Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Kinetics of shelterin genes expression during HHV-6A/B infection. HSB-2 cells (A-G) and Molt3 cells (H-M) were respectively infected with HHV-6A or HHV-6B. At various time post infection, total RNA was extracted and analyzed by reverse transcriptase QPCR for TRF1, TRF2, POT1, RAP1, TIN2, TPP1, GAPDH and U90 genes expression. Shelterin genes expression was normalized relative to GAPDH gene expression while U90 was analyzed to demonstrate infection. Results represent data from 4-6 independent experiments expressed as mean +/-SD gene expression relative to that of uninfected cells. *p<0.05.

Article Snippet: The TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the MBP coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Expressing, Infection

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: TRF2 expression during productive HHV-6A/B infections. Mock, HHV-6A- or HHV-6B-infected cells were analyzed for TRF2 expression by flow cytometry. Uninfected and 5 days old HHV-6A-infected HSB-2 cells (A-B) and HHV-6B-infected Molt3 cells (C) were fixed, permeabilized and stained for TRF2, P41 and gp102 proteins expression. Numbers in the top and bottom left quadrants indicate mean relative TRF2 fluorescence intensities. Results are representative of two independent experiments. D) Western blot analysis of TRF2 expression in HHV-6A/B infected. Tubulin was used as loading controls and IE1 to demonstrate HHV-6A/B infection. Numbers represent TRF2 expression levels relative to mock-infected cells after normalization with tubulin.

Article Snippet: The TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the MBP coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Expressing, Infection, Flow Cytometry, Staining, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Increased TRF2 expression in HHV-6A-infected U2OS cells. U2OS cells were infected with HHV-6A and analyzed for TRF2 and IE2 expression at 24h, 48h and 72h post-infection by dual color immunofluorescence. A) Representative TRF2 and IE2 expression in bystander and IE2 expressing cells at 48h post infection. B) Mean relative TRF2 expression + SD in uninfected (white), IE2-(green-uninfected bystander) or IE2+ (red-infected) cells at 24h, 48h and 72h post infection. Each symbol represents the relative TRF2 expression from a single cell.

Article Snippet: The TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the MBP coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Expressing, Infection, Immunofluorescence

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Binding of TRF2 to HHV-6 viral DNA. Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled telomeric dsDNA (A) and binding was assessed by EMSA. Excess of unlabeled telomeric and non-telomeric dsDNA were added as competitors. Samples were migrated on non-denaturing acrylamide gel, dried and exposed to X-ray films. B) Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled non-telomeric dsDNA and binding was assessed by EMSA. C) Recombinant MBP and MBP-TRF2 were coated to the wells of a 96 well-plate and incubated with HaeIII digested DIG-labeled HHV-6A DNA (25 ng/condition) in the presence or absence of competitors. After washing, bound DNA was quantified by adding peroxidase-labeled anti-DIG antibodies and substrate. Results are expressed as mead absorbance +SD of triplicate values. Experiment is representative of two additional experiments. *** P<0.001.

Article Snippet: The TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the MBP coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Binding Assay, Recombinant, Incubation, Labeling, Acrylamide Gel Assay

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Colocalization of shelterin complex proteins and HHV-6A IE2 protein at viral and cellular telomeres. A) U2OS cells were infected for 48h with HHV-6A after which cells were processed for IF-FISH. Telomeres were labeled in blue, p41 in green and IE2 in red. These images demonstrate colocalization of IE2 with P41, a viral protein that associates with viral DNA during infection, and diffuse telomeric signals (arrows). B) Telomeres were labeled in magenta, TRF2 in green and IE2 in red. The panels in the middle row show images of cells productively infected (minority of cells) with HHV-6A. Large diffuse telomeric signals (viral replication compartments) where TRF2 and IE2 accumulates (rectangles) are represented. The panels in the third row represent infected cells that do not actively replicate viral DNA with TRF2 and IE2 colocalizing (dashed squares) at distinct telomeres. C) Colocalization of HHV-6A IE2 protein at telomeres in the absence of viral DNA. U2OS cells were transfected with an empty vector or an IE2 expression vector. Forty-eight hours later cells were processed for dual color immunofluorescence. TRF2 was labeled in green and IE2 in red. Examples of IE2 colocalizing with TRF2 are presented (dashed squares). D) U2OS cells were transfected with WT IE2 or IE2 Δ1290-1500 expression vectors. Forty-eight hours later cells were processed for IF-FISH. Telomeres were labeled in cyan, IE2 in red and nuclei in blue. Examples of IE2 colocalizing with TRF2 are presented (dashed squares). E) Uninfected and HHV-6A-infected U2OS cells were transfected with an empty vector or a myc tagged TRF1 expression vector. Forty-eight hours later cells were processed for IF-FISH. Telomeres were labels in cyan, TRF1 in green and IE2 in red. Examples of TRF1 localizing at telomeres (dashed squares) in uninfected cells are shown in the top row. Examples of IE2 colocalizing with TRF1 and telomeres in infected cells are presented in the bottom row (dashed squares). F) Uninfected and HHV-6A-infected U2OS cells were transfected with an empty vector or a myc tagged POT1 expression vector. Forty-eight hours later cells were processed for IF-FISH. Telomeres were labels in cyan, POT1 in green and IE2 in red. Examples of POT1 localizing at telomeres (dashed squares) in uninfected cells are shown in the top row. Examples of IE2 colocalizing with POT1 and telomeres in infected cells are presented in the bottom row (dashed squares).

Article Snippet: The TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the MBP coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Infection, Labeling, Transfection, Plasmid Preparation, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Binding of TRF2 to viral DNA during HHV-6A/B infection. A) Schematic representation of the HHV-6A/B genome. The DR6 probe used for hybridization is shown in red. Uninfected and HHV-6A-infected HSB-2 cells (B-C) or uninfected and HHV-6B-infectd Molt3 cells (D-E) were analyzed for TRF2 binding to viral DNA using ChIP. The input was hybridized with Alu probe to assess quantity of starting material. Anti-IgG (negative control) or TRF2 antibodies were used for immunoprecipitation. Eluted DNA was serially diluted and hybridized with 32 P-labeled telomeric (TTAGGG) 3 or HHV-6 (DR6) probes. After hybridization the membranes were washed and exposed to X-ray films. The quantity of TRF2 bound to telomeric and viral DNA is measured relative to the input. Results are of 3 independent experiments.

Article Snippet: The TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the MBP coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Binding Assay, Infection, Hybridization, Negative Control, Immunoprecipitation, Labeling

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: IE2 localized to VRC in the absence of TRF2. U2OS cells were transduced with a lentiviral vector coding for a Dox inducible shRNA against TRF2. Transduced cells were selected with puromycin for a week. A) Half of the cultures was treated with Dox for seven days to induce TRF2 knockdown (KD), as determined by western blot. B) Control (-Dox) and TRF2 KD (+Dox) cells were infected with HHV-6A for 48h and processed for IF-FISH. Telomeres were labeled in cyan, TRF2 in green and IE2 in red. As show in the–Dox condition, TRF2 colocalized with IE2 as well as diffuse (dashed square) and punctate (dashed circle) telomeric signals. In the +Dox condition, TRF2 KD was confirmed with IE2 colocalizing with diffuse telomere signals (dashed squares). C) DDR at telomeres as a consequence of TRF2 knockdown. U2OS cells were treated or not with Dox and infected with HHV-6A as in . Cells were then processed for IF-FISH. Telomeres were labeled in cyan, IE2 in red, 53BP1 (as marker of DDR) in green and nuclei in blue.

Article Snippet: The TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the MBP coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Transduction, Plasmid Preparation, shRNA, Western Blot, Infection, Labeling, Marker

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Knockdown of TRF2 does not affect HHV-6A/B replication. SUP-T1 cells were transduced with a lentiviral vector coding for a Dox inducible shRNA against TRF2. A) Transduced cells were selected with puromycin for two weeks. TRF2 knockdown (KD) was induced by adding Dox to the culture medium for three weeks and confirmed by western blot. B-C) Control (-Dox) and TRF2 KD (+Dox) SUP-T1 cells were infected with HHV-6A (B) or HHV-6B (C). Whole cell DNA was isolated at various time points and the relative number of HHV-6A/B genomes determined and normalized against cellular DNA.

Article Snippet: The TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the MBP coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Transduction, Plasmid Preparation, shRNA, Western Blot, Infection, Isolation

Journal: Molecular cell

Article Title: Nuclear acetyl-CoA production by ACLY promotes homologous recombination

doi: 10.1016/j.molcel.2017.06.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLPC TRF2 deltaB delta M vector (addgene plasmid #18008) was obtained and viral supernatant was generated using standard protocol with PhxA cells.

Techniques: Western Blot, Immunohistochemistry, Recombinant, Clone Assay, Expressing, CRISPR, shRNA, Plasmid Preparation, Construct, Software