© 2019. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML

deficiency

Mingqi Han1*, Christine E. Napier1**, Sonja Frölich2, Erdahl Teber3, Ted Wong3, Jane R.

Noble1, Eugene H. Y. Choi1, Roger D. Everett4, Anthony J. Cesare2, Roger R. Reddel1

1Cancer Research Unit, Children’s Medical Research Institute, Faculty of Medicine and

Health, The University of Sydney, Westmead, NSW 2145 Australia

2Genome Integrity Unit, Children’s Medical Research Institute, Faculty of Medicine and

Health, The University of Sydney, Westmead, NSW 2145 Australia

3Bioinformatics Group, Children’s Medical Research Institute, Faculty of Medicine and

Health, The University of Sydney, Westmead, NSW 2145 Australia

4MRC - Centre for Virus Research, 464 Bearsden Road, G61 1QH, Scotland, UK

Keywords: Sarcoma/soft-tissue malignancies, Telomeres and telomerase,

Posttranscriptional and translational control, Oncolytic virus, ATRX, PML

Summary statement: Cancer cells with ATRX deficiency are highly sensitive to lysis by

mutant herpes simplex virus-1 due to downregulated PML levels.

Financial support: Kids Cancer Alliance Scholarship to M.H., Cure Cancer Australia

Foundation Project Grant 1062240 to C.E.N., Cancer Council New South Wales (NSW)

Research Grant RG 15-12, National Health and Medical Research Council of Australia

Project Grant 1053195 and Cancer Institute NSW 11/FRL/5-02 to A.J.C., and Cancer

Council NSW Program Grant PG11-08 and National Health and Medical Research Council

of Australia Project Grant 1088646 to R.R.R.

*Current address: Department of Pulmonary and Critical Care Medicine, David Geffen

School of Medicine, University of California, Los Angeles, CA 90095, USA

**Current address: Cancer Division, Garvan Institute of Medical Research, Darlinghurst,

NSW 2010 Australia

Corresponding author:

Roger R Reddel

Children's Medical Research Institute

Faculty of Medicine and Health, The University of Sydney

214 Hawkesbury Road

Westmead, NSW 2145 Australia

Telephone: +61-2-8865-2901

Fax: +61-2-8865-2860

Email: rreddel@cmri.org.au

The authors declare no conflicts of interest

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

JCS Advance Online Article. Posted on 11 February 2019

ABSTRACT

Cancers that utilize the Alternative Lengthening of Telomeres (ALT) mechanism for

telomere maintenance are often difficult to treat and have a poor prognosis. They are also

commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a

component of PML nuclear bodies (PML NBs) which are required for intrinsic immunity to

various viruses. Here we asked whether ATRX-deficiency creates a vulnerability in ALT

cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell

types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that

degrades PML NB components including ATRX, was ten- to one thousand-fold more

effective in killing ATRX-deficient cells. Infection of co-cultured primary and ATRX-null

cancer cells revealed that mutant HSV-1 selectively killed ATRX-null cells. Sensitivity to

mutant HSV-1 infection also correlated inversely with PML protein levels, and we showed

that ATRX upregulates PML expression at both the transcriptional and post-transcriptional

levels. These data provide a basis for predicting, based on ATRX or PML levels, which

tumors will respond to a selective oncolytic herpesvirus.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

INTRODUCTION

Normal cells can divide a limited number of times, whereas cancer cell populations usually

acquire an unlimited proliferative capacity. Telomeres are the nucleoprotein structures at

the termini of chromosomes, which, due to the end replication problem, shorten with each

cell division. Most human tumors activate a telomere lengthening mechanism, either

telomerase (TEL) or alternative lengthening of telomeres (ALT), to counteract telomere

shortening and thereby enable unlimited cellular proliferation (Kim et al., 1994; Bryan et

al., 1995). Approximately 85-90% of tumors activate telomerase, usually through

dysregulated expression of its catalytic component, telomerase reverse transcriptase

(TERT), which is caused, for example, by activating mutations in the promoter region of

the TERT gene (Shay and Bacchetti, 1997; Zhang et al., 2000; Horn et al., 2013; Huang et

al., 2013). ALT is activated in many of the remaining 10-15% of cancers, and is common

in various cancers including osteosarcomas, several soft tissue sarcoma subtypes, and

astrocytomas including pediatric glioblastoma (Bryan et al., 1997; Henson et al., 2005;

Heaphy et al., 2011). Loss of the chromatin remodeling protein α-thalassemia mental

retardation X-linked (ATRX) or its heterodimeric binding partner, death domain-associated

protein 6 (DAXX) has been identified in a significant proportion of tumors and cell lines

that utilize ALT (Heaphy et al., 2011; Bower et al., 2012; Jiao et al., 2012; Lovejoy et al.,

2012).

ATRX and DAXX are constitutive components of promyelocytic leukemia nuclear

bodies (PML NBs), and these subnuclear structures are indispensable for intrinsic immunity

(Xue et al., 2003; Bieniasz, 2004). PML NBs act as a first line of defense against viral

infection, specifically by associating with and silencing viral genes (Tavalai and

Stamminger, 2008). Incomplete PML NBs generated by knockdown of one or more

constitutive PML NB proteins, such as PML, Sp100, ATRX or DAXX, did not hinder

replication of wild-type herpes simplex type 1 (WT HSV-1) in human cells (Everett et al.,

2006; Everett et al., 2008; Lukashchuk and Everett, 2010; Glass and Everett, 2013). The

HSV-1 immediate early protein ICP0, which is an E3 ubiquitin ligase (Boutell and Everett,

2003; Lilley et al., 2010), is involved in counteracting this intrinsic immunity, and ICP0-

null HSV-1 proliferates very poorly in cells with intact PML NBs (Stow and Stow, 1986;

Cai and Schaffer, 1989). However, disruption of PML NBs by knockdown of ATRX alone,

DAXX alone, DAXX and PML, or DAXX, PML and Sp100, facilitates replication of ICP0-

null HSV-1 (Everett et al., 2008; Lukashchuk and Everett, 2010; Glass and Everett, 2013).

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Here, we have investigated whether the deficiency of ATRX protein expression

which is common in ALT-dependent cancers creates an opportunity for a synthetic-lethal

treatment strategy (Kaelin, 2005). Specifically, we asked whether ICP0-null HSV-1, which

is unable to effectively infect cells with intact PML NBs, is able to infect and kill ATRX-

deficient cancer cells. We found that increased infectivity of this mutant virus (by 10- to

1,000-fold) correlates with ATRX deficiency, and also with PML deficiency. Moreover, we

found for the first time that ATRX regulates PML expression, and that this occurs at both

the transcriptional and post-transcriptional levels. These data indicate that ATRX and/or

PML levels may be used to predict response to this oncolytic virus.

RESULTS

ATRX deficiency enhances infectivity of ICP0-null HSV-1

Intrinsic immunity to viral infection involves translocation of PML NB components to the

nuclear periphery to inhibit viral replication (Everett and Murray, 2005). Using an HSV-1

with an inactivating deletion in ICP0, we compared the infectivity of WT and ICP0-null

(Mutant) HSV-1 in two pairs of closely-related cell lines. One pair consisted of a TEL-

positive cell line and its subline generated by inactivating ATRX by gene targeting (Fig.

1A). The other pair of cell lines was derived from one fibroblast line by two different

spontaneous immortalization events, with one being an ALT-positive cell line containing a

spontaneous inactivating mutation in ATRX, and the other being a TEL-positive line

expressing ATRX (Fig. 1B). We found that expression of viral proteins, including

immediate early proteins involved in replication compartment assembly (ICP4, ICP8 and

ICP27) and the capsid protein expressed at late stage (VP5), was strongly limited in WT

ATRX cells infected with mutant as compared to WT HSV-1 (Fig. 1C,D, left panels). In

contrast, WT and mutant virus produced similar levels of viral proteins in cells lacking

ATRX (Fig. 1C,D, right panels).

To determine the impact of ICP0-null HSV1 infection on cell viability we

determined plaque formation in a panel of cell lines, including the two cell line pairs (Fig.

2A,B). Each cell line's coefficient of resistance was calculated as the ratio of plaques formed

by WT versus mutant virus. The cell line panel comprised WT ATRX and ATRX-deficient

cell lines that were further subcategorized by telomere length maintenance mechanism

(ALT or TEL; Table S1). ATRX-deficient cells were 10 to 1,000 times less resistant to

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

mutant HSV-1 infection than WT ATRX cells, regardless of telomere length maintenance

mechanism. However, of ten ATRX-deficient ALT cell lines, three lines (JFCF-

6/T.1J/1.3C, IIICF/c and JFCF-6/T.1J/5H) exhibited greater than 60-fold resistance to viral

infection than the remaining seven. On average, however, these three ATRX-deficient ALT

cell lines were four times less resistant than the ATRX-positive panel of 15 cell lines.

We analyzed the cell cycle profile of the ATRX-positive and -negative cancer cell

line pair, HCT116 and HCT116 ATRXN/O, growing exponentially and asynchronously, and

found similar cell cycle profiles (percentages of cells in the G1, S and G2/M compartments

were 37.8 ± 0.4, 28.1 ± 0.3, 31.5 ± 0.5; and 34.4 ± 0.5, 27.3 ± 0.5 and 33.9 ± 1.1,

respectively; mean±s.e.m., n=3). Thus, there was no evidence that a change in cell cycle

parameters accounted for the difference in anti-viral resistance following knock-out of the

ATRX gene in this cell line.

PML NB count correlates with resistance to mutant HSV-1

Because ATRX is a constitutive component of PML NBs, we examined these nuclear

structures in the cell line panel. Automated quantitation of PML NBs using

immunofluorescent staining of PML and Sp100 proteins revealed that the resistant ATRX-

deficient/ALT cell lines contain more PML NBs than cell lines that were sensitive to mutant

virus infection (Fig. 2C; Fig. S1A). A plot of the coefficient of resistance against the number

of PML NBs/cell shows a clear distinction between cell lines permissive to infection

(defined here as having a coefficient of resistance <60) and a smaller number of PML NBs

versus cell lines that are resistant to infection and have a greater PML NB count (Fig. 2D).

These data demonstrate that resistance to the mutant virus infection segregates with the

number of PML NBs.

Inactivation of ATRX diminishes cellular PML levels

We further investigated the relationship between ATRX, PML and PML NBs and found

that the number of PML NBs, the intensity of PML immunofluorescent staining inside PML

NBs, and PML protein expression as determined by Western blot significantly correlated

with ATRX status (Fig. 3A,B; Fig. S1B). For the majority of ATRX-positive cell lines, there

was a strong correlation between ATRX and PML levels. The correlation for all cell lines

was R2 = 0.05 (Fig. S1C), but when two outliers (SK-LU-1 and JFCF-6/T.1J/6B) with high

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

ATRX expression and one (MeT-4A) with high PML were removed, R2 = 0.75 (Fig. S1D).

To determine if the relationship between ATRX and PML expression is causal, we depleted

ATRX in HT1080 fibrosarcoma cells using siRNA treatment and examined PML protein

expression. PML protein levels were significantly decreased after ATRX depletion relative

to control cells (Fig. 3C,D; Fig. S1E), demonstrating that ATRX positively regulates PML

expression. Furthermore, we confirmed this result in five ATRX-positive cell lines,

including three ALT cell lines (SK-LU-1, IIICF-E6E7/C4 and G292) and two TEL cell lines

(HeLa and Fre80-3T-sc2) (Fig. S2A). Consistent with the result demonstrated in HT1080

cells, depletion of ATRX reduced PML protein levels by up to 50% when compared to

control siRNA treated samples (Fig. S2B).

To determine whether ATRX loss of function affects the subset of PML NBs which

have telomeric content (i.e., ALT-associated PML NBs [APBs]), we quantitated APBs in

three ATRX-positive (SK-LU-1, IIICF-E6E7/C4 and IVG-bf/LXSN) and three ATRX-

deficient (GM847, IIICF/c and Saos-2) cell lines (>600 nuclei in each group). The number

of APBs per nucleus was 3.97 ± 0.14 and 3.79 ± 0.14 (mean±s.e.m.) for the ATRX-positive

and -deficient cell lines, respectively. Therefore, we found no evidence that ATRX status

affects APB numbers.

ATRX regulates PML expression at the level of both transcription and protein stability

We then investigated the mechanism whereby ATRX regulates PML, and found that PML

transcription was reduced in response to siATRX treatment (Fig. 4A). Assaying PML

degradation kinetics using the protein synthesis inhibitor cycloheximide showed that PML

protein was degraded more rapidly in siATRX-treated HT1080 cells as compared to control

cells (Fig. 4B,C). Furthermore, inhibition of the proteasome with MG132 in siATRX-treated

HT1080 cells partially stabilized PML protein (Fig. 4D,E). These data indicate that ATRX

regulates PML expression, and protects PML protein from proteasome-dependent

degradation thus resulting in the accumulation of PML protein in control cells. These data

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

indicate that ATRX controls PML expression at both the transcriptional and post-

translational level.

Modification of ATRX and/or PML expression influences sensitivity to mutant HSV-

1

We next determined whether manipulating PML expression altered the viral resistance of

the three ATRX-deficient ALT cell lines with elevated numbers of PML NBs (JFCF-

6/T.1J/5H, JFCF-6/T.1J/1.3C and IIICF/c). As expected, siPML treatment reduced PML

protein expression, and it also decreased the number of PML NBs and significantly reduced

the resistance of these cell lines to mutant HSV-1 infection (Fig. 5A; Fig. S3A,B). To further

confirm our findings that PML and ATRX expression are intimately linked to mutant HSV-

1 resistance, we depleted PML and/or ATRX in HT1080 cells. Depletion of either, or both,

proteins caused a significant reduction in PML NB numbers and a significantly reduced

resistance to mutant HSV-1 infection (Fig. 5B; Fig. S3C). These results further demonstrate

that cellular PML NB levels correlate with resistance to mutant HSV-1 infection (Fig. 5C).

Normal cells are resistant to mutant HSV-1

To gain insight into the feasibility of using the mutant HSV-1 as a therapy for ATRX-

deficient tumors, we confirmed that mutant HSV-1 replicates ~1,000-fold more effectively

in ATRX-deficient cells than in normal fibroblast or epithelial cells (Fig. 6A,B). We

obtained further evidence that mutant HSV-1 selectively targets ATRX-deficient cells by

infecting fluorescently-tagged normal and ATRX-deficient cell co-cultures with WT or

mutant HSV-1. Mutant HSV-1 infection resulted in a 61% decrease (P=0.016) in the number

of ATRX-deficient cells after only 36 hours, whereas the effect on normal cells was not

significant (Fig. 6C; Fig. S4). In contrast, WT HSV-1 infection caused equivalent decreases

in the numbers of ATRX-deficient and normal cells. The data provide conclusive evidence

that mutant HSV-1 replicates more effectively in ATRX-deficient cells than in normal cells.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

DISCUSSION

Here we have uncovered additional complexity in the relationship between the PML and

ATRX proteins and the ALT mechanism. ALT is an homologous recombination-dependent,

break-induced telomere synthesis mechanism (Reddel et al., 1997; Dunham et al., 2000;

Dilley et al., 2016; Garcia-Exposito et al., 2016), and there is some evidence that PML NBs

may play a role in this process (Pickett and Reddel, 2015). A subset of the PML NBs in

ALT cells contain telomeric DNA, some of which is extrachromosomal, together with

shelterin proteins and proteins involved in homologous recombination; because these are

highly characteristic of cancer cells that use ALT, they are referred to as ALT-associated

PML bodies (APBs) (Yeager et al., 1999). It has been observed that telomeres move into

PML bodies in ALT cells (Molenaar et al., 2003) and that this is enhanced by telomere DNA

breaks (Cho et al., 2014). PML is thought to promote clustering and recombination of

telomeres within APBs (Draskovic et al., 2009; Chung et al., 2011). Moreover, a common

genetic change in ALT cancers and cell lines is a loss-of-function mutation in ATRX

(Heaphy et al., 2011; Bower et al., 2012; Jiao et al., 2012; Lovejoy et al., 2012), which is

normally a constitutive component of PML NBs (Xue et al., 2003; Bieniasz, 2004). This is

consistent with ATRX being a suppressor of ALT (Clynes et al., 2015; Napier et al., 2015).

The mechanism whereby ATRX suppresses ALT is unknown, but one speculation

is that loss of ATRX function allows PML NBs to participate in telomere lengthening,

whereas this function is suppressed when ATRX is present. We were therefore initially

surprised to find here that the number and size of PML foci, and the PML protein content,

were reduced in ATRX-deficient ALT cells. We found that the decrease in PML levels was

a direct result of the loss of ATRX-mediated upregulation of PML levels at both the

transcriptional and post-translational levels. Moreover, we found no correlation between

ATRX status and number of APBs per nucleus. It is therefore clear that the reduced level of

PML resulting from loss of ATRX function is compatible with APB formation and ALT

activity. Three of the ATRX-deficient cell lines (JFCF-6/T.1J/1.3C, IIICF/c and JFCF-

6/T.1J/5H) did not have a reduction in PML levels. Moreover, the correlation coefficient for

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

PML and ATRX in ATRX-positive cell lines is consistent with the hypothesis that, in

addition to ATRX, other factors contribute to the control of PML levels.

Although investigating mechanisms of controlling PML level apart from ATRX is

outside the scope of this study, we looked for loss-of-function mutations in E3 ubiquitin

ligase genes in these ATRX-deficient cell lines that could potentially result in a reduced rate

of PML degradation. Sequencing of the CBX4, UBE3A, HECTD1, HECTD2, MDM2,

PARK2, PIAS1, PIN1, RNF4 and SMURF1 genes in these three cell lines identified a

deletion of 160,305bp in PARK2 in JFCF-6/T.1J/1.3C and RNA-Seq showed that this was

associated with a 3-fold decrease in PARK2 expression (data not shown). PARK2

expression was also reduced (2-fold) in IIICF/c cells. Other factors that could

counterbalance the effect of ATRX loss on PML levels potentially include upregulated

JAK-STAT signaling (Hubackova et al., 2012).

Many tumors that depend on ALT are difficult to treat and have a poor prognosis

(Henson and Reddel, 2010). Given the association between ALT and ATRX loss-of-

function mutations, and the role of ATRX in intrinsic resistance to viral infection, we

examined whether this difference between normal and ALT cells creates a vulnerability in

ALT cancers which could be exploited for synthetic lethality. We found, as expected, that

ATRX-deficiency results in selective sensitivity to infection with ICP0-null HSV-1.

Another mutant HSV-1, talimogene laherparepvec, was approved by the FDA in 2015 for

the treatment of advanced melanoma (Andtbacka et al., 2015), and is in clinical trials for

other solid tumors including of the bladder, brain, breast, bronchus, colon, head and neck,

liver, ovary, pancreas, rectum and skin (melanoma and non-melanoma, including Merkel

cell carcinoma) and soft tissue sarcomas (https://clinicaltrials.gov/, accessed December 15,

2018). The testing done to obtain regulatory approval for this virus, and the experience

gained with its subsequent clinical use, will most likely facilitate the development of other

HSV-1 oncolytic viruses as cancer therapeutics.

In addition to inactivating the ICP0 protein, further modifications could be made to

HSV-1 to enhance its selectivity. For example, HSV-1 also expresses high levels of

microRNA (miR)-H1 during infection, and one of its targets is the 3’ UTR of ATRX, and

the HSV-1 tegument protein, virion-associated host shutoff (Vhs) which is an

endoribonuclease, was shown to efficiently facilitate degradation of ATRX mRNA (Jurak

et al., 2012). Mutations in HSV-1 that inactivate gene products such as these could further

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

hamper its ability to replicate in normal cells, and therefore enhance its selectivity for

cytolysis of cells with genetic lesions resulting in decreased intrinsic immunity.

We also demonstrate here that total PML content, and PML NB number correlates

with resistance to the mutant virus, and that depleting cells of PML protein decreases this

resistance. The relationship between ATRX deficiency and decreased PML in ALT cells

was found to be causal: we showed that ATRX upregulates PML by increasing transcription

of its mRNA and decreasing its proteasome-mediated degradation. It has been observed in

a number of studies that PML expression is completely or substantially lost, via unknown

mechanisms, in many tumor types that are not known to be ATRX-deficient (Zhang et al.,

2000; Gurrieri et al., 2004; Lee et al., 2007; Reineke et al., 2008), and a more extensive

survey of PML expression in cancer could potentially reveal many more. In contrast, most

normal tissues display high levels of PML protein and PML NB immunostaining (Gurrieri

et al., 2004), indicating that normal tissues should be relatively resistant to ICP0-null HSV-

1. Therefore, although the vulnerability to ICP0-null HSV-1 was found through testing an

hypothesis regarding ATRX-deficient ALT cancer cells, the additional findings regarding

PML may have uncovered a target for selective oncolytic viral therapy in a wider array of

tumor types, namely, low PML levels, regardless of the cause of the downregulation.

MATERIALS AND METHODS

Cell culture

Baby hamster kidney-21 (BHK21) cells were grown in Glasgow Modified Eagle’s Medium

(GMEM; ThermoFisher, Melbourne, Australia), 10% fetal bovine serum (FBS; Sigma

Aldrich, Castle Hill, Australia) and 10% tryptose phosphate broth (TPB). TPB consists of

20 g L-1 tryptose, 2 g L-1 dextrose, 5 g L-1 NaCl, 2.5 g L-1 disodium phosphate. JFCF-6/T.1R,

U-2 OS, SUSM-1, Saos-2, JFCF-6/T.1M, GM847, JFCF-6/T.1/P-sc2, JFCF-6/T.1J/1.3C,

IIICF/c, JFCF-6/T.1J/5H, G292, LFS-05F-24, IIICF-E6E7/C4, SK-LU-1, IVG-bf/LXSN,

HeLa, JFCF-6/T.1F, A549, HT1080, HT1080-6TG, Fre80-3T-sc2, JFCF-6/T.1J/6B, JFCF-

6/T.1/P-sc1, Fre16s, Fre98, MEF, MRC-5 and IMR-90 were cultured in Dulbecco’s

Modified Eagle’s Medium (DMEM; ThermoFisher) supplemented with 10% FBS. MeT-4A

cells were grown in DMEM and 5% FBS. Fre80-3T-shATRX-3 and Fre80-3T-shATRX-7

cells were cultured in DMEM, 10% FBS and 0.5 µg mL-1 puromycin. HCT116 cells were

grown in McCoy’s 5A medium (ThermoFisher), 10% FBS and 5 mM L-glutamine. HCT116

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

ATRXN/O cells were grown in McCoy’s 5A medium, 10% FBS, and 5 mM L-glutamine

supplemented with 1 mg mL-1 G418. WI-38 cells were grown in minimum essential medium

(ThermoFisher), 10% FBS and 5 mM L-glutamine. Bre101 cells were cultured in MCDB

170 medium (ThermoFisher). All cell lines were cultured at 37oC in 10% CO2 and

atmospheric O2, with the exception of Bre101 cells, which were grown in 5% CO2.

BHK21 cells were obtained from CellBank Australia (Sydney, Australia). The

JFCF-6/T- and Fre80-3T-derived cell lines are individual immortalization events following

SV40 transfection of a mass population (Lovejoy et al., 2012; Napier et al., 2015). ATRX

was inactivated in the HCT116 ATRXN/O cell line using rAAV targeting exon 5 (Napier et

al., 2015). All other cell lines were constructed or obtained as described (Huschtscha et al.,

2012; Lovejoy et al., 2012; Napier et al., 2015). The identity of all cell lines was confirmed

by short tandem repeat DNA analyses at CellBank Australia.

siRNAs, vectors and antibodies

All siRNAs used in this study were purchased from QIAGEN (Melbourne, Australia). RNAi

transfections (40 nM) were performed by Lipofectamine RNAiMax (ThermoFisher) using

a forward transfection, according to the manufacturer’s instructions. The individual siRNA

duplexes were: negative control (5’-AATTCTCCGAACGTGTCACGT-3’), PML (5’-

AACGACAGCCCAGAAGAGGAA-3’) (Xu et al., 2003), ATRX-5 (5’-

ACCGCTGAGCCC ATGAGTGAA-3’), ATRX-6 (5’-

AGCAGCTACAGTGACGACTAA-3’), ATRX-7 (5’-CCC

AGCAATCACAGAAGCCGA-3’) and ATRX-8 (5’-CTCCAGTGCATTTCTATCGTA-

3’).

U-2 OS cells were transfected with psi-mH1-mCherry (GeneCopoeia, Rockville,

MD, USA) and subjected to long-term culturing in the presence of 0.8 µg mL-1 puromycin.

U-2 OS cells with mCherry fluorescence within the highest 20% were sorted on a BD Influx

at the Westmead Institute for Medical Research (Sydney, Australia) and used for subsequent

experiments.

We used the following primary antibodies: ICP4 (1:300 dilution; Santa Cruz,

Tingalpa, Australia, sc-69809), ICP8 (1:500; Abcam, Melbourne, Australia, ab20194),

ICP27 (1:300; Santa Cruz, sc69807), VP5 (1:200; Santa Cruz, sc13525), ATRX (1:333;

Sigma Aldrich, HPA001906), PML (Santa Cruz, sc5621 [1:200; Western blotting] and

sc9862 [1:300; immunofluorescence]), Sp100 (1:500; Sigma Aldrich, HPA016707) and

actin (1:1000; Sigma Aldrich, A2066). The secondary antibodies used were: goat anti-

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

mouse IgG conjugated to horseradish peroxidase (HRP; Dako, Kingsgrove, Australia,

P0447), goat anti-rabbit IgG HRP (Dako, P0448), donkey anti-rabbit conjugated to Alexa

Fluor 488 (ThermoFisher, A21206) and donkey anti-goat Alexa Fluor 594 (ThermoFisher,

A11058), all at 1:1000 dilution for both Western blotting and immunofluorescence.

Viral infections and plaque assay

WT HSV-1 (in1863) and ICP0-null mutant HSV-1 (dl1403) contain the lacZ gene under

control of the HCMV promoter (Stow and Stow, 1986). Viruses were propagated in BHK21

cells and titrated on U-2 OS cells (Yao and Schaffer, 1995; Everett et al., 2004). For testing

viral gene expression, sub-confluent cells were infected with WT or mutant HSV-1 at a

multiplicity of infection (MOI) of 2. Cells were agitated every 7 min for 1 h for virus

adsorption and then overlaid with GMEM with 10% FBS and 10% TPB. Cells were

harvested at the indicated time (h) post infection (h. p. i.).

For the plaque assay, cells were seeded in 24-well plates at a density that yielded

confluent wells 12 h post-seeding. Cells were infected with sequential 3-fold dilutions of

WT or mutant HSV-1, and the plates were agitated every 7 min for 1 h for virus adsorption

and overlaid with medium containing 1% human serum (Lonza, Mt Waverley, Australia).

We detected β-galactosidase-positive plaques 30 h after infection using the Senescence-

Associated β-Galactosidase Staining Kit (Cell Signaling Technology, Arundel, Australia)

following the manufacturer’s protocol. The coefficient of resistance was calculated as the

ratio of the WT plaque forming units (PFU) to the mutant PFU.

Automated PML NB and APB detection and quantification

Cells were seeded on coverslips stained with Alcian Blue (1 mg mL-1, Sigma Aldrich), fixed

in 2% paraformaldehyde and further permeabilized with KCM buffer (120 mM KCl, 20 mM

NaCl, 10 mM Tris pH 7.5, 0.1% Triton X-100). After blocking for 1 h in antibody dilution

buffer (ABDIL: 20 mM Tris pH 7.5, 2% BSA, 0.2% fish gelatin, 150 mM NaCl, 0.1% Triton

X-100, 0.1% sodium azide), cells were incubated with PML and Sp100 antibodies diluted

in ABDIL overnight at 4°C. Following extensive washing with phosphate buffered saline

with 0.1% Tween-20 (PBST), cells were incubated with fluorescently labeled secondary

antibodies for 1 h at room temperature, and washed again in PBST. After incubating cells

with 50 ng mL-1 4',6-diamidino-2'-phenylindole hydrochloride (DAPI; D9542, Sigma

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Aldrich) for 10 min, cells were mounted on slides with ProLong Gold Antifade Solution

(P36930, ThermoFisher).

Immunofluorescence with anti-PML and SP100 antibodies had sufficiently low

background that quantitation of co-localizing foci was able to be automated using Metafer4

software (Metasystems GmbH, North Ryde, Australia) on an Axioplan 2 microscope (Zeiss,

North Ryde, Australia), with a 63X NA (1.4 Plan-Apochromat) oil objective, and

appropriate filter cubes (Supplementary Figure S5). We captured interphase cells in 10 Z-

planes in 0.25 µm increments. DAPI stained nuclei were identified and background

subtraction, image sharpening and TopHat transformation was applied to the PML and

Sp100 immunofluorescence channels. PML and Sp100 immunofluorescence foci were each

identified as foci of > 0.2 µm diameter, > 20% intensity over background, separated by a

minimum distance of 0.5 µm. Co-localizations were events where the center of a PML and

an Sp100 focus were ≤ 0.3 µm apart in three dimensions.

APBs were visualized as described for PML NBs, with the modifications being that

Sp100 antibody staining was omitted, and hybridization to a peptide nucleic acid (PNA)

probe was added. PML staining as described above was followed by dehydration of the

slides in an ice-cold ethanol series (75% v/v, 85% and 100% ethanol, 2 min each), air drying

of the slides, and hybridization with 0.06 mL PNA probe (0.3 µg mL-1 Alexa488-OO-

(CCCTAA)3) (F1004, Panagene, Daejeon, Republic of Korea). Slides were heated to 80oC

for 12 min, incubated in a humidified chamber overnight at room temperature, rinsed in

distilled water, then washed in 1X SSC/50% formamide (15 min, 37oC) and 1X SSC (15

min, 37oC). Slides were rinsed briefly in distilled water, incubated for 5 min at room

temperature with DAPI (50 ng mL-1), and mounted with ProLong Gold Antifade Solution.

A Zeiss Axio Imager was used to acquire images, which were analyzed using CellProfiler

image analysis software (Broad Institute, Cambridge MA, USA). The criterion for

automated scoring of an APB was a minimum of 50% overlap between foci detected by

PML immunostaining and PNA hybridization.

Quantitative RT-PCR

RNA was extracted using the QIAGEN RNAeasy Mini Kit, and cDNA was synthesized

with SuperScript III Reverse Transcriptase (ThermoFisher) following standard protocols.

cDNA was amplified using FastStart Essential DNA Green Master (Roche, North Ryde,

Australia) and analyzed on a Roche LightCycle 96 machine. Gene expression was

normalized to GAPDH. Primer sequences were: PML forward 5’-

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

GATGGCTTCGACGAGTTCAA-3’, PML reverse 5’-GGGCAGGTCAACGTCAATAG-

3’, GAPDH forward 5’-ACCCACTCCTCCACCTTTG

-3’, and GAPDH reverse 5’-CTCTTGTGCTCTTGCTGGG-3’.

Protein extraction and Western blotting

In order to detect viral proteins, total protein was extracted in lysis buffer as described

(Bower et al., 2012), and to detect PML and ATRX expression lysates were prepared by

lysing cells in 4x LDS (106 mM Tris-HCl, 141 mM Tris-Base, 2% SDS, 10% glycerol,

0.75% SERVA Blue G50, 0.25% Phenol Red) containing benzonase (Merck Millipore,

Bayswater, Australia) and β-mercaptoethanol. Proteins were separated, probed and analyzed

as indicated (Bower et al., 2012). PML expression was quantified as the signal between 60

and 100 kD, covering the predicted molecular weights of PML isoforms localized to the

nucleus (Nisole et al., 2013).

Flow cytometry

Fre-16s-eGFP and U-2 OS-mCherry cells were mixed at a ratio that yielded an equivalent

number of cells 12 h after plating, and then infected with WT or mutant HSV-1 at an MOI

of 0.96 for 36 h. Cells were fixed in 2% formaldehyde for 10 min at 37°C, chilled on ice for

1 min and washed extensively with FACS buffer (1% FBS in PBS). Samples were analyzed

on a BD LSRFortessa (BD, North Ryde, Australia) at the Westmead Institute for Medical

Research (Sydney, Australia).

Competing interests

No competing interests declared.

ACKNOWLEDGEMENTS

Microscopy was performed at the Australian Cancer Research Foundation Telomere

Analysis Centre (ATAC) and flow cytometry at the Westmead Research Hub Flow

Cytometry Facility. We thank Dr Elizabeth Sloan (University of Glasgow) and Dr Monica

Miranda Saksena (Westmead Institute for Medical Research) for many helpful discussions,

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

and Dr Christine Smyth for assistance with FACS. The work was supported by a Kids

Cancer Alliance Scholarship to M.H., Cure Cancer Australia Foundation Project Grant

1062240 to C.E.N., Cancer Council New South Wales (NSW) Research Grant RG 15-12,

National Health and Medical Research Council of Australia Project Grant 1053195 and

Cancer Institute NSW 11/FRL/5-02 to A.J.C., and Cancer Council NSW Program Grant

PG11-08 and National Health and Medical Research Council of Australia Project Grant

1088646 to R.R.R.

References

Andtbacka, R. H., Kaufman, H. L., Collichio, F., Amatruda, T., Senzer, N., Chesney,

J., Delman, K. A., Spitler, L. E., Puzanov, I., Agarwala, S. S., et al. (2015). Talimogene

Laherparepvec improves durable response rate in patients with advanced melanoma. J.

Clin. Oncol. 33, 2780-2788.

Bieniasz, P. D. (2004). Intrinsic immunity: a front-line defense against viral attack. Nat.

Immunol. 5, 1109-1115.

Boutell, C. and Everett, R. D. (2003). The herpes simplex virus type 1 (HSV-1)

regulatory protein ICP0 interacts with and ubiquitinates p53. J. Biol. Chem 278, 36596-

36602.

Bower, K., Napier, C. E., Cole, S. L., Dagg, R. A., Lau, L. M., Duncan, E. L., Moy, E.

L. and Reddel, R. R. (2012). Loss of wild-type ATRX expression in somatic cell hybrids

segregates with activation of Alternative Lengthening of Telomeres. PLoS ONE 7,

e50062.

Bryan, T. M., Englezou, A., Dalla-Pozza, L., Dunham, M. A. and Reddel, R. R. (1997). Evidence for an alternative mechanism for maintaining telomere length in human

tumors and tumor-derived cell lines. Nat. Med 3, 1271-1274.

Bryan, T. M., Englezou, A., Gupta, J., Bacchetti, S. and Reddel, R. R. (1995).

Telomere elongation in immortal human cells without detectable telomerase activity.

EMBO J. 14, 4240-4248.

Cai, W. Z. and Schaffer, P. A. (1989). Herpes simplex virus type 1 ICP0 plays a critical

role in the de novo synthesis of infectious virus following transfection of viral DNA. J

Virol 63, 4579-4589.

Cho, N. W., Dilley, R. L., Lampson, M. A. and Greenberg, R. A. (2014).

Interchromosomal homology searches drive directional ALT telomere movement and

synapsis. Cell 159, 108-121.

Chung, I., Leonhardt, H. and Rippe, K. (2011). De novo assembly of a PML nuclear

subcompartment occurs through multiple pathways and induces telomere elongation. J.

Cell Sci 124, 3603-3618.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Clynes, D., Jelinska, C., Xella, B., Ayyub, H., Scott, C., Mitson, M., Taylor, S., Higgs,

D. R. and Gibbons, R. J. (2015). Suppression of the alternative lengthening of telomere

pathway by the chromatin remodelling factor ATRX. Nat. Commun 6, 7538.

Dilley, R. L., Verma, P., Cho, N. W., Winters, H. D., Wondisford, A. R. and

Greenberg, R. A. (2016). Break-induced telomere synthesis underlies alternative

telomere maintenance. Nature 539, 54-58.

Draskovic, I., Arnoult, N., Steiner, V., Bacchetti, S., Lomonte, P. and Londono-

Vallejo, A. (2009). Probing PML body function in ALT cells reveals spatiotemporal

requirements for telomere recombination. Proc. Natl. Acad. Sci. U. S. A 106, 15726-

15731.

Dunham, M. A., Neumann, A. A., Fasching, C. L. and Reddel, R. R. (2000). Telomere

maintenance by recombination in human cells. Nat. Genet 26, 447-450.

Everett, R. D., Boutell, C. and Orr, A. (2004). Phenotype of a herpes simplex virus type

1 mutant that fails to express immediate-early regulatory protein ICP0. J. Virol 78, 1763-

1774.

Everett, R. D. and Murray, J. (2005). ND10 components relocate to sites associated

with herpes simplex virus type 1 nucleoprotein complexes during virus infection. J. Virol.

79, 5078-5089.

Everett, R. D., Parada, C., Gripon, P., Sirma, H. and Orr, A. (2008). Replication of

ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100. J.

Virol 82, 2661-2672.

Everett, R. D., Rechter, S., Papior, P., Tavalai, N., Stamminger, T. and Orr, A. (2006). PML contributes to a cellular mechanism of repression of herpes simplex virus

type 1 infection that is inactivated by ICP0. J. Virol. 80, 7995-8005.

Garcia-Exposito, L., Bournique, E., Bergoglio, V., Bose, A., Barroso-Gonzalez, J.,

Zhang, S., Roncaioli, J. L., Lee, M., Wallace, C. T., Watkins, S. C., et al. (2016).

Proteomic profiling reveals a specific role for translesion DNA Polymerase eta in the

Alternative Lengthening of Telomeres. Cell Rep 17, 1858-1871.

Glass, M. and Everett, R. D. (2013). Components of promyelocytic leukemia nuclear

bodies (ND10) act cooperatively to repress herpesvirus infection. J. Virol 87, 2174-2185.

Gurrieri, C., Capodieci, P., Bernardi, R., Scaglioni, P. P., Nafa, K., Rush, L. J.,

Verbel, D. A., Cordon-Cardo, C. and Pandolfi, P. P. (2004). Loss of the tumor

suppressor PML in human cancers of multiple histologic origins. J. Natl. Cancer Inst. 96,

269-279.

Heaphy, C. M., de Wilde, R. F., Jiao, Y., Klein, A. P., Edil, B. H., Shi, C., Bettegowda,

C., Rodriguez, F. J., Eberhart, C. G., Hebbar, S., et al. (2011). Altered telomeres in

tumors with ATRX and DAXX mutations. Science 333, 425.

Heaphy, C. M., Subhawong, A. P., Hong, S. M., Goggins, M. G., Montgomery, E. A.,

Gabrielson, E., Netto, G. J., Epstein, J. I., Lotan, T. L., Westra, W. H., et al. (2011).

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism

in human cancer subtypes. Am. J. Pathol 179, 1608-1615.

Henson, J. D., Hannay, J. A., McCarthy, S. W., Royds, J. A., Yeager, T. R.,

Robinson, R. A., Wharton, S. B., Jellinek, D. A., Arbuckle, S. M., Yoo, J., et al. (2005). A robust assay for alternative lengthening of telomeres in tumors shows the

significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clin.

Cancer Res 11, 217-225.

Henson, J. D. and Reddel, R. R. (2010). Assaying and investigating Alternative

Lengthening of Telomeres activity in human cells and cancers. FEBS Lett. 584, 3800-

3811.

Horn, S., Figl, A., Rachakonda, P. S., Fischer, C., Sucker, A., Gast, A., Kadel, S.,

Moll, I., Nagore, E., Hemminki, K., et al. (2013). TERT promoter mutations in familial

and sporadic melanoma. Science 339, 959-961.

https://clinicaltrials.gov/ (Accessed December 15, 2018).

Huang, F. W., Hodis, E., Xu, M. J., Kryukov, G. V., Chin, L. and Garraway, L. A. (2013). Highly recurrent TERT promoter mutations in human melanoma. Science 339,

957-959.

Hubackova, S., Krejcikova, K., Bartek, J. and Hodny, Z. (2012) Interleukin 6 signaling

regulates promyelocytic leukemia protein gene expression in human normal and cancer

cells. J. Biol Chem 287, 26702-26714.

Huschtscha, L. I., Napier, C. E., Noble, J. R., Bower, K., Au, A. Y., Campbell, H. G.,

Braithwaite, A. W. and Reddel, R. R. (2012). Enhanced isolation of fibroblasts from

human skin explants. Biotechniques 53, 239-244.

Jiao, Y., Killela, P. J., Reitman, Z. J., Rasheed, A. B., Heaphy, C. M., de Wilde, R. F.,

Rodriguez, F. J., Rosemberg, S., Oba-Shinjo, S. M., Marie, S. K., et al. (2012).

Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant

gliomas. Oncotarget 3, 709-722.

Jurak, I., Silverstein, L. B., Sharma, M. and Coen, D. M. (2012). Herpes simplex virus

is equipped with RNA- and protein-based mechanisms to repress expression of ATRX, an

effector of intrinsic immunity. J. Virol 86, 10093-10102.

Kaelin, W. G. (2005). The concept of synthetic lethality in the context of anticancer

therapy. Nat. Rev. Cancer 5, 689-698.

Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L.,

Coviello, G. M., Wright, W. E., Weinrich, S. L. and Shay, J. W. (1994). Specific

association of human telomerase activity with immortal cells and cancer. Science 266,

2011-2015.

Lee, H. E., Jee, C. D., Kim, M. A., Lee, H. S., Lee, Y. M., Lee, B. L. and Kim, W. H. (2007). Loss of promyelocytic leukemia protein in human gastric cancers. Cancer Lett.

247, 103-109.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Lilley, C. E., Chaurushiya, M. S., Boutell, C., Landry, S., Suh, J., Panier, S., Everett,

R. D., Stewart, G. S., Durocher, D. and Weitzman, M. D. (2010). A viral E3 ligase

targets RNF8 and RNF168 to control histone ubiquitination and DNA damage responses.

EMBO J 29, 943-955.

Lovejoy, C. A., Li, W., Reisenweber, S., Thongthip, S., Bruno, J., de Lange, T., De,

S., Petrini, J. H., Sung, P. A., Jasin, M., et al. (2012). Loss of ATRX, genome

instability, and an altered DNA damage response are hallmarks of the Alternative

Lengthening of Telomeres pathway. PLoS Genet. 8, e1002772.

Lukashchuk, V. and Everett, R. D. (2010). Regulation of ICP0-null mutant herpes

simplex virus type 1 infection by ND10 components ATRX and hDaxx. J. Virol. 84, 4026-

4040.

Molenaar, C., Wiesmeijer, K., Verwoerd, N. P., Khazen, S., Eils, R., Tanke, H. J. and

Dirks, R. W. (2003). Visualizing telomere dynamics in living mammalian cells using

PNA probes. EMBO J 22, 6631-6641.

Napier, C. E., Huschtscha, L. I., Harvey, A., Bower, K., Noble, J. R., Hendrickson, E.

A. and Reddel, R. R. (2015). ATRX represses alternative lengthening of telomeres.

Oncotarget 6, 16543-16558.

Nisole, S., Maroui, M. A., Mascle, X. H., Aubry, M. and Chelbi-Alix, M. K. (2013).

Differential Roles of PML Isoforms. Front Oncol 3, 125.

Pickett, H. A. and Reddel, R. R. (2015). Molecular mechanisms of activity and

derepression of alternative lengthening of telomeres. Nat Struct Mol Biol 22, 875-880.

Reddel, R. R., Bryan, T. M. and Murnane, J. P. (1997). Immortalized cells with no

detectable telomerase activity. A review. Biochemistry (Mosc. ) 62, 1254-1262.

Reineke, E. L., Lam, M., Liu, Q., Liu, Y., Stanya, K. J., Chang, K. S., Means, A. R.

and Kao, H. Y. (2008). Degradation of the tumor suppressor PML by Pin1 contributes to

the cancer phenotype of breast cancer MDA-MB-231 cells. Mol. Cell. Biol. 28, 997-1006.

Shay, J. W. and Bacchetti, S. (1997). A survey of telomerase activity in human cancer.

Eur. J. Cancer 33, 787-791.

Stow, N. D. and Stow, E. C. (1986). Isolation and characterization of a herpes simplex

virus type 1 mutant containing a deletion within the gene encoding the immediate early

polypeptide Vmw110. J. Gen. Virol. 67 ( Pt 12), 2571-2585.

Tavalai, N. and Stamminger, T. (2008). New insights into the role of the subnuclear

structure ND10 for viral infection. Biochim. Biophys. Acta 1783, 2207-2221.

Xu, Z. X., Timanova-Atanasova, A., Zhao, R. X. and Chang, K. S. (2003). PML

colocalizes with and stabilizes the DNA damage response protein TopBP1. Mol. Cell. Biol

23, 4247-4256.

Xue, Y., Gibbons, R., Yan, Z., Yang, D., McDowell, T. L., Sechi, S., Qin, J., Zhou, S.,

Higgs, D. and Wang, W. (2003). The ATRX syndrome protein forms a chromatin-

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies.

Proc. Natl. Acad. Sci. U.S.A. 100, 10635-10640.

Yao, F. and Schaffer, P. A. (1995). An activity specified by the osteosarcoma line U2OS

can substitute functionally for ICP0, a major regulatory protein of herpes simplex virus

type 1. J Virol 69, 6249-6258.

Yeager, T. R., Neumann, A. A., Englezou, A., Huschtscha, L. I., Noble, J. R. and

Reddel, R. R. (1999). Telomerase-negative immortalized human cells contain a novel

type of promyelocytic leukemia (PML) body. Cancer Res 59, 4175-4179.

Zhang, A., Zheng, C., Lindvall, C., Hou, M., Ekedahl, J., Lewensohn, R., Yan, Z.,

Yang, X., Henriksson, M., Blennow, E., et al. (2000). Frequent amplification of the

telomerase reverse transcriptase gene in human tumors. Cancer Res 60, 6230-6235.

Zhang, P., Chin, W., Chow, L. T., Chan, A. S., Yim, A. P., Leung, S. F., Mok, T. S.,

Chang, K. S., Johnson, P. J. and Chan, J. Y. (2000). Lack of expression for the

suppressor PML in human small cell lung carcinoma. Int. J. Cancer 85, 599-605.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Figures

Fig. 1. Loss of ATRX increases viral gene expression when infected with mutant HSV-

1. (A and B) ATRX protein expression by Western blotting in two cell line pairs: wild-type

and ATRX-knockout HCT116, and JFCF-6/T.1/P-sc1 (WT ATRX) and JFCF-6/T.1/P-sc2

(ATRX-null). (C and D) Expression of viral proteins during infection. The cell line pairs

were infected with WT or Mutant HSV-1, and harvested at the indicated times [hours post

infection (h. p. i.)]. The antibodies used are indicated to the left of each panel.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Fig. 2. ATRX expression and PML NB count correlate with sensitivity to mutant HSV-

1. (A) Representative images of plaque forming assays used to calculate the coefficient of

resistance. Cells were infected with serially diluted viral preparations (1 in 3 dilutions from

left to right). Infected cells express β-galactosidase and plaques were detected by X-gal

staining. (B) Viral plaques were counted 30 h after infection of the indicated cell lines and

the coefficient of resistance (WT plaque forming units [PFU]/mutant PFU) was plotted.

Bars: mean±s.e.m. of three biologic replicates; ***P<0.001, Mann-Whitney test grouping

ATRX-positive (ATRX+) versus ATRX-deficient (ATRX-) cell lines. The box around three

ALT/ATRX- cell lines indicates the resistant cell lines. (C) Representative images of PML

NB staining: PML protein, red; Sp100 protein, green; DNA counterstained with DAPI, blue.

The scale bar indicates 10 μm. (D) Coefficient of resistance segregates with PML NB

number/cell. Data represent mean values for individual cell lines from three biologic

replicates. Permissive cell lines are defined as having less than six PML NBs per cell and a

coefficient of resistance less than 60, whereas resistant cell lines have greater than six PML

NBs per cell and a coefficient of resistance greater than 60.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Fig. 3. ATRX status correlates with PML expression levels. (A) ATRX and PML protein

expression was analyzed by Western blot in the panel of cell lines. (B) PML NBs per cell

(left plot) and PML fluorescence intensity within PML NBs (center plot) were quantitated

using automated imaging. Each data point represents a cell line, >200 nuclei counted per

cell line, mean±s.e.m., n=3 independent experiments. PML protein abundance was also

quantitated by Western blot and expressed relative to actin and normalized to the JFCF-

6/T.1M cell line (right plot). Cell lines are grouped by ATRX status; **P<0.01, Mann-

Whitney test. (C) HT1080 cells were treated with control siRNA (siNC) or siATRX for the

time indicated, and Western blotting performed at the indicated timepoints. (D) Quantitation

of ATRX and PML expression subsequent to siNC or siATRX transfection. Expression was

normalized to actin, and then to the siNC-treated sample at each time point. Data are

expressed as the mean±s.e.m. of three biologic replicates; *P<0.05, **P<0.01, paired two-

tailed t-test.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Fig. 4. ATRX regulates PML expression at the RNA and protein levels. (A) HT1080

cells were depleted of ATRX for the indicated times and real-time PCR analysis of PML

expression was performed at the indicated time points. Data were normalized to siNC.

*P<0.05, **P<0.01, paired two-tailed t-test. (B and C) HT1080 cells were treated with siNC

or siATRX for 24 h, followed by cycloheximide (30 ng µL-1) for the indicated times.

Abundance of ATRX and PML was analyzed by Western blot. Data are expressed as the

ratio of PML to actin, and the ratio of each time point was then normalized to the non-

cycloheximide control (0 h). *P<0.05, paired two-tailed t-test. (D and E) HT1080 cells were

treated with siNC, siATRX and/or MG132 (5 µM) as indicated above each lane. PML/actin

ratio was calculated and normalized to the untreated control. *P<0.05, paired two-tailed t-

test.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Fig. 5. Depletion of PML increases sensitivity to cytolytic virus. (A and B) The three

virus-resistant ALT cell lines (A) and the HT1080 (TEL) cell line (B) were treated with

siNC or siPML (and/or siATRX for HT1080) for 72 h, and depletion of PML was confirmed

by Western blot. PML NB number and coefficient of resistance to viral infection are also

shown; mean±s.e.m., n=3 independent experiments; *P<0.05, **P<0.01 using a paired two-

tailed t-test. (C) Coefficient of resistance versus PML NB numbers was plotted for untreated

and PML and/or ATRX depleted (“siPML/siATRX”) cells, with the three resistant ALT

lines and HT1080 cells indicated in colored circles; the remaining data points are from Fig.

2D.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Fig. 6. Mutant HSV-1 is selectively toxic to ATRX-null cells. (A) Plaque assays for

MRC-5 human fibroblasts and U-2 OS (ATRX-deficient/ALT) cells infected with a 1:3

serial dilution of mutant HSV-1. (B) Primary (WI-38, Fre98, MEF, MRC-5, IMR-90 and

Bre101) and ALT-positive (U-2 OS and Saos-2) cells were infected with mutant HSV-1;

plaque forming units are shown as mean±s.e.m., n=3 independent experiments. (C) eGFP-

labeled normal fibroblasts (Fre-16s) and U-2 OS-mCherry cells were mixed in a ratio that

yielded an equivalent number of cells 12 h after plating, and then infected with WT or

mutant HSV-1 at an MOI of 0.96. The percentage (left plot) and the number (right plot) of

eGFP- and mCherry-labeled cells remaining 30 h after mock or viral infection as determined

by FACS is graphed as mean±s.e.m., n=3 independent experiments. *P<0.05, paired two-

tailed t-test.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Fig. S1. Correlation between ATRX and PML protein levels. (A) Representative images of PML NB staining for the indicated panel of cell lines. PML protein is stained in red, Sp100 protein in green and DNA counterstained with DAPI in blue. The scale bar denotes 10 μM. (B) The number of PML NBs in the cell line panel was graphed as mean±s.e.m. Data were collected from three independent experiments and >200 nuclei were counted per experiment. PML NB counts were obtained using automated imaging. (C and D) Correlation between ATRX and PML expression in ATRX+ cell lines as quantitated by Western blotting in Fig. 3A and fitted with a linear regression line, with (C) and without (D) data for cell lines with very high ATRX or PML expression. (E) Depletion of ATRX results in decreased PML protein. HT1080 cells were treated with control siRNA (siNC) or four different siATRX sequences for 54 h, and the abundance of ATRX and PML protein was analyzed by Western blot.

J. Cell Sci.: doi:10.1242/jcs.222349: Supplementary information

Jour

nal o

f Cel

l Sci

ence

• S

uppl

emen

tary

info

rmat

ion

Fig. S2. Alteration in PML levels responding to ATRX knock down. (A) Western blot showing PML, ATRX and actin protein expression in three ALT/ATRX+ cell lines (SK-LU-1, IIICF-E6E7/C4 and G292) and two TEL/ATRX+ cell lines (HeLa and Fre80-3T-sc2) subsequent to treatment with siNC or siATRX for 60 h. (B) Quantification of PML expression, normalized to actin and then the siNC. Bars represent mean±s.e.m. of three independent experiments. *P<0.05, **P<0.01, paired two-tailed t-test.

J. Cell Sci.: doi:10.1242/jcs.222349: Supplementary information

Jour

nal o

f Cel

l Sci

ence

• S

uppl

emen

tary

info

rmat

ion

Fig. S3. Depletion of PML increases sensitivity to mutant virus. (A) The number of PML NBs decreased following PML knockdown. Representative images of PML NB staining are shown for three cell lines (indicated above each column) treated with siNC or siPML. PML is indicated in red, Sp100 as green and DAPI counterstain in blue. Co-localizations are indicated in yellow. (B) siPML treatment results in decreased resistance to mutant virus. Example of a plaque assay performed in untreated IIICF/c cells or after transfection with siNC or siPML. (C) Reduction of PML NB numbers by siATRX and/or siPML. HT1080 cells were treated with siNC, siPML, siATRX or siATRX and siPML. Red staining indicates PML protein, green staining represents Sp100 and blue fluorescence is the DNA counterstain DAPI. The scale bars indicate 10 μM.

J. Cell Sci.: doi:10.1242/jcs.222349: Supplementary information

Jour

nal o

f Cel

l Sci

ence

• S

uppl

emen

tary

info

rmat

ion

Fig S4. ATRX-negative cells are more sensitive to mutant HSV-1. Representative FACS plots of co-cultured eGFP-labeled Fre-16s primary human fibroblasts and mCherry-labeled U-2 OS ATRX-negative ALT osteosarcoma cells infected for 30 h as indicated above each plot. The double-negative and double-positive compartments presumably represent cells that have ceased expressing the marker, and ungated doublets, respectively. Data are plotted in Fig. 6C.

J. Cell Sci.: doi:10.1242/jcs.222349: Supplementary information

Jour

nal o

f Cel

l Sci

ence

• S

uppl

emen

tary

info

rmat

ion

J. Cell Sci.: doi:10.1242/jcs.222349: Supplementary information

Jour

nal o

f Cel

l Sci

ence

• S

uppl

emen

tary

info

rmat

ion

Fig. S5. Development and optimization of high content screening for assaying PML NBs. (A) Summary of automated detection and quantitation of PML NBs. PML NBs are labeled with antibodies to PML (red) and Sp100 (green), and interphase nuclei are counterstained with DAPI (blue). Images are acquired as z-stacks and analyzed using a high-content wide-field microscope. DAPI staining is used as nuclear mask to mark the regions of interest (ROIs), Sp100 and PML foci inside this mask are identified, and then their sizes and relative intensities are quantified. Co-localized Sp100 and PML foci are identified by measuring distance between individual spots and the number of PML bodies determined as co-localized Sp100 and PML foci within 0.3 µm. Data are reported as PML NBs per nuclei. (B) Wide-field fluorescent images of JFCF-6/T.1J/6B, GM847 and Saos-2 cells labeled with antibodies against PML and Sp100 proteins, and counterstained with DAPI. (C) Quantitation of co-localizations between PML and Sp100 foci in cells from (B) completed manually, or using the automated algorithm from (A) on identical nuclei (n=30 nuclei, mean±s.e.m.). (D) Western blots of whole cell lysates from JFCF-6/T.1J/6B and GM847 cells treated with control (siNC) or siPML. (E) Corresponding immunofluorescent images of JFCF-6/T.1J/6B cells from (D) treated as indicated to the left of each row. (F) Automated quantitation of JFCF-6/T.1J/6B and GM847 cells from (D) (n=334 nuclei, displayed as a Tukey box plot with the mean indicated by “+”). The scale bars indicate 10 μM.

J. Cell Sci.: doi:10.1242/jcs.222349: Supplementary information

Jour

nal o

f Cel

l Sci

ence

• S

uppl

emen

tary

info

rmat

ion

Cell line TLM AATRX status Method of immortalization p53 status

U-2 OS ALT negative tumor WTSaos-2 ALT negative tumor NSUSM-1 ALT negative chemical M IIICF/c ALT negative spontaneous N GM847 ALT negative SV40 transformation T ag JFCF-6/T.1/P-sc2 ALT negative SV40 transformation T ag JFCF-6/T.1M ALT negative SV40 transformation T ag JFCF-6/T.1R ALT negative SV40 transformation T ag JFCF-6/T.1J/5H ALT negative SV40 transformation T ag JFCF-6/T.1J/1.3C ALT negative SV40 transformation T ag

SK-LU-1 ALT positive tumor MG292 ALT positive tumor MLFS-05F-24 ALT positive spontaneous N IVG-bf/LXSN ALT positive spontaneous N IIICF-E6E7/C4 ALT positive HPV N MeT-4A ALT positive SV40 transformation T ag

HCT116 ATRXN/O TEL negative tumor WTFre80-3T-shATRX-3 TEL negative SV40 transformation T ag Fre80-3T-shATRX-7 TEL negative SV40 transformation T ag

HCT116 TEL positive tumor WTHT1080 TEL positive tumor WTHT1080-6TG TEL positive tumor MHeLa TEL positive tumor WTA549 TEL positive tumor WTJFCF-6/T.1F TEL positive SV40 transformation T ag JFCF-6/T.1J/6B TEL positive SV40 transformation T ag Fre80-3T-sc2 TEL positive SV40 transformation T ag JFCF-6/T.1/P-sc1 TEL positive SV40 transformation T ag

Table S1. Details regarding the telomere lengthening mechanism (TLM), ATRX status, immortalization method of the cell line panel, and p53 status (WT, wild-type; N, null; M, mutant; T ag, p53 inactivation by SV40 large T antigen). Note that all of the ATRX-deficient/TEL cell lines were created by experimental manipulation, whereas spontaneous ATRX deficiency is common in ALT cell lines.

J. Cell Sci.: doi:10.1242/jcs.222349: Supplementary information

Jour

nal o

f Cel

l Sci

ence

• S

uppl

emen

tary

info

rmat

ion