutants. The cells were labeled with [ 35 S]methionine-cysteine from 6 to 9 h

    Article Snippet: The cells were washed, permeabilized with 0.2% Tween 20, and stained with rabbit anti-HSV-1 serum (Dako, Copenhagen, Denmark) followed by donkey anti-rabbit immunoglobulin G (IgG) antibodies conjugated with horseradish peroxidase (Amersham), and plaques were visualized by the addition of a peroxidase substrate, 3,3′-diaminobenzidine hydrochloride (Sigma).

    Techniques: Immunoprecipitation, Infection, Labeling

    A, Representative images (phase contrast or fluorescence, 10X) of BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed virus reconstitution following pBAC-BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed-pA electroporation into BEK or BEK expressing cre recombinase. B, Time after electroporation (hours) employed by pBAC-BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed-pA and pBAC-BoHV-4 to get viral reconstitution and plaque formation, following electroporation into BEK expressing cre cells (The data presented are the means ± standard errors of 3 electroporation for each BAC). C, Replication kinetics of BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed compared with BoHV-4-A. The data presented are the means ± standard errors of triplicate measurements ( P > .05 for all time points as measured by Student's t test). D, Representative images (10X) of plaque morphology and relative plaque size of BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed and BoHV-4-A on Vero cells. E, The plaque sizes (μm 2 ) software program. Bars represent means ± standard errors of 50 plaques for each virus; P > .05.

    Journal: Neuro-Oncology

    Article Title: Herpes simplex virus type 1 thymidine kinase-armed bovine herpesvirus type 4-based vector displays enhanced oncolytic properties in immunocompetent orthotopic syngenic mouse and rat glioma models

    doi: 10.1093/neuonc/nor219

    Figure Lengend Snippet: A, Representative images (phase contrast or fluorescence, 10X) of BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed virus reconstitution following pBAC-BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed-pA electroporation into BEK or BEK expressing cre recombinase. B, Time after electroporation (hours) employed by pBAC-BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed-pA and pBAC-BoHV-4 to get viral reconstitution and plaque formation, following electroporation into BEK expressing cre cells (The data presented are the means ± standard errors of 3 electroporation for each BAC). C, Replication kinetics of BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed compared with BoHV-4-A. The data presented are the means ± standard errors of triplicate measurements ( P > .05 for all time points as measured by Student's t test). D, Representative images (10X) of plaque morphology and relative plaque size of BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed and BoHV-4-A on Vero cells. E, The plaque sizes (μm 2 ) software program. Bars represent means ± standard errors of 50 plaques for each virus; P > .05.

    Article Snippet: Membranes were incubated with monoclonal anti-HSV-1 TK (clone sc-28037 [vN-20]; Santa Cruz Biotechnology), probed with horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (Sigma), and visualized by enhanced chemiluminescence (ECL kit; Pierce).

    Techniques: Fluorescence, Electroporation, Expressing, BAC Assay, Software

    Representative BoHV-4-A-TK HSV-1 -IRES-dsRed infected cell line (10X) at different time post infection (24, 48, and 72 h) expressing red fluorescent protein (fluorescence and phase contrast microscope images) and HSV-1-TK (Western immunoblotting). Negative control was made with a cell extract coming from uninfected cells (-).

    Journal: Neuro-Oncology

    Article Title: Herpes simplex virus type 1 thymidine kinase-armed bovine herpesvirus type 4-based vector displays enhanced oncolytic properties in immunocompetent orthotopic syngenic mouse and rat glioma models

    doi: 10.1093/neuonc/nor219

    Figure Lengend Snippet: Representative BoHV-4-A-TK HSV-1 -IRES-dsRed infected cell line (10X) at different time post infection (24, 48, and 72 h) expressing red fluorescent protein (fluorescence and phase contrast microscope images) and HSV-1-TK (Western immunoblotting). Negative control was made with a cell extract coming from uninfected cells (-).

    Article Snippet: Membranes were incubated with monoclonal anti-HSV-1 TK (clone sc-28037 [vN-20]; Santa Cruz Biotechnology), probed with horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (Sigma), and visualized by enhanced chemiluminescence (ECL kit; Pierce).

    Techniques: Infection, Expressing, Fluorescence, Microscopy, Western Blot, Negative Control

    A, Phase contrast microscope images of RD-4 cells (10X) infected with BoHV-4-A-TK HSV-1 -IRES-dsRed (BoHV-4 TKHSV1 ) or BoHV-4-A and treated (GCV+) or untreated (GCV−) with GCV. B, Infection and treatment quantified by MTT assay. (The data presented are the means ± standard errors of three different experiments). C, Representative images of apoptotic cell death detected by DNA laddering in cells infected with BoHV-4-A-TK HSV-1 -IRES-dsRed (BoHV-4-TK HSV1 ) and treated with GCV but not with the other combinations of infection/treatment.

    Journal: Neuro-Oncology

    Article Title: Herpes simplex virus type 1 thymidine kinase-armed bovine herpesvirus type 4-based vector displays enhanced oncolytic properties in immunocompetent orthotopic syngenic mouse and rat glioma models

    doi: 10.1093/neuonc/nor219

    Figure Lengend Snippet: A, Phase contrast microscope images of RD-4 cells (10X) infected with BoHV-4-A-TK HSV-1 -IRES-dsRed (BoHV-4 TKHSV1 ) or BoHV-4-A and treated (GCV+) or untreated (GCV−) with GCV. B, Infection and treatment quantified by MTT assay. (The data presented are the means ± standard errors of three different experiments). C, Representative images of apoptotic cell death detected by DNA laddering in cells infected with BoHV-4-A-TK HSV-1 -IRES-dsRed (BoHV-4-TK HSV1 ) and treated with GCV but not with the other combinations of infection/treatment.

    Article Snippet: Membranes were incubated with monoclonal anti-HSV-1 TK (clone sc-28037 [vN-20]; Santa Cruz Biotechnology), probed with horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (Sigma), and visualized by enhanced chemiluminescence (ECL kit; Pierce).

    Techniques: Microscopy, Infection, MTT Assay, DNA Laddering

    Percentage (mean ± standard error of the mean) of apoptosis (A, C, E, and G) and necrosis (B, D, F, and H) induced by different BoHV-4 based treatments in different glioma cells: GL261 (A and B), F98 (C and D), GLI36 (E and F) and P26 (G and H). The black bar represents the treatment with BoHV-4-A-TK HSV-1 -IRES-dsRed (BoHV-4-TK HSV1 ) + GCV and it has been compared to each of the other conditions with t test. In A, B, C, D, E, and G the induced cell death is higher in BoHV-4 TKHSV1 + GCV than in each of the other treatment. In F and H the induced necrosis was significantly lower if compared to the other BoHV-4 groups and significantly higher if compared to the GCV and control groups (*** t test, P

    Journal: Neuro-Oncology

    Article Title: Herpes simplex virus type 1 thymidine kinase-armed bovine herpesvirus type 4-based vector displays enhanced oncolytic properties in immunocompetent orthotopic syngenic mouse and rat glioma models

    doi: 10.1093/neuonc/nor219

    Figure Lengend Snippet: Percentage (mean ± standard error of the mean) of apoptosis (A, C, E, and G) and necrosis (B, D, F, and H) induced by different BoHV-4 based treatments in different glioma cells: GL261 (A and B), F98 (C and D), GLI36 (E and F) and P26 (G and H). The black bar represents the treatment with BoHV-4-A-TK HSV-1 -IRES-dsRed (BoHV-4-TK HSV1 ) + GCV and it has been compared to each of the other conditions with t test. In A, B, C, D, E, and G the induced cell death is higher in BoHV-4 TKHSV1 + GCV than in each of the other treatment. In F and H the induced necrosis was significantly lower if compared to the other BoHV-4 groups and significantly higher if compared to the GCV and control groups (*** t test, P

    Article Snippet: Membranes were incubated with monoclonal anti-HSV-1 TK (clone sc-28037 [vN-20]; Santa Cruz Biotechnology), probed with horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (Sigma), and visualized by enhanced chemiluminescence (ECL kit; Pierce).

    Techniques:

    A, Diagram exemplifying the experimental strategy used. B, Body weight gain in healthy mice treated with BoHV-4-A-TK HSV-1 -IRES-dsRed and GCV, BoHV-4-A-TK HSV-1 -IRES-dsRed, GCV or PBS. The curves were analyzed with one way ANOVA and no differences were found.

    Journal: Neuro-Oncology

    Article Title: Herpes simplex virus type 1 thymidine kinase-armed bovine herpesvirus type 4-based vector displays enhanced oncolytic properties in immunocompetent orthotopic syngenic mouse and rat glioma models

    doi: 10.1093/neuonc/nor219

    Figure Lengend Snippet: A, Diagram exemplifying the experimental strategy used. B, Body weight gain in healthy mice treated with BoHV-4-A-TK HSV-1 -IRES-dsRed and GCV, BoHV-4-A-TK HSV-1 -IRES-dsRed, GCV or PBS. The curves were analyzed with one way ANOVA and no differences were found.

    Article Snippet: Membranes were incubated with monoclonal anti-HSV-1 TK (clone sc-28037 [vN-20]; Santa Cruz Biotechnology), probed with horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (Sigma), and visualized by enhanced chemiluminescence (ECL kit; Pierce).

    Techniques: Mouse Assay

    A, hCMVie-TK HSV-1 -IRES-dsRed-pA expression cassette diagram (not on scale) containing the human cytomegalovirus immediate early promoter (CMVp; blue), the HSV-1 -TK ORF (TK HSV-1 ; yellow), an internal ribosomal entry site (IRES; white), the red fluorescent protein ORF (RFP; red) and a polyadenylation signal (pA; white). B, Representative image of cells transiently transfected with hCMVie-TK HSV-1 -IRES-dsRed-pA, expressing the RFP as visualized by a fluorescence microscope with a red filter (TRITC) and their nuclei counterstained with DAPI, which co-localized with red cells when overimposed (MERGE). C, Western immunoblotting of the same hCMVie-TK HSV-1 -IRES-dsRed-pA transiently transfected cells expressing HSV-1-TK (+). A negative control was performed with cells transiently transfected with an empty vector (-). D, Schematic diagram (not on scale) of the retargeting strategy employed on BAC-BoHV-4-A genome targeted at the TK locus with a KanaGalK selectable cassette (pBAC-BoHV-4-A-TK-KanaGalK-TK). The Kana/GalK cassettes were removed via heat-inducible homologous recombination and replaced with hCMVie-TK HSV-1 -IRES-dsRed-pA expression cassette. E, The selected colonies were tested through HindIII restriction enzyme analysis, agar gel electrophoresis and Southern blotting. The retargeted clones were detected through the disappearance of the 2.6 kb band (indicated by a white arrow) and the appearance of a 2.2 kb band detected by Southern blotting. F, pBAC-BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed-pA clonal stability in Escherichia coli SW102 cells, passaged for 21 consecutive days and analyzed by HindIII digestion and agarose gel electrophoresis. The unretargeted pBAC-BoHV-4-A-TK-KanaGalK-TK was used as a control.

    Journal: Neuro-Oncology

    Article Title: Herpes simplex virus type 1 thymidine kinase-armed bovine herpesvirus type 4-based vector displays enhanced oncolytic properties in immunocompetent orthotopic syngenic mouse and rat glioma models

    doi: 10.1093/neuonc/nor219

    Figure Lengend Snippet: A, hCMVie-TK HSV-1 -IRES-dsRed-pA expression cassette diagram (not on scale) containing the human cytomegalovirus immediate early promoter (CMVp; blue), the HSV-1 -TK ORF (TK HSV-1 ; yellow), an internal ribosomal entry site (IRES; white), the red fluorescent protein ORF (RFP; red) and a polyadenylation signal (pA; white). B, Representative image of cells transiently transfected with hCMVie-TK HSV-1 -IRES-dsRed-pA, expressing the RFP as visualized by a fluorescence microscope with a red filter (TRITC) and their nuclei counterstained with DAPI, which co-localized with red cells when overimposed (MERGE). C, Western immunoblotting of the same hCMVie-TK HSV-1 -IRES-dsRed-pA transiently transfected cells expressing HSV-1-TK (+). A negative control was performed with cells transiently transfected with an empty vector (-). D, Schematic diagram (not on scale) of the retargeting strategy employed on BAC-BoHV-4-A genome targeted at the TK locus with a KanaGalK selectable cassette (pBAC-BoHV-4-A-TK-KanaGalK-TK). The Kana/GalK cassettes were removed via heat-inducible homologous recombination and replaced with hCMVie-TK HSV-1 -IRES-dsRed-pA expression cassette. E, The selected colonies were tested through HindIII restriction enzyme analysis, agar gel electrophoresis and Southern blotting. The retargeted clones were detected through the disappearance of the 2.6 kb band (indicated by a white arrow) and the appearance of a 2.2 kb band detected by Southern blotting. F, pBAC-BoHV-4-A-hCMVie-TK HSV-1 -IRES-dsRed-pA clonal stability in Escherichia coli SW102 cells, passaged for 21 consecutive days and analyzed by HindIII digestion and agarose gel electrophoresis. The unretargeted pBAC-BoHV-4-A-TK-KanaGalK-TK was used as a control.

    Article Snippet: Membranes were incubated with monoclonal anti-HSV-1 TK (clone sc-28037 [vN-20]; Santa Cruz Biotechnology), probed with horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (Sigma), and visualized by enhanced chemiluminescence (ECL kit; Pierce).

    Techniques: Expressing, Transfection, Fluorescence, Microscopy, Western Blot, Negative Control, Plasmid Preparation, BAC Assay, Homologous Recombination, Nucleic Acid Electrophoresis, Southern Blot, Clone Assay, Agarose Gel Electrophoresis

    Human MxB is a herpesvirus restriction factor. (a) U87MG cells expressing HA-tagged Mx proteins were infected (MOI of 0.5) with GFP-encoding HIV-1 VSV-G pseudotyped lentiviral vector, HSV-1, MCMV, MHV68, Ad5, MVA, VSV, or IAV (H7N7) and subjected to fluorescence-activated cell sorter (FACS) analysis 20 hpi or 6 hpi (only VSV) or 48 hpi (only HIV-1). The percentage of GFP + cells relative to nontransduced control cells (NT) is shown. For Western blot analysis, Mx was detected by anti-HA antibody. Actin served as a control. (b) A549 cells expressing untagged Mx proteins were infected (MOI of 50) with GFP-encoding HCMV. Data represent percentages of GFP + cells relative to cells transduced with empty vector (ctrl) and the relative values of the mean fluorescence intensities (MFI). For Western blot analysis, MxB was detected by anti-MxB antibody. Actin served as a control. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. ***,

    Journal: Journal of Virology

    Article Title: Human MxB Protein Is a Pan-herpesvirus Restriction Factor

    doi: 10.1128/JVI.01056-18

    Figure Lengend Snippet: Human MxB is a herpesvirus restriction factor. (a) U87MG cells expressing HA-tagged Mx proteins were infected (MOI of 0.5) with GFP-encoding HIV-1 VSV-G pseudotyped lentiviral vector, HSV-1, MCMV, MHV68, Ad5, MVA, VSV, or IAV (H7N7) and subjected to fluorescence-activated cell sorter (FACS) analysis 20 hpi or 6 hpi (only VSV) or 48 hpi (only HIV-1). The percentage of GFP + cells relative to nontransduced control cells (NT) is shown. For Western blot analysis, Mx was detected by anti-HA antibody. Actin served as a control. (b) A549 cells expressing untagged Mx proteins were infected (MOI of 50) with GFP-encoding HCMV. Data represent percentages of GFP + cells relative to cells transduced with empty vector (ctrl) and the relative values of the mean fluorescence intensities (MFI). For Western blot analysis, MxB was detected by anti-MxB antibody. Actin served as a control. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. ***,

    Article Snippet: The following viruses were used: HSV-1 (McIntyre) (ATCC, VR-539D); HSV-1 (F) ( ); HSV-1 GFP (17+ ) ( ); HSV-2 (MS) (ATCC, VR-734); HCMV (TB40-SE-EGFP) ( ); MCMV Δ1Δ6 GFP (generated by inserting a GFP expression cassette into a ΔMCMV bacterial artificial chromosome [BAC] via its FLP recombination target [FRT] site [ ]); wild-type MCMV rescued from BAC pSM3fr-MCK-2fl clone 3.3 ( ); MHV68 GFP ( ); Ad5 GFP ( ); MVA GFP ( ); VSV GFP ( ); and IAV GFP (A/Seal/MA/1/80-SC35M, H7N7) ( ).

    Techniques: Expressing, Infection, Plasmid Preparation, Fluorescence, FACS, Western Blot, Transduction