codon-optimized rcmv ie1 ( rat ie1) template cdna  (BioCat GmbH)

Journal: PLoS Pathogens

Article Title: Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers

doi: 10.1371/journal.ppat.1009863

Figure Lengend Snippet: (A) In silico disorder prediction analysis of human ( hum ), rhesus ( rhes ) and rat ( rat ) cytomegalovirus IE1 sequences using IUPred2A . The disorder score for all three proteins suggest a globular domain with disordered N- and C-termini (scores ≥ 0.5 indicate disorder). (B) Limited proteolysis of recombinant rat IE1. Purified rat IE1 was incubated with subtilisin (1 mU protease per mg rat IE1) for different times, and samples were analyzed by SDS-PAGE and Coomassie blue staining. (C) CD spectroscopy of hum IE1 14–382, rat IE1 1–392 and rat IE1 30–392. The spectra were normalized at 207 nm as suggested by Raussens and coworkers .

Article Snippet: The codon-optimized RCMV IE1 ( rat IE1) template cDNA (strain RCMV-E, sequence based on Uniprot K7XWE8) was obtained from Biocat GmbH gene synthesis service.

Techniques: In Silico, Recombinant, Purification, Incubation, SDS Page, Staining, Spectroscopy

Journal: PLoS Pathogens

Article Title: Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers

doi: 10.1371/journal.ppat.1009863

Figure Lengend Snippet: (A) Schematic overview of full-length rat PML and deletion mutants. (B) Efficient interaction of rat IE1 CORE with rat PML in co-immunoprecipitation analysis. HEK293T cells were co-transfected with expression plasmids encoding FLAG-tagged rat IE1 or ra tIE1core (residues 1–392) and Myc-tagged rat PML variants. After cell lysis, immunoprecipitation was performed with an anti-FLAG antibody. Co-precipitated rat PML proteins (IP), precipitated rat IE1 proteins, and proteins within the cell lysate (input) were analyzed by Western blotting as indicated. (C) Binding of rat IE1 CORE to rat PML requires both the coiled-coil and the RING domain. HEK293T cells were co-transfected with expression plasmids encoding FLAG-tagged rat PML variants and Myc-tagged rat IE1 CORE (residues 1–382) as indicated. Upper two panels: Western blot detection of rat IE1 and rat PML after immunoprecipitation using an anti-FLAG antibody. Lower two panels: detection of rat IE1 and rat PML in cell lysates before precipitation (input). (D) Inhibition of rat PML SUMOylation by rat IE1 expression. HEK293T cells were transfected with expression plasmids encoding Myc- rat PML, HA-SUMO2 and FLAG- rat IE1 as indicated. After cell harvest, rat PML and SUMOylated rat PML were visualized by Western blotting using anti-Myc and anti-HA antibodies, respectively. Expression of IE1 was analyzed with an anti-FLAG antibody and β-actin was included as internal control. (E) Impact of RCMV infection on rat PML SUMOylation. Rat embryonic fibroblast (REF) cells were infected with RCMV at an MOI of 1.5 or mock infected, and were harvested at indicated times for Western Blot analysis of rat PML (upper panel), rat IE1 (middle panel), and β-actin (lower panel) as loading control. (F) Impact of RCMV infection on rat PML-NB integrity. REF cells were infected with RCMV at an MOI of 0.7 or mock infected, and were harvested at indicated times for immunofluorescence analysis of rat IE1 (left panel) or rat PML (right panel). Cell nuclei were stained with DAPI. F, FLAG; M, Myc; R, RING domain; B, B-boxes; CC, coiled-coil domain.

Article Snippet: The codon-optimized RCMV IE1 ( rat IE1) template cDNA (strain RCMV-E, sequence based on Uniprot K7XWE8) was obtained from Biocat GmbH gene synthesis service.

Techniques: Immunoprecipitation, Transfection, Expressing, Lysis, Western Blot, Binding Assay, Inhibition, Infection, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers

doi: 10.1371/journal.ppat.1009863

Figure Lengend Snippet: (A) Analysis of PML-NB integrity in rat fibroblasts after HCMV infection. REF cells were infected with HCMV strain AD169 (MOI = 0.5) or mock infected. Cells were harvested at indicated times after infection to analyze the subcellular localization of rat PML (left panel) and hum IE1 (right panel). Cell nuclei were stained with DAPI. (B) Species-specific binding of IE1 proteins to rat PML in co-immunoprecipitation analysis. HEK293T cells were co-transfected with expression plasmids coding for the TRIM motif of rat PML fused to a myc-tag ( rat PML RBCC) and either FLAG- rat IE1 CORE (residues 1–392), FLAG- hum IE1 CORE (residues 1–382) or an empty plasmid (pcDNA3). Afterwards, immunoprecipitation was performed with an anti-FLAG antibody. Left panels: Western blot detection of precipitated IE1 proteins and co-precipitated rat PML RBCC (IP). Right panels: detection of IE1 proteins and rat PML RBCC in cell lysates before precipitation (input). (C) Analysis of PML-NB integrity in human fibroblasts after RCMV infection. HFF cells were infected with RCMV-E (MOI = 0.5) or mock infected. Cells were fixed at indicated times for immunofluorescence analysis of hum PML and rat IE1. Cell nuclei were visualized by DAPI staining. (D) Species-specific binding of IE1 proteins to hum PML in co-immunoprecipitation analysis. HEK293T cells were co-transfected with expression plasmids encoding myc-tagged hum PML and either FLAG- rat IE1 CORE (residues 1–392), FLAG- hum IE1 CORE (residues 1–382) or an empty plasmid (pcDNA3). After immunoprecipitation of IE1 with an anti-FLAG antibody, co-precipitated hum PML (left panels) as well as proteins in the lysate before precipitation (right panels) were detected by Western blotting.

Article Snippet: The codon-optimized RCMV IE1 ( rat IE1) template cDNA (strain RCMV-E, sequence based on Uniprot K7XWE8) was obtained from Biocat GmbH gene synthesis service.

Techniques: Infection, Staining, Binding Assay, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers

doi: 10.1371/journal.ppat.1009863

Figure Lengend Snippet: (A, B) Increased initiation of RCMV gene expression in hum IE1-expressing HFF. HFF with doxycycline-inducible expression of FLAG-tagged hum IE1 (HFF/ hum IE1) or control cells (HFF/control) were treated with doxycycline (+ Dox) or mock treated (- Dox) for 24 h and subsequently infected with RCMV-E (MOI = 0.1). At 8 h post-infection (hpi), cells were harvested for Western Blot analysis of rat IE1 as well as hum IE1 with an anti-FLAG antibody and β-actin as loading control (A) or for immunofluorescence detection of rat IE1, hum IE1 (FLAG), and cell nuclei by DAPI staining (B). The percentage of rat IE1-positive cells was determined from triplicate samples. (C) Release of infectious RCMV particles from hum IE1-expressing HFF. HFF/control and HFF/ hum IE1 were infected with RCMV-E at an MOI of 0.01 after 24 h of doxycycline treatment. Supernatants were harvested at 6 d post infection and titrated on REF cells. Values are derived from triplicate samples and represent mean values ± SD. P-values were calculated using two-tailed Student’s t-test. ***, p ≤ 0.001. (D) Multistep growth curve analysis of RCMV in hum IE1-expressing HFF. HFF/control, HFF/ hum IE1 and HFF/ hum IE1 CORE , which express residues 1–382 of hum IE1, were treated with doxycycline for 24 h and subsequently infected with RCMV-E at an MOI of 0.01. Supernatants were harvested at indicated times after infection and analyzed for genome equivalents by RCMV gB-specific quantitative real-time PCR. (E) Increased initiation of RCMV gene expression in PML-depleted human fibroblasts. HFF expressing a control shRNA (HFF/shControl) or a shRNA directed against PML (HFF/shPML) were infected with RCMV-E (MOI = 0.1). At 8 hpi, cells were fixed for immunofluorescence detection of rat IE1 and hum PML. Cell nuclei were visualized by DAPI staining. The percentage of rat IE1-positive cells was quantified from triplicate samples. (F) Multistep growth curve analysis of RCMV in PML-knockdown HFF. HFF/shControl and HFF/shPML infected with RCMV-E at an MOI of 0.01. Supernatants were harvested at indicated times after infection and analyzed for genome equivalents by RCMV gB-specific quantitative real-time PCR. (G) Colocalization of RCMV genomes with PML-NBs in human fibroblasts. HFF cells were infected with RCMV-EdC at an MOI of 0.05 or were mock infected. At 8 hpi, cells were fixed for click labeling to visualize RCMV genomes (vDNA) in combination with immunofluorescence detection of rat IE1 and hum PML. DAPI staining was performed to visualize cell nuclei. Arrows in the merged PML-vDNA image indicate RCMV genomes colocalizing with PML-NBs. Dashed lines indicate the position of the cell nuclei. (H) Increased initiation of HCMV gene expression in rat IE1-expressing REF. REF/control and REF/ rat IE1 were treated with doxycycline for 24 h and subsequently infected with HCMV strain AD169 (MOI = 0.1). At 24 hpi, cells were harvested for immunofluorescence analysis of hum IE1, followed by quantification of hum IE1-positive cells from triplicate samples. Rat IE1 expression was confirmed by staining with an anti-FLAG antibody and cell nuclei were detected with DAPI. (I, J) Release of infectious HCMV particles from rat IE1-expressing REF. REF/control and REF/ rat IE1 were treated with doxycycline for 24h and subsequently infected with HCMV strain AD169 (I) or TB40/E (J) at an MOI of 0.1. Supernatants were harvested at 6 d post infection and directly subjected to titration on HFF cells. Values are derived from triplicate samples and represent mean values ± SD. P-values were calculated using two-tailed Student’s t-test. **, p ≤ 0.01.

Article Snippet: The codon-optimized RCMV IE1 ( rat IE1) template cDNA (strain RCMV-E, sequence based on Uniprot K7XWE8) was obtained from Biocat GmbH gene synthesis service.

Techniques: Expressing, Infection, Western Blot, Immunofluorescence, Staining, Derivative Assay, Two Tailed Test, Real-time Polymerase Chain Reaction, shRNA, Labeling, Titration