Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT

Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide transport protein CERT. CERT mediates non- vesicular transport of ceramide from the ER to the trans-Golgi network, whereupon ceramide is converted to sphingomyelin and other sphingolipids that play important roles in cell proliferation, cell signalling and membrane trafficking. Using click chemistry to profile the kinetics of sphingolipid metabolism in cultured cells, we show that pUL21-mediated dephosphorylation activates CERT and increases the rate of ceramide to sphingomyelin conversion. Purified pUL21 and full-length CERT interact with sub-micromolar affinity and we map the domains responsible for the interaction. Solving the solution structure of the pUL21 C-terminal domain in complex with the CERT PH and START domains using small-angle X-ray scattering allows us to identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. Sphingolipid profiling demonstrates that ceramide to sphingomyelin conversion is severely diminished in the context of HSV- 1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. However, virus replication and spread are not significantly altered when pUL21-mediated CERT dephosphorylation is abolished, highlighting that dephosphorylation of other cellular and/or viral targets underpins the important role of pUL21 in HSV-1 biology. Significance Herpes simplex virus (HSV)-1 causes a life-long dormant infection of neurons, sporadically reactivating to manifest as cold-sores or genital herpes. While the impact of HSV-1 upon the protein content of infected cells has been well studied, we know relatively little about its impact upon cellular lipids. Using bioorthogonal labelling in cultured cells we show that HSV-1 protein pUL21 activates the key cellular lipid transport protein CERT to accelerate the conversion of ceramide to sphingomyelin. HSV-1 infection dramatically alters the kinetics of ceramide metabolism, leading to ceramide accumulation. Mutation of HSV-1 pUL21 to prevent CERT activation further enhances ceramide accumulation but this does not alter the replication or spread of HSV-1, highlighting that other cellular and/or viral proteins represent the critical targets of pUL21-mediated dephosphorylation in cultured cells.

pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple 23 cellular and virus proteins, including the cellular ceramide transport protein CERT. CERT mediates non-24 vesicular transport of ceramide from the ER to the trans-Golgi network, whereupon ceramide is 25 converted to sphingomyelin and other sphingolipids that play important roles in cell proliferation, cell 26 signalling and membrane trafficking. Using click chemistry to profile the kinetics of sphingolipid 27 metabolism in cultured cells, we show that pUL21-mediated dephosphorylation activates CERT and 28 increases the rate of ceramide to sphingomyelin conversion. Purified pUL21 and full-length CERT 29 interact with sub-micromolar affinity and we map the domains responsible for the interaction. Solving 30 the solution structure of the pUL21 C-terminal domain in complex with the CERT PH and START 31 domains using small-angle X-ray scattering allows us to identify a single amino acid mutation on the 32 surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. Sphingolipid profiling 33 demonstrates that ceramide to sphingomyelin conversion is severely diminished in the context of HSV-34 1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 35 that lacks the ability to activate CERT. However, virus replication and spread are not significantly altered 36 when pUL21-mediated CERT dephosphorylation is abolished, highlighting that dephosphorylation of 37 other cellular and/or viral targets underpins the important role of pUL21 in HSV-1 biology. 38 Significance 39 Herpes simplex virus (HSV)-1 causes a life-long dormant infection of neurons, sporadically reactivating 40 to manifest as cold-sores or genital herpes. While the impact of HSV-1 upon the protein content of 41 infected cells has been well studied, we know relatively little about its impact upon cellular lipids. Using 42 bioorthogonal labelling in cultured cells we show that HSV-1 protein pUL21 activates the key cellular 43 lipid transport protein CERT to accelerate the conversion of ceramide to sphingomyelin. HSV-1 infection 44 dramatically alters the kinetics of ceramide metabolism, leading to ceramide accumulation. Mutation of 45

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Human cells typically maintain lipidome homeostasis via robust feedback and control mechanisms, 119 which respond to perturbations via local activation of specific signalling pathways [39,40]. We 120 hypothesised that pUL21, expressed at late points of infection [3], would likely alter the rate of 121 sphingolipid synthesis and thereby affect lipid-mediated signalling events, rather than altering the 122 overall steady-state lipid composition of infected cells. Synthetic lipids with alkyne-containing acyl 123 chains are efficiently processed by mammalian lipid-modifying enzymes and represent powerful tools 124 to probe lipid metabolism [41]. The impact of pUL21-directed CERT dephosphorylation on sphingolipid 125 metabolism was therefore probed using a clickable analogue of sphingosine (Sph), alkyne-Sph, to 126 monitor the rate of sphingomyelin biogenesis in immortalized human keratinocyte (HaCaT) cells. 127 Exogenous Sph is efficiently incorporated into cellular metabolic pathways, being rapidly converted into 128 ceramide (Cer) and then sphingomyelin (SM) or hexosylceramides (HexCer) like glucosylceramide or 129 galactosylceramide [42]. It is also converted into phosphatidylcholine (PC) via a so-called 'salvage' 130 pathway ( Fig 1A) that directs sphingosine to palmitoyl-CoA which serves as substrate for re-acetylation 131 of lysophosphatidylcholine [43]. 132 To monitor Sph metabolism, HaCaT cells, either parental or stably expressing pUL21 (HaCaT21), were 133 incubated with alkyne-Sph for five minutes (pulse) and the rate of alkyne-Sph incorporation into the 134 competing metabolic pathways was monitored for two hours (chase) by high performance thin layer 135 chromatography (HPTLC) separation and detection of lipids conjugated to coumarin-azide via a 'click' 136 reaction (Fig 1B and 1C). Alkyne-Sph was very efficiently converted to alkyne-Cer in both cell types, 137 the levels of alkyne-Cer remaining relatively stable throughout the chase, and for both cell types 138 synthesis of alkyne-PC and alkyne-SM was observed, but not synthesis of alkyne-HexCer. HaCaT21 139 cells exhibited significantly reduced rates of alkyne-Sph conversion and the level of alkyne-Cer is 140 significantly lower (Fig S1). The rate of alkyne-PC synthesis was also reduced, although this reduction 141 was not significant owing to the high inter-experiment variability of the alkyne-PC signal for the parental 142 HaCaT cells. Taken together, these results indicated that lipid metabolism is altered in HaCaT cells 143 constitutively expressing pUL21. However, it remained unclear whether these differences reflected 144 adaptations of cellular lipid metabolism in response to constitutive pUL21 expression rather than a direct 145 effect upon CERT activity. Since CERT-mediated non-vesicular transport of Cer from the ER to the 146 TGN defines the rate of Cer-to-SM conversion [21-23], pUL21-directed CERT dephosphorylation (and 147 thus activation) should increase the rate of SM synthesis. Consistent with this hypothesis, the 148 abundance alkyne-SM as a fraction of total alkyne-Cer plus alkyne-SM was significantly higher in 149 HaCaT21 versus HaCaT cells at early time points (0-30 min) during the chase (Fig 1D). By the 60 min 150 chase time point the rate of alkyne-SM accumulation slows and the difference in relative abundance 151 between cell lines was diminished, consistent with the alkyne-SM levels in both cell types approaching 152 equilibrium. This increase in alkyne-SM accumulation as a fraction of alkyne-Cer plus alkyne-SM 153 abundance was observed despite similar overall signal for alkyne-SM and alkyne-Cer in HaCaT21 cells 154 when compared to parental cells at early time points (0-30 min), the abundance of these lipids being 155 significantly lower in HaCaT21 cells at later time points (Fig 1E). Reduced overall levels of SM plus Cer 156 that H6-pUL21C forms an equimolar complex with H6-miniCERTL with a dissociation constant (KD) of 234 4.3 ± 1.2 μM (Fig 3B; Table 1). The approximately 4-fold reduction in miniCERTL binding by pUL21C 235 versus full-length pUL21 is consistent with previous immunoprecipitation results, where transfected 236 pUL21C-GFP captured endogenous CERT slightly less efficiently than did pUL21-GFP in HEK293T 237 cells [18]. These ITC experiments confirm that the C-terminal domain of pUL21 is the major determinant 238 of CERT binding. 239 To probe the structural basis of the CERT recruitment by pUL21, a pre-formed complex of H6-240 miniCERTL and H6-pUL21C was subjected to SEC with inline SAXS measurement (SEC-SAXS, Fig  241   3C). SAXS data were processed by averaging frames with a consistent calculated radius of gyration 242 (Rg) and then subtracting averaged buffer frames to yield the H6-miniCERTL:H6-pUL21C complex 243 scattering profile (Fig 3D). The probable frequency of real-space distances (p(r) profile) of the complex 244 is moderately asymmetric (Fig 3E), in contrast to the highly symmetric p(r) profiles of H6-pUL21C (Fig  245   S2B) or H6-miniCERT ( Fig 2H) alone, suggesting a less spherical particle, and the dimensions of the 246 complex (Rg 3.5 nm, Dmax 13.6 nm) are substantially larger than for H6-pUL21C (Rg 2.2 nm, Dmax 8.5 247 nm) or H6-miniCERT (Rg 2.7 nm, Dmax 9.1 nm). The peak of the dimensionless Kratky plot is slightly 248 higher, with its peak away from sRg = √3 (Fig 3F), suggesting some flexibility in the system granted 249 either by modest dissociation of the complex or some mobility of the H6-miniCERTL domains with 250 respect to each other and H6-pUL21C. Ab initio modelling using GASBOR indicated an elongated 251 molecule ( Fig 3G). While initial pseudo-atomic models of the complex generated using a fixed 252 conformation of H6-miniCERTL did not fit the SAXS profile acceptably, allowing the PH and STARTL 253 domains freedom to move with respect to each other and to H6-pUL21C yielded three pseudo-atomic 254 models with high-quality fits to the SAXS profile ( Fig 3H and Fig S3). In all three top models miniCERTL 255 binds 'end-on' to the pUL21 molecule, forming an ellipsoidal 'rugby ball' like particle. In two of the top 256 three models miniCERTL binds the 'left wing' of the dragonfly-shaped pUL21C domain [50], whereas in 257 the other it binds the 'right wing' (Fig S3). These models all have a similar overall shape, and thus all 258 explain the SAXS scattering data well, but the relative orientations of H6-miniCERTL and H6-pUL21C 259 differ. All three top models were thus used to design specific pUL21 mutations that might disrupt 260 (mini)CERT binding. 261 Mutations in pUL21C were designed to identify whether miniCERTL binds the left or right wing of this 262 domain. Amino acids in helix α4 or the subsequent loop of pUL21C were mutated to test binding to the 263 left wing (Fig 3H, Fig S3), whereas amino acids in helices α5 and α9 were used to test binding to the 264 right wing (Fig S3B). Immunoprecipitation experiments in transfected HEK293T cells that had been 265 infected with HSV-1 lacking pUL21 expression (HSV-1 ΔpUL21) demonstrated that 4 of 5 substitution 266 in the left wing of pUL21C (P365D, V368E, R373E and V382E) disrupted the ability of CERT to co-267 precipitate with pUL21-GFP (Fig 4A), whereas none in the pUL21C right wing disrupted CERT co-268 precipitation ( Fig S3C). These results are consistent with CERT binding the left wing of pUL21C. The 269 proximity of the pUL21C amino terminus to miniCERTL in these models is also consistent with 270 observations that CERT binding is lost when pUL21C has a bulky N-terminal GFP tag, but retained 271 when the GFP tag is C-terminal [18]. 272 Of the pUL21 substitutions that disrupted CERT binding (Fig 4A), V382E appeared to cause the largest 273 decrease in CERT binding while maintaining the ability of pUL21 to co-precipitate its other known 274 binding partners, PP1 [18] and pUL16 [10]. H6-pUL21 V382E was purified following bacterial expression. 275 Differential scanning fluorimetry (a.k.a. Thermofluor) showed pUL21 V382E to be well folded as its thermal 276 stability is similar to wild-type H6-pUL21 ( Fig 4B). ITC analysis demonstrated that H6-pUL21 V382E has 277 approximately 8-fold reduced binding affinity for H6-miniCERTL when compared to wild-type pUL21 ( Fig  278   4C; Table 1). The effect of the V382E substitution upon the ability of pUL21 to promote CERT 279 dephosphorylation, converting CERT P to CERT O , was probed using an in vitro dephosphorylation assay 280 with all-purified reagents (Fig 4D and E) infected cells (Fig 5A,B). In addition to CERT, pUL21 expression reduces the phosphorylation of 290 multiple substrates of the viral kinase pUS3 in HSV-1 infected cells [18], the phosphorylated forms of 291 these substrates being detectable using an antibody that recognises phosphorylated substrates of the 292 cellular kinase Akt [53]. While infection with ΔpUL21 HSV-1 causes a dramatic increase in the 293 abundance of multiple phosphorylated pUS3 substrates, the abundance of these phosphoforms is 294 indistinguishable between wild-type and pUL21 V382E HSV-1 (Fig 5A). This confirms that the V382E 295 substitution specifically disrupts CERT dephosphorylation, rather than generally inhibiting the ability of 296 pUL21 V382E to recruit PP1 to substrates. Similar changes in CERT dephosphorylation, but not in the 297 dephosphorylation of other pUS3 substrates, are observed when Vero cells are infected with pUL21 V382E 298 HSV-1 (Fig S4A,B). Immunocytochemistry confirms that both wild-type and V382E pUL21 have the 299 similar subcellular localisation, being observed predominantly at the nuclear rim of infected Vero cells 300 ( Fig 5C). 301 Metabolic labelling was used to monitor the impact of pUL21-mediated CERT dephosphorylation on 302 sphingolipid biogenesis during infection. A pulse-chase experiment was performed where HaCaT cells 303 infected with wild-type or pUL21 V382E HSV-1, or mock infected, were incubated for 5 min with alkyne-304 Sph at 14 hours post-infection (hpi) and its metabolites were monitored for two hours. Cer accumulates 305 and the rate of SM synthesis is significantly decreased in cells infected with wild-type and pUL21 V382E 306 HSV-1 when compared to uninfected cells (Fig 5D). Although a substantial decrease in the rate of SM 307 synthesis is seen for both wild-type and mutant HSV-1 infection, the defect is significantly larger in cells 308 infected with HSV-1 pUL21 V382E (Fig 5E). The overall abundance of SM plus Cer is similar between 309 infected and uninfected cells, suggesting that Cer to SM conversion is specifically impaired rather than 310 influx of click-Sph into the SM biogenesis pathway being defective (Fig 5F). Taken together, this 311 suggests that pUL21-mediated activation of CERT accelerates Cer to SM conversion during infection, 312 albeit from a much lower base than in uninfected cells. 313 Having confirmed that pUL21 V382E HSV-1 specifically lacks the ability to stimulate CERT 314 dephosphorylation, and that HSV-1 encoding pUL21 V382E has a reduced rate of Cer to SM conversion, 315 the impact of this deficit upon virus replication and spread in cultured cells was assessed. Wild-type 316 and pUL21 V382E HSV-1 form similar sized plaques on HaCaT and Vero cells (Fig 6A), suggesting that 317 CERT dephosphorylation is dispensable for efficient viral cell-to-cell spread. A single-step growth curve, 318 where Vero and HaCaT cells are infected at a high multiplicity of infection (MOI) and the production of 319 infectious progeny is monitored over time, was used to compare the replication of wild type and 320 pUL21 V382E HSV-1 (Fig 6B). Both viruses produce similar abundance of infectious progeny by 24 hours 321 post infection. In two biologically independent experiments performed for each cell type the kinetics of 322 virus replication was consistently accelerated, with higher titres of pUL21 V382E HSV-1 being observed 323 between 6-12 hours post-infection, but the difference in growth rate is not statistically significant for 324 either cell line (two-way ANOVA with Sidak's multiple comparisons test). 325 While HSV-1 preferentially remains cell-attached, spreading via direct cell:cell contacts, the cell-free 326 secretion of virions from infected cells is altered when CERT is depleted or overexpressed [54]. Cell-327 free release of pUL21 V382E HSV-1 was determined by quantitating the amount of infectivity secreted into 328 the medium as a percentage of overall infectivity (secreted plus cell-associated), normalised for each 329 independent replicate to the secretion of a wild-type virus in control experiments performed at the same 330 time. HaCaT cells infected for 12 h with various MOI of pUL21 V382E HSV-1 did not release significantly 331 more or less infectivity when compared to WT HSV-1 infection (Fig 6C). Similarly, the cell-free release 332 of infectivity at 12 hpi did not differ between Vero cells infected with pUL21 V382E or WT HSV-1 (Fig S4C). and activation of the Cer transport protein CERT (Fig 5). This is the first observation of a viral protein 342 directly binding CERT and altering its activity. While pUL21-mediated CERT activation causes an 343 apparent modest acceleration in the rate of virus replication, this effect is not statistically significant and 344 CERT activation does not contribute to replication or cell-to-cell spread of HSV-1 in cultured 345 keratinocytes or epithelial cells (Fig 6). 346 Our previous work showed that pUL21 is a phosphatase adaptor with multiple different targets [18]. 347 Mutation of the pUL21 TROPPO motif, required for PP1 binding and thus stimulation of 348 dephosphorylation, dramatically reduced the replication and spread of HSV-1. While the TROPPO motif 349 was identified via its conservation across alphaherpesvirus sequences, the molecular basis of CERT 350 recruitment remained unknown. Biochemical mapping and SAXS structural characterisation allowed us 351 to identify a specific mutation, V382E, that decreases the affinity of pUL21 for miniCERTL by 352 approximately 8-fold ( Fig 4C). While this reduction in affinity is moderate when assayed in dilute 353 biochemical solution, it is sufficient to disrupt co-precipitation of CERT from transfected cells (Fig 4A), 354 it abolishes pUL21-mediated dephosphorylation of CERT during infection (Fig 5A,

Mutagenesis of viral genomes and generation of recombinant HSV-1 543
All HSV-1 strain KOS viruses used in this study were reconstituted from a bacterial artificial 544 chromosome (BAC) [76] and the mutated strain was generated using the two-step Red recombination 545 method [52] with the following primers: 546 Forward: 5′-CGGCTCGTAGGCCGGTACACACAGCGCCACGGCCTGTACGAACCTCGGCCCGACG 547 ACCCAGTAGGATGACGACGATAAGTAGGG 548 Reverse: 5′-CGTTGATGGCATCGGCCAAGACTGGGTCGTCGGGCCGAGGTTCGTACAGGCCGTG 549 GCGCTGTCAACCAATTAACCAATTCTGATTAG 550 The generation of pUL21 deletion mutant (ΔpUL21) was described previously [18]. To generate the P0 551 stocks, Vero cells were transfected with the recombinant BAC DNA together with pGS403 encoding 552 Cre recombinase (to excise the BAC cassette) using TransIt-LT1 (Mirus) following the manufacturer's 553 instructions. After 3 days the cells were scraped into the media, sonicated at 50% power for 30 s in a 554 cup-horn sonicator (Branson), and titrated on Vero cell monolayers. The subsequent stocks were 555 generated by infecting either Vero (HSV-1 WT) or HaCaT pUL21 cells (HSV-1 mutants) at MOI of 0.01 556 for 3 days. The cells were then scraped and isolated by centrifugation at 1,000×g for 5 min. Pellets were 557 resuspended in 1 mL of complete DMEM supplemented with 100 U/mL penicillin, 100 μg/mL 558 streptomycin and freeze/thawed thrice at -70°C before being aliquoted, titered on Vero cell monolayers, 559 and stored at -70°C until required. The presence of the desired mutation in the reconstituted virus 560 genomes was confirmed by sequencing the pUL21 gene. 561

Metabolic labelling and lipid extraction for thin layer chromatography 562
Metabolic labelling was performed using sub-confluent (60-80% confluence) HaCaT or HaCaT21 cells 563 grown in a 6-well plate. For analysis of stable cell lines, the cells were pre-treated for 30 min with 564 complete DMEM containing 1 μM N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-565 12, Tokyo Chemical Industry) dissolved in 0.1% dimethyl sulfoxide (DMSO, Merck), or 0.1% DMSO 566 alone, and HPA-12 or DMSO were retained at the same concentrations throughout the subsequent 567 incubation steps. For infection, cells were infected (below) 14 hours before metabolic labelling. 568 For metabolic labelling, wells were washed twice with warm PBS before incubation in 500 μL pre-569 warmed DMEM with 1% (v/v) Nutridoma (Merck) supplemented with 5 μM clickable sphingosine 570 (Cayman Chemical) for 5 min (pulse). Next, cells were washed twice with warm PBS and 1 mL of pre-571 warmed DMEM with 1% (v/v) Nutridoma was added to each well, followed by incubation at 37°C for the 572 indicated times of chase. Click-Sph was stored as a 3.3 mM ethanolic stock solution at -20°C. 573 At the indicated times of chase, the plate with cells was transferred onto the ice, washed twice with 1 574 mL ice-cold PBS, scraped into 300 μL of ice-cold PBS and transferred into appropriate 1.5 mL 575 microcentrifuge tubes containing 600 μL of methanol. To each tube 150 μL of chloroform was added, 576 followed by vigorous vortexing. The precipitated protein was pelleted (20,000×g, 1 min) at room 577 temperature (RT). The organic supernatant was transferred to separate 2 mL tubes containing 300 μL 578 chloroform. 600 μL of 0.1% acetic acid in H2O was subsequently added to each tube to induce formation 579 of two phases. Following extensive vortexing, the phases were separated by centrifugation (20,000×g, 580 1 min, RT) and the lower phase was transferred to a new 1.5 mL microcentrifuge tube. This lipid-581 containing solvent phase was dried in a UniVapo centrifugal vacuum concentrator (UniEquip) at 30°C 582 for 20 min. Guinier approximation (lnI(s) vs s 2 , for sRg < 1.3). The maximum particle dimension, Dmax, was estimated 670 based on the probable distribution of real-space distances p(r) which was calculated using GNOM [83]. 671 A concentration-independent estimate of molecular weight was determined using a Bayesian 672 consensus method [84]. All structural parameters are reported in Table S1. 673 Ab initio modelling was performed using GASBOR [49] and figures show the models that best fit their 674 corresponding SAXS profiles (lowest χ 2 ). Pseudo-atomic modelling was performed using CORAL [85]. 675

Virus release assays 736
Monolayers of HaCaT or Vero cells were infected as described above for single-step virus growth 737 assays, infections being performed in technical duplicate or triplicate for each independent experiment. 738 At 12 hpi, the media (500 μL) were harvested to 1.5 mL Eppendorf tubes, spun down for 5 min at 1000 739 g to remove any detached cells, 300 μL of the supernatant was carefully transferred to fresh tubes and 740 stored at -70°C until titration. The cells were overlaid with 500 μL of fresh DMEM and the plates were 741 immediately frozen at -70°C. Titrations were performed on monolayers of Vero cells as described 742 above. Statistical tests were performed using Prism 7 (GraphPad Software). 743 Immunocytochemistry 744 Cells grown on #1.5 coverslips were infected at an MOI of 1 as described above. At 14 hpi, cells were 745 washed with PBS and incubated with freezing cold (-20°C) methanol for 5 min at -20°C. Coverslips 746 were washed with PBS, followed by incubation with blocking buffer (1% (w/v) BSA in PBS) for 30 min 747 at RT. Primary antibodies (above) were diluted in blocking buffer and incubated with coverslips for 1 h. 748 Coverslips were washed ten times with PBS before incubation for 45 min with the secondary antibodies 749 (above) diluted in blocking buffer. Coverslips were washed ten times in PBS and ten times in ultrapure 750 water before mounting on slides using Mowiol 4-88 (Merck) containing 200 nM 4′,6-diamidino-2-751 phenylindole (DAPI). Images were acquired using an inverted Olympus IX81 widefield microscope with 752 a 60× Plan Apochromat N oil objective (numerical aperture 1.42) (Olympus) and Retiga EXi Fast1394 753 interline CCD camera (QImaging). 754 Samples were harvested at the indicated times and titres determined by plaque assay using Vero cells. 916 Data are presented as mean values ± SEM of technical duplicates from one representative experiment. 917 Difference in replication kinetics across two biological replicates is not statistically significant (two-way 918 ANOVA with Sidak's multiple comparisons test). (C) Virus release into the culture supernatant from 919 HaCaT cells infected with WT or pUL21 V382E HSV-1 at various MOIs. Samples were harvested at 12 hpi 920 and virus infectivity in the cells versus the culture medium was measured by titration on Vero cells. The 921 fold change in secretion of infectivity into the culture medium for pUL21 V382E versus WT HSV-1 is shown 922 as mean values ± SEM of two (MOI = 1 or 3-5) or three (MOI = 10) independent experiments. For MOI 923 3-5, the data represent one independent experiment performed at MOI = 3 and one at MOI = 5. For 924 each MOI, the extent of virus secretion was compared using an unpaired t-test (ns, non-significant). 925 926