Promyelocytic Leukemia (PML) Nuclear Bodies (NBs) Induce Latent/Quiescent HSV-1 Genomes Chromatinization Through a PML-NB/Histone H3.3/H3.3 Chaperone Axis

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact implication is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML-NBs, leading to the formation of viral DNA-containing PML-NBs (vDCP-NBs). Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP-NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP-NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP-NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP-qPCR performed on fibroblasts stably expressing tagged H3.3 (e-H3.3) or H3.1 (e-H3.1) show that latent/quiescent viral genomes are chromatinized almost exclusively with e-H3.3, consistent with an interaction of the H3.3 chaperones with multiple viral loci. Depletion by shRNA of single proteins from the H3.3 chaperone complexes only mildly affects H3.3 deposition on the latent viral genome, suggesting a compensation mechanism. In contrast, depletion (by shRNA) or absence of PML (in mouse embryonic fibroblast (MEF) pml−/- cells) significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. Consequently, the study demonstrates a specific epigenetic regulation of latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML-NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML-NB/histone H3.3/H3.3 chaperone axis. Author summary An understanding of the molecular mechanisms contributing to the persistence of a virus in its host is essential to be able to control viral reactivation and its associated diseases. Herpes simplex virus 1 (HSV-1) is a human pathogen that remains latent in the PNS and CNS of the infected host. However, the latency is unstable, and frequent reactivations of the virus are responsible for PNS and CNS pathologies. It is thus crucial to understand the physiological, immunological and molecular levels of interplay between latent HSV-1 and the host. Promyelocytic leukemia (PML) nuclear bodies (NBs) play a major role in controlling viral infections by preventing the onset of lytic infection. In previous studies, we showed a major role of PML-NBs in favoring the establishment of a latent state for HSV-1. A hallmark of HSV-1 latency establishment is the formation of PML-NBs containing the viral genome, which we called “viral DNA-containing PML-NBs” (vDCP-NBs). The genome entrapped in the vDCP-NBs is transcriptionally silenced. This naturally occurring latent/quiescent state could, however, be transcriptionally reactivated. Therefore, understanding the role of PML-NBs in controlling the establishment of HSV-1 latency and its reactivation is essential to design new therapeutic approaches based on the prevention of viral reactivation.

neuron level, a strict, transcriptionally silent, quiescence can be observed, and vDCP-NB-containing 122 neurons are major contributors of this latent/quiescent HSV-1 state. In humans, vDCP-NB-like 123 structures have also been observed in latently infected TG neurons [16], suggesting that vDCP-NBs 124 are probably molecular hallmarks of the HSV-1 latency process, including in the natural host. 125 Another essential feature of HSV-1 latency is the potent chromatinization of its 150-kb 126 genome, which enters the nucleus of the infected cells as a naked/non-nucleosomal dsDNA [25][26][27]. 127 Once the viral genome is injected into the nucleus of the infected neuron, it circularizes, associates 128 with nucleosomes to become chromatinized, and remains as an episome that is unintegrated into 129 the host cell genome [28]. Although latent viral genomes sustain epigenetic regulation, essentially linking PML-NBs with the chromatin assembly pathway independently of replication [35][36][37]. 146 Because vDCP-NBs contain DAXX and ATRX [15,16,38], their involvement in the chromatinization 147 of incoming HSV-1 genomes and/or long-term maintenance of chromatinized HSV-1 genomes is 148 thus plausible. 149 Human primary fibroblasts or adult mouse primary TG neuron cultures infected through 150 their cell body with a replication-defective HSV-1 virus, in1374, which is unable to synthesize 151 functional ICP4 and ICP0 under specific temperature conditions, enable the establishment of a 152 latent/quiescent state for HSV-1 [16, [38][39][40]. The latent/quiescent state of HSV-1 in human primary 153 fibroblasts has also been reproduced using engineered HSV-1 unable to express major immediate 154 early genes [41,42]. We have shown that this latent/quiescent state is linked to the formation of 155 vDCP-NBs, mimicking, at least concerning this particular structural aspect, the latency observed in a 156 subset of neurons in mouse models and in humans [15,16]. Here, using the in1374-based in cellula 157 model of infection, we showed that vDCP-NBs contained not only the DAXX and ATRX proteins but 158 also all the components of the HIRA complex and H3.3 itself. HIRA was also detected co-localizing 159 with vDCP-NBs in neurons of TG harvested from HSV-1 wild type infected mice. Both DAXX/ATRX 160 and HIRA complex components were found interacting with multiple viral loci by chromatin 161 immunoprecipitation (ChIP). Using the same approaches, we showed that latent/quiescent viral 162 genomes were almost exclusively chromatinized with H3.3. Most interestingly, we found that H3.3 163 chromatinization of the viral genomes was dependent on intact PML-NBs, demonstrating that PML-164 NBs contribute to an essential part of the chromatinization of the latent/quiescent HSV-1 genomes. 165 Overall, this study shows that the chromatinization of latent HSV-1 involves a PML-NB/histone 166 H3.3/histone H3.3 chaperone axis that confers and probably maintains epigenetic marks on viral 167

genomes. 168
Results 169 170 The HIRA complex components accumulate in the vDCP-NBs 171 The formation of vDCP-NBs is a molecular hallmark of HSV-1 latency, and vDCP-NBs are 172 present in infected neurons from the initial stages of latency establishment to latency per se in 173 mouse models [15,16]. Using a previously established in vitro latency system [39] consisting of 174 human primary fibroblast cultures infected by a replication-deficient virus (hereafter called 175 in1374) unable to express functional VP16, ICP4 and ICP0, we and others were able to reproduce 176 the formation of vDCP-NBs [16,38]. We first verified that vDCP-NBs induced in human foreskin 177 fibroblast (BJ) and other human primary cells infected by in1374 at a non-permissive temperature 178 of 38.5°C, contained, in addition to PML, the proteins constitutively found in the PML-NBs, i.e., 179 Sp100, DAXX, ATRX, SUMO-1 and SUMO-2/3 ( Fig. S1Ai to vi, and Table S1). The DAXX/ATRX 180 complex is one of the two chaperones of the histone variant H3.3 involved in the replication-181 independent chromatinization of specific, mostly heterochromatic, genome loci [43]. Interestingly, 182 HSV-1 enters the nucleus of the infected cell as a naked/non-nucleosomal dsDNA and remains 183 during latency as a circular chromatinized episome unintegrated in the host genome [28,44]. It is 184 thus tempting to speculate that the presence of DAXX/ATRX in the vDCP-NBs could be linked to a 185 process of initiation and/or maintenance of chromatinization of the latent/quiescent viral genome. 186 The other H3.3 chaperone is known as the HIRA complex and was initially described as specific for 187 the replication-independent chromatinization of euchromatin regions [31,45]. Remarkably, 188 proteins of the HIRA complex are able to bind in a sequence-independent manner to a naked/non-189 nucleosomal DNA [46], suggesting that the HIRA complex could also participate in the recognition 190 and chromatinization of the incoming naked HSV-1 genome. We thus investigated the localization 191 of all members of the HIRA complex and found that they co-localize with the latent/quiescent HSV-192 1 genomes at 2 days post-infection (dpi) in BJ and other human primary cells (Figure 1 Ai to iv, 193 Table S1). To confirm that the co-localization of members of the HIRA complex with the 194 latent/quiescent HSV-1 could be reproduced in neuronal cells, adult mouse TG neuron cultures 195 were infected with in1374 for 2 days before performing immuno-FISH. Mouse Hira, which was the 196 only protein of the HIRA complex detectable in mouse cells, showed a clear co-localization with a 197 subset of viral genomes (Fig. 1B). To analyze whether this co-localization was also reproducible in 198 vivo, immuno-FISH was performed on TG samples from HSV-1-infected mice. Hira was found to co-199 localize with HSV-1 genomes with the "multiple acute"/vDCP-NB pattern (see [16,47] in TG 200 neurons from infected mice at 6 dpi ( Fig. 1C) but not with the "single"/vDCP-NB pattern (see 201 [15,47] at 28 dpi (Fig. 1D), suggesting a dynamic association of this protein with the vDCP-NBs. 202 To analyze this dynamic association, co-localizations between incoming HSV-1 genomes and 203 proteins of the PML-NBs or of the HIRA complex were quantified at early times from 30 min pi to 6 204 hpi using a synchronized infection procedure ( Fig. 1E and Table S2). Except for the proteins of the 205 HIRA complex, the percentages of co-localization increased with time. Interestingly, at 30 min pi, 206 the percentage of co-localization of HSV-1 genomes with HIRA was significantly higher than with 207 PML (41±7% vs 23±5%, p value = 0.03, Student's t-test, Table S2). Although DAXX and ATRX also 208 showed, on average, a greater percentage of co-localization with HSV-1 genomes (36±7% and 209 34±5% at 30 min, respectively) compared with PML, the data were not significant (Table S2). The 210 absence of co-localization of mouse Hira with viral genomes with the "single"/vDCP-NB pattern in 211 mouse TG neurons at 28 dpi suggests that longer infection times could lead to loss of proteins of the 212 HIRA complex from the vDCP-NBs. Infection of BJ cells were reiterated as above, but this time 213 quantifications were performed from 24 hpi to 7 dpi. Strikingly, whereas all the proteins 214 permanently present in the PML-NBs remained co-localized with a maximum of 100% of the 215 latent/quiescent HSV-1 genome from 2 dpi until 7 dpi, proteins of the HIRA complex peaked at 2 216 dpi, and then their co-localization decreased at longer times pi, confirming the temporary 217 association of the HIRA complex with the vDCP-NBs ( Figure 1F, and Table S3). 218 To definitively show that proteins of the HIRA complex were present in vDCP-NBs, immuno-219 FISH were performed on BJ cells infected for 2 days with in1374 to detect either member of the 220 HIRA complex, HSV-1 genomes, and PML. Strikingly, whereas in non-infected cells proteins of the 221 HIRA complex showed predominant nucleoplasmic staining (Fig. 2i, iii, v, vii), in infected cells all 222 the proteins clearly and systematically accumulated in PML-NBs (Fig. 2ii, iv, vi, viii). Consequently, 223 HIRA, UBN1, CABIN1 and ASF1a co-localized with the latent/quiescent HSV-1 genomes in vDCP-224 NBs (arrows in Fig. 2ii, iv, vi, viii). Altogether, these data show that both DAXX/ATRX and HIRA 225 complexes are present within vDCP-NBs in neuronal and non-neuronal cells, suggesting a role for 226 these two complexes in latent/quiescent HSV-1 chromatinization. 227 228 Histone H3.3 chaperones interact with incoming viral genome 229 The co-localization of proteins of the DAXX/ATRX and HIRA complexes with the incoming 230 HSV-1 genomes and their presence in the vDCP-NBs suggested an interaction of these proteins with 231 the viral genome, which we tested by chromatin immunoprecipitation (ChIP) assays. Since DAXX, 232 HIRA, and UBN1 antibodies were not efficient in the ChIP experiments, we constructed cell lines 233 stably expressing myc-DAXX, HIRA-HA, or HA-UBN1 by transduction of BJ cells with the respective 234 lentivirus-expressing vectors (Fig. S2). Cells were infected with in1374 at 38.5°C and harvested to 235 perform ChIP-qPCR on multiple loci spread over the HSV-1 genome (Fig. 3A). Significant 236 enrichments compared to controls were detected for HIRA and UBN1 on several loci, confirming 237 the interaction of these proteins with latent/quiescent HSV-1 genomes. Although ATRX bound to 238 the viral genome, DAXX was not found significantly enriched, but we cannot exclude an alteration of 239 the chromatin binding capacities of the tagged protein. Overall the ChIP-qPCR data confirmed the 240 interaction of proteins of both the DAXX/ATRX and HIRA complexes with incoming vDCP-NBs-241 associated HSV-1 latent/quiescent genomes. [36], Fig. S3A and B). We confirmed that ectopic expression of e-H3.3 led to its accumulation in 250 PML-NBs unlike e-H3.1 (Fig. S3C) [35,36]. In1374 infection of BJ e-H3.1/3-expressing cells led to 251 the co-localization of viral genomes almost exclusively with e-H3.3 ( Fig. 4Ai and ii, and B). 252 Importantly, e-H3.3 co-localized with HSV-1 genomes together with PML in vDCP-NBs (Fig. 4C). The 253 lack of co-localization of viral genomes with e-H3.1 was in accordance with the absence of detection 254 of either H3.1 CAF-1 chaperone subunits (p150, p60, p48) in the vDCP-NBs (Fig. 4D, Table S1). To 255 confirm that e-H3.3, unlike e-H3.1, interacted with HSV-1 genomes, ChIP experiments followed by 256 qPCR were conducted on the same loci than those analyzed above. As expected, e-H3.3, but not e-257 H3.1, was highly enriched on the viral genome independently of the examined locus (Fig. 4E). To 258 confirm that this discrepancy between e-H3.3 and e-H3.1 binding to viral genomes was not due to 259 the ectopic expression of either protein, we performed a similar experiment using antibodies 260 against native proteins. One specific antibody for H3.3 and working in ChIP experiments has been 261 protein quantities in BJ cells ( Fig. S5A and B). None of the shRNA impacted the detection of PML-274 NBs, suggesting that PML-NBs were potentially functional in the absence of either protein (Fig. S6). 275 We first measured the impact of the depletion of each protein on the co-localization of HSV-1 276 genomes with PML. Both shRNAs for each protein provided similar results, i.e., a significant 277 decrease in the co-localization between HSV-1 genomes and PML and thus a decrease in the 278 formation and/or stability of the vDCP-NBs ( Fig. 5A and B, Table S4). The absence of HIRA had a 279 much weaker effect compared to the three others. These data showed that the inactivation of either The above experiments were conducted in a context where the cells, although deficient for 297 the activity of one H3.3 chaperone complex at a time, still contained intact PML-NBs accumulating 298 e-H3.3 ( Fig. S6 and S8). Therefore, we hypothesized that the accumulation of H3.3 within the PML-299 NBs could be one of the key events acting upstream of the H3.3 chaperone complex activity for the 300 induction of chromatinization of the latent/quiescent HSV-1 by H3.3. Thus, we analyzed the HSV-1 301 chromatinization in cells lacking PML-NBs. We had previously analyzed the impact of PML 302 depletion on the co-localization of the DAXX/ATRX and HIRA chaperone complexes with the HSV-1 303 genomes. In a previous study conducted in HSV-1 latently infected PML KO mice, we showed that 304 the absence of PML significantly impacted the number of latently infected TG neurons showing the 305 "single"/vDCP-NB HSV-1 pattern and favored the detection of neurons containing the "multiple-306 latency" pattern prone to LAT expression [15,47]. We analyzed the very few neurons showing a 307 "single"/vDCP-NB-like pattern in the latently infected PML KO mice for the co-localization of DAXX 308 and ATRX with the viral genomes. We could not detect any of the two proteins co-localizing with 309 the latent HSV-1 genomes ( Fig. 6Ai to vi). Although informative, these in vivo studies did not allow The HSV-1 genome enters the nucleus of infected neurons, which support HSV-1 latency as a 349 naked/non-nucleosomal DNA. Many studies have described the acquisition of chromatin marks on 350 the viral genome concomitantly to the establishment, and during the whole process, of latency. 351 Paradoxically, although it is undisputable that these chromatin marks will predominantly be 352 associated with latency and reactivation, few data are available for the initiation of the 353 chromatinization of the incoming viral genome. Here, we demonstrate the essential contribution of with HIRA compared with PML at very early times pi (30 min). These data suggest that the HIRA 368 complex could also be involved to some extent in the establishment of HSV-1 latency by the initial 369 recognition of the incoming naked/non-nucleosomal viral DNA and the chromatinization of non-370 replicative HSV-1 genomes intended to become latent. In this respect, a recent study suggested an 371 anti-viral activity associated with HIRA against HSV-1 and murine cytomegalovirus lytic cycles [51]. 372 Interestingly, all the proteins of the HIRA complex have been previously shown to be able to 373 directly bind to naked DNA in a sequence-independent manner, in contrast to DAXX and ATRX 374 proteins [46]. Nevertheless, our ChIP data highlighted some specific viral genome loci that interact 375 at least with ATRX. Thus, it is likely that ATRX, unlike HIRA and UBN1, indirectly binds to the viral 376 genome. The gamma-interferon-inducible protein 16 (IFI16), a member of the PYHIN protein 377 family, has been described as a nuclear sensor of incoming herpesviruses genomes and suggested 378 to promote the addition of specific chromatin marks that contribute to viral genome silencing [52- Given the particular structure formed by the latent/quiescent HSV-1 genome with the PML-398 NBs, our study raises the question of the possible acquisition of chromatin marks in the vDCP-NBs. 399 The depletion of H3.3, which almost exclusively participates in latent/quiescent HSV-1 genome 400 chromatinization, does not prevent the formation of vDCP-NBs and is rather in favor of such a 401 scenario (Fig. S11). It is unlikely that a possible replacement of H3.3 by the canonical H3.1 for the 402 chromatinization of the incoming HSV-1 genomes could occur prior to the association with vDCP-403 NBs. Indeed, our multiple immuno-FISH and ChIP assays failed to detect H3.1 and/or H3.1 404 chaperones that associate or co-localize with viral genomes. Nonetheless, we cannot rule out a 405 possible replacement of H3.3 with another H3 variant. 406 Even if a process of viral genome chromatin assembly and/or maintenance occurs in the 407 vDCP-NBs, some of our data tend to show that it unlikely represents the only pathway for 408 chromatinization of the incoming naked HSV-1 genomes. Indeed, the depletion of DAXX, ATRX, 409 UBN1 and to a lesser extent HIRA, significantly impacts the co-localization of the latent/quiescent 410 HSV-1 genomes with PML, and hence the formation of vDCP-NBs, but only mildly affects the H3.3 411 association with the analyzed viral genome loci. However, beyond a compensatory mechanism 412 between the two complexes that could bypass the requirement of the vDCP-NBs, we cannot exclude

Frozen sections 498
Frozen sections of mouse TG were generated as previously described [69]. Mice were 499 anesthetized at 6 or 28 d.p.i., and before tissue dissection, mice were perfused intra-cardially with a 500 solution of 4% formaldehyde, 20% sucrose in 1X PBS. Individual TG were prepared as previously 501 thawed, rehydrated in 1x PBS and permeabilized in 0.5% Triton X-100. Heat-based unmasking was 510 performed in 100 mM citrate buffer, and sections were post-fixed using a standard methanol/acetic 511 acid procedure and dried for 10 min at RT. DNA denaturation of the section and probe was 512 performed for 5 min at 80°C, and hybridization was carried out overnight at 37°C. Sections were 513 washed 3 x 10 min in 2 x SSC and for 3 x 10 min in 0.2 x SSC at 37°C, and nuclei were stained with 514 Hoechst 33258 (Invitrogen). All sections were mounted under coverslips using Vectashield 515 mounting medium (Vector Laboratories) and stored at 4°C until observation. 516 For immuno-DNA FISH, cells or frozen sections were treated as described for DNA-FISH up to the 517 antigen-unmasking step. Tissues were then incubated for 24 h with the primary antibody. After 518 three washes, secondary antibody was applied for 1 h. Following immunostaining, the cells were 519 post-fixed in 1% PFA, and DNA FISH was carried out from the methanol/acetic acid step onward. 520 The same procedures were used for infected neuronal cultures except that the cells were fixed in 521 2% PFA before permeabilization.

553
ChIP and quantitative PCR 554 Cells were fixed with formaldehyde 2% for 5 min at RT, and then glycine 125 mM was added 555 to arrest fixation for 5 min. After two washes with ice-cold PBS, the cells were resuspended in 556 buffer A (100 mM Tris HCl pH 9.4, 10 mM DTT) and subjected to two 15 min-incubations on ice 557 then at 30°C. The cells were subsequently lysed in lysis buffer B (10 mM Hepes pH 6.5, 0.25 % 558 Triton, 10 mM EDTA, 0.5 mM EGTA) for 5 min at 4°C to recover the nuclei. Nuclei were incubated 559 for 5 min at 4°C in buffer C (10 mM Hepes pH 6.5, 2 M NaCl, 10 mM EDTA, 0.5 mM EGTA) and then 560 The following primary antibodies were used: 589 All secondary antibodies were HRP-conjugated and were raised in goats (Sigma). 597       Table S2 for data).  Table S3 for data).        Table   670 S4 for data).

671
(C) ChIP-qPCR for the detection of e-H3.3 associated with HSV-1 genomes and performed in e-H3.3-expressing BJ cells 672 infected with in1374 for 24 h and previously transduced with a lentivirus expressing a control shRNA (shCTRL, blue) or a targeted shRNA (red). Anti-HA antibody was used for the ChIP experiments. The analyzed viral loci were described 674 previously. Data represent means from three independent experiments ± SD. The Student's t-test was applied to assess the 675 significance of the results. * = p< 0.05 (see Table S5 for data).   Table S7 for data).

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Data represent means from three independent experiments ± SD. The Student's t-test was applied to assess the significance 697 of the results. * = p< 0.05 (See Table S6 for data).     Figure S1. Latent/quiescent HSV-1 genomes co-localize with PML and PML-NB-associated proteins in vDCP-NBs.                             Cohen et al. Figure 6 A.  C.