KSHV lytic reactivation induces the double strand break response, and the formation of DNA breaks.

<p>(<b>A</b>), TREx BCBL Rta cells remained unreactivated or were reactivated for 24 hours and fixed for immunofluorescence. Cells were then stained using a monoclonal antibody to ORF57 to show reactivation and a monoclonal antibody to γH2A.x to show sites of DNA damage. (<b>B</b>) A time-course of protein expression in reactivated TREx BCBL Rta cells over 0, 8, 16 and 24 hours post induction. A monoclonal antibody to GAPDH was used to show equal loading, and monoclonal antibodies to Myc for Rta and to ORF57 were used to show KSHV reactivation. To demonstrate DNA damage, a polyclonal antibody was used to show total levels of H2A.x and a monoclonal antibody for the phosphorylated form, γH2A.x. (<b>C</b>) For comet assays, TREx BCBL Rta cells were reactivated for 24 hours and reactivation assessed by western blot using monoclonal antibodies to Myc for Rta and to ORF57. A monoclonal antibody to GAPDH was also used to show equal loading. (<b>D</b>). Comet assays were performed and scored using CometScore and tail moments calculated (<b>E</b>); n- and <i>P</i>-values are represented in the figure and error bars show the standard error from the mean.</p

Model of how sequestration of hTREX by ORF57 leads to R-loops and genome instability.

<p>In a healthy cell, components of hTREX are recruited to cellular pre-mRNA during the inter-linked processes of transcription and splicing. These components then act to stabilise the newly transcribed mRNA. In situations when hTREX is rendered non-functional through mutation or siRNA, the newly transcribed mRNA can become unstable and anneal to the template strand of DNA forming R-loops and leading to an increase in DNA strand breaks. During KSHV infection or exogenous ORF57 expression, ORF57 recruits the hTREX complex, essentially replicating a system of mutated hTREX and leading to an increase in genome instability.</p

Model of how sequestration of hTREX by ORF57 leads to R-loops and genome instability.

<p>In a healthy cell, components of hTREX are recruited to cellular pre-mRNA during the inter-linked processes of transcription and splicing. These components then act to stabilise the newly transcribed mRNA. In situations when hTREX is rendered non-functional through mutation or siRNA, the newly transcribed mRNA can become unstable and anneal to the template strand of DNA forming R-loops and leading to an increase in DNA strand breaks. During KSHV infection or exogenous ORF57 expression, ORF57 recruits the hTREX complex, essentially replicating a system of mutated hTREX and leading to an increase in genome instability.</p

ORF57 expression induces the DNA damage response through phosphorylation of H2A.x.

<p>(<b>A</b>) iORF57 293 cells were either uninduced, treated with 50 µM etoposide for 30 minutes to induce DSBs, or induced to express ORF57, fixed on coverslips and visualised by confocal microscopy. A polyclonal antibody against Flag was used to detect ORF57 and a monoclonal antibody against γH2A.x was used to demonstrate cells with DSBs. (<b>B</b>) iORF57 293 cells were either uninduced, treated with 50 µM etoposide for 30 minutes, or induced for 48 hours. Cell lysates were collected and western blots performed to analyse protein levels. A monoclonal antibody to GAPDH was used to show equal loading, a polyclonal antibody to Flag to show ORF57 expression and a monoclonal antibody to γH2A.x to demonstrate DSBs. (<b>C</b>) iORF57 293 cells were either uninduced or induced for 48 hours and alkaline comet assays were performed. (<b>D</b>) Comets were scored using CometScore and tail moments calculated. n- and <i>P</i>-values are represented in the figure and error bars show the standard error from the mean.</p

ORF57-induced double strand breaks are the result of R-loop formation and sequestration of hTREX.

<p>(<b>A</b>) Alkaline comet assays were performed on HEK 293T cells and protein levels for each assay were assessed by western blot using a monoclonal antibody to GAPDH as a loading control; monoclonal antibody to Myc for Myc-Aly; monoclonal antibody to GFP for ORF57pMut, EGFP-RNaseH1 and EGFP-AID; polyclonal antibody to RFP for mCherry and mCherry-ORF57. (<b>B</b>) Cells were transfected with mCherry or mCherry-ORF57, or treated with 50 µM etoposide for 30 minutes as a positive control, visualised by fluorescence microscopy to confirm transfection efficiency (<b>i</b>); comet assays performed and scored using CometScore (<b>ii</b>); and tail moments calculated (<b>iii</b>). (<b>C</b>) To demonstrate R-loop formation, cells were co-transfected with mCherry-ORF57 and either pMSCVgfp::AID or pEGFP-RNH, visualised by fluorescence microscopy to confirm transfection efficiency (<b>i</b>); comet assays performed and scored using CometScore (<b>ii</b>); and tail moments calculated (<b>iii</b>). (<b>D</b>)To demonstrate sequestration of hTREX by ORF57 as the cause of R-loop formation, cells were either transfected with pEGFP-57pmut or co-transfected with mCherry-ORF57 and Myc-Aly visualised by fluorescence microscopy to confirm transfection efficiency (<b>i</b>); comet assays performed and scored using CometScore (<b>ii</b>); and tail moments calculated (<b>iii</b>). n- and <i>P</i>-values are represented for all data in the figure and error bars show the standard error from the mean.</p

Chromosome instability in ORF57 expressing cells.

<p>(<b>A</b>) iORF57 293 cells remained uninduced or induced for 24 hours, then fixed on coverslips and DNA-stained with DAPI. Confocal microscopy was used to identify and image mitotic cells. Three representative images are shown for uninduced and induced cells and merged images include the phase contrast image. Chromosome lagging in the induced samples is highlighted by white arrows. (<b>B</b>) HEK 293T cells mock transfected or transfected with EGFP or EGFP-ORF57 were fixed onto coverslips and stained with DAPI for imaging of mitotic cells. Chromosome lagging in the EGFP-ORF57 expressing cell is highlighted by a white arrow.</p

SILAC analysis demonstrates the induction of double strand breaks in ORF57 expressing cells.

<p>Fold-increases of proteins involved in NHEJ upon ORF57 expression are presented along with the number of peptide hits for each protein. UniProt numbers are shown for reference.</p

HSV-1 ICP27 induces R-loop mediated DNA damage due to hTREX recruitment.

<p>(<b>A</b>) HEK 293T cells were transfected with EGFP, EGFP-ICP27, EGFP-ICP27WRL or a dual transfections of EGFP-ICP27 along with Myc-Aly, EGFP-RNaseH1 or pMSCVgfp::AID for 24 hours alkaline comet assays were performed. (<b>B</b>) Comet tails were scored using CometScore and n- and <i>P</i>-values are represented for all data and error bars show the standard error from the mean.</p

ORF57 induces double strand breaks in a neutral comet assay.

<p>Neutral comet assays were performed to assess double-strand breaks. (<b>A</b>) Cells were either treated with 50 µM etoposide for 15 minutes as a positive control, transfected with mCherry, or transfected with mCherry-ORF57 and analysed by fluorescence microscopy to assess transfection efficiency. (<b>B</b>) Protein expression was assessed by western blot using a monoclonal antibody to GAPDH as a loading control and polyclonal antibody to RFP to detect mCherry and mCherry-ORF57 expression. (<b>C</b>) Comets were performed and scored using CometScore and (<b>D</b>) tail moments calculated. N- and p- values are shown and error bars represent the standard error from the mean.</p

Lytically replicating KSHV cells demonstrate the presence of R-loops.

<p>(<b>A</b>) 293T rKSHV.219 cells were left either unreactivated, or reactivated for 36 hours and transfected with Myc-Aly or EGFP-RNaseH1 and alkaline comet assays were performed. (<b>B</b>) Comet tails were scored using CometScore and n- and <i>P</i>-values are represented for all data and error bars show the standard error from the mean.</p