{"paper_id":"b47a76aa-15e6-4c9c-bf0e-543125fc1918","body_text":"Alphaherpesvirus pUL21 homologues use non-canonical motifs to 1 \ncompete with cellular adaptors for protein phosphatase 1 binding 2 \nHolly Monkhouse1,2,a, Daniela S. Carter-Lopez1,a, Tomasz H. Benedyk1,b, Janet E. Deane2, 3 \nStephen C. Graham1 4 \n 5 \n1Department of Pathology, University of Cambridge, Cambridge CB2 1QP , UK 6 \n2Cambridge Institute for Medical Research, University of Cambridge, Cambridge Biomedical 7 \nCampus, Cambridge, UK 8 \naThese authors contributed equally to this work 9 \nbCurrent address: Department of Biology and Biochemistry, University of Houston, Houston, TX 10 \n77204  11 \nCorresponding author 12 \nStephen C. Graham <scg34@cam.ac.uk> 13 \nKeywords 14 \nVZV , RIPPO, PIP , regulatory subunit, PPP1R15A 15 \nORCIDs: 16 \nTHB 0000-0001-6420-3665; JED 0000-0002-4863-0330; SCG 0000-0003-4547-4034 17 \nRunning title:  18 \nHerpes competes for PP1 binding using novel motifs 19 \nContributions: 20 \nConceptualization: SCG; Data Curation: HM, DSC, SCG; Funding Acquisition: JED, SCG; 21 \nInvestigation: HM, DSC, THB, OST, SCG; Project Administration: SCG, JED; Supervision: SCG; 22 \nVisualization: HM, DSC, SCG; Writing – Original Draft Preparation: SCG; Writing – Review & 23 \nEditing: HM, DSC, THB, JED, SCG.   24 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n2 \n \nAbstract  25 \nProtein phosphatase 1 (PP1) is a key regulator of cellular phosphorylation and its activity is 26 \nregulated via binding to cellular regulatory proteins via conserved short linear motifs (SLiMs). 27 \nThe herpes simplex virus (HSV)-1 protein pUL21 binds PP1 via the TROPPO motif, which lacks 28 \nsequence similarity to canonical PP1-binding SLiMs. Here, we combine structure prediction, 29 \nmutagenesis, and biophysical assays to elucidate the molecular basis of this interaction. 30 \nAlphaFold2-Multimer structural models suggest that the TROPPO motifs of pUL21 and of 31 \npORF38, the varicella-zoster virus homologue of pUL21, bind the same hydrophobic groove on 32 \nPP1 as RVxF and ϕϕ[xF] motifs, forming an extended β-sheet that bridges PP1 and the N-33 \nterminal domain of pUL21 or pORF38. Site-directed mutagenesis of both pUL21 and PP1 34 \nconfirms key predicted interactions. Competition fluorescence polarisation confirms that 35 \npUL21 competes directly with cellular PP1 RvXF motifs for PP1 binding, albeit with lower affinity. 36 \nSubstituting key residues of the pUL21 TROPPO motif to resemble a canonical RVxF sequence 37 \nincreases PP1 binding, suggesting that alphaherpesviruses have evolved suboptimal motifs to 38 \nfine-tune phosphatase recruitment that may balance viral kinase and phosphatase activities 39 \nduring infection. Our findings reveal a novel mechanism of PP1 recruitment by viral proteins and 40 \nsuggest that many other PP1 regulators may possess non-canonical binding motifs and thus 41 \nremain undiscovered. 42 \nIntroduction 43 \nPhosphorylation is critical to the control of most cellular processes. Viruses have learnt to 44 \nexploit this, encoding kinases that regulate cell cycle progression, modify gene expression, 45 \novercome innate immune restriction and prevent apoptosis (1, 2). Similarly, multiple viruses 46 \nmodulate the dephosphorylation of both cellular and viral target proteins within infected cells. 47 \nHerpes simplex virus (HSV)-1 protein ICP34.5 (a.k.a. γ134.5) counteracts the antiviral innate 48 \nimmune response by stimulating dephosphorylation of eIF2α to relieve host transcriptional 49 \nshutdown (3, 4). The HIV Tat protein interacts with the cellular enzyme protein phosphatase 1 50 \n(PP1) to promote viral gene transcription (5, 6). Additionally, the measles virus V protein 51 \nsuppresses innate immune signalling by sequestering PP1, thereby preventing 52 \ndephosphorylation of the cytosolic RNA sensor MDA5 that is required for its activation (7). Given 53 \nthe multitude of cellular signalling pathways that are regulated by protein phosphorylation, 54 \ninterfering with virus-mediated protein dephosphorylation represents a promising new avenue 55 \nfor the development of potent antiviral therapies (6, 8). 56 \nPP1 is a highly abundant cellular serine/threonine phosphatase. PP1 has low intrinsic 57 \nspecificity and it recruits its substrates via regulatory proteins (9, 10), also known as adaptor 58 \nproteins, PP1 Interacting Proteins (PIPs) or Regulators of Protein Phosphatase One (RIPPOs) 59 \n(11). These regulatory proteins utilise small linear motifs (SLiMs) to bind different surfaces of 60 \nPP1 (9, 10). The most abundant and well characterised of these is the RVxF motif, which binds 61 \nto a hydrophobic groove on the surface of PP1. Over 90% of cellular PP1 adaptors have an RVxF 62 \nmotif (12), which has the consensus sequence [KR][KR][VI]ψ[FW], where ψ is any amino acid 63 \nexcept FIMYDP (13). Many PP1 regulatory proteins possess more than one PP1-interacting SLiM 64 \n(reviewed in (10)) allowing combinatorial control of PP1 binding (14). One example of an 65 \n‘accessory’ SLiM is the ϕϕ[xF] motif, first identified via structural characterisation of PP1 in 66 \ncomplex with spinophilin (15), which lies approximately 6–8 residues downstream of the RVxF 67 \nmotif and is present alongside the RVxF motif in most PP1 regulatory proteins (16). Like their 68 \ncellular counterparts, viral PP1 regulatory proteins commonly contain an RVxF motif that is 69 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n3 \n \ncritical for PP1 binding (5, 7, 17), but the presence of additional SLIMs in these adaptors is less 70 \nwell explored. 71 \nHSV-1 pUL21 was recently identified as a novel viral PP1 regulatory protein that promotes virus 72 \nreplication and spread. HSV-1 pUL21 and its homologues across the alphaherpesviruses 73 \ncomprise two folded domains (18, 19) that are linked by a flexible linker domain (20).  PP1 binds 74 \npUL21 via a novel short linear motif, termed the Twenty-one Recruitment Of Protein 75 \nPhosphatase One (TROPPO) motif, that is present in the linker region and is conserved across 76 \nalphaherpesviruses. Mutation of the TROPPO motif abolishes the ability of either HSV-1 pUL21 77 \nor the Varicella-Zoster virus (VZV) homologue (pORF38) to bind PP1. Loss of PP1 binding results 78 \nin hyperphosphorylation of multiple proteins, both viral and cellular, in HSV-1 infected cells. 79 \npUL21 binding to PP1 regulates phosphorylation of components of the viral nuclear egress 80 \ncomplex (20), which remodels the nuclear membrane to support virus maturation (21, 22). The 81 \npUL21:PP1 interaction also leads to dephosphorylation and thus hyperactivation of the 82 \nceramide transport protein CERT, altering sphingolipid metabolism in infected cells (23). This 83 \nTROPPO motif does not bear obvious relation to known PP1 interacting motifs and pUL21 does 84 \nnot have any other known PP1 binding motifs such as an RVxF motif. The molecular basis of 85 \npUL21 binding to PP1 was thus unclear. 86 \nWhile inhibition of the ubiquitous and highly active enzyme PP1 is toxic, disrupting specific PP1 87 \nactivities via blocking of specific interactions is a promising avenue for the design of new 88 \ntherapies (24). The lack of homology with ‘RVxF’ motifs used by cellular PP1-binders suggested 89 \nthat the viral TROPPO motif might bind a novel surface on PP1 and thus could be specifically 90 \ntargeted by compounds that would inhibit virus replication and spread with minimal side-91 \neffects, as cellular functions of PP1 would remain uninhibited. We thus sought to determine 92 \nhow the TROPPO motif of HSV-1 pUL21 and its homologues bind PP1. 93 \nMethods 94 \nPlasmids 95 \nBacterial expression plasmids encoding the mouse PP1γ catalytic domain (residues 7–300)-H6 96 \nand pUL21-H6, and mammalian expression vectors encoding pUL21(FV242AA)-GFP , pORF38-97 \nGFP and pORF38(FV255AA)-GFP , was described previously (20). We note that the mouse and 98 \nhuman PP1γ catalytic domains share 100% amino acid identity. pORF38-NLT-H6 was generated 99 \nby sub-cloning residues 1–263 of ORF38 into the vector pOPTnH (25). pUL21-RVxF-H6 was 100 \ngenerated by inserting the mutations A237V and V239F into pUL21-H6 by QuikChange site-101 \ndirected mutagenesis. Human PP1α was cloned from HeLa cell cDNA into a vector derived from 102 \npF5K (Promega) with an N-terminal Myc epitope tag via restriction-based cloning, and point 103 \nmutations were introduced into Myc-PP1α by QuikChange mutagenesis. A plasmid encoding 104 \npUL21-GFP was generated by sub-cloning a codon-optimised synthetic UL21 gene (GeneArt) 105 \ninto pEGFP-N1 (Clontech), yielding an identical pUL21-GFP amino acid sequence as used in 106 \n(20). pUL21-ΔL, -ΔR and -ΔLR were generated from this vector via inverse PCR, and point 107 \nmutations were introduced via QuikChange mutagenesis. 108 \nPeptides 109 \nPeptides were commercially synthesised to >95% purity (GenScript) as follows: GADD34 110 \nRVxF+ϕϕ[xF] (residues 552–568) with fluorescein isothiocyanate (FITC) attached via an N-111 \nterminal aminohexanoic (Ahx) linker (sequence: [FITC-Ahx]-KARKVRFSEKVTVHFLA); pUL21 112 \nTROPPO (residues 234–250, sequence: AKRATVSEFVQVKHIDR); pOPF38 TROPPO (residues 113 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n4 \n \n246–263; sequence: KSDHITLSNFVQIRTIPR). TROPPO motif peptides were dissolved in aqueous 114 \nbuffers and GADD34 RVxF+ϕϕ[xF] was dissolved in dimethyl sulfoxide (DMSO). 115 \nRecombinant protein purification 116 \nProteins were expressed using Escherichia coli T7 Express lysY/Iq cells (New England Biolabs) 117 \ngrown in 2×TY medium at 37°C to OD600 0.8–1.2 before cooling to 22°C and induction of protein 118 \nexpression using 0.4 mM IPTG. Cells were harvested at 16–20 hours post-induction and pellets 119 \nstored at -70°C until required. Cells were resuspended in chilled lysis buffer (20 mM Tris pH 7.5, 120 \n20 mM imidazole, 500 mM NaCl, 1.4 mM β-mercaptoethanol 0.5 mM MgCl2 and 0.05% TWEEN-121 \n20 [plus 1 mM MnCl2 for PP1]) that was supplemented with 200 µL EDTA-free protease inhibitor 122 \ncocktail (Merck) and 400 U bovine DNase I (Merck). Cells were lysed using a TS series cell 123 \ndisruptor (Constant Systems) at 24 kpsi and lysates were clarified (40,000×g, 30min, 4 °C) 124 \nbefore incubation with Ni-NTA agarose (Qiagen) for 1 h at 4°C. The resin was washed with ≥20 125 \ncolumn volumes of wash buffer (20 mM Tris, 20 mM imidazole and 500 mM NaCl [plus 1 mM 126 \nMnCl2 for PP1]) at pH 7.5 (PP1γ and pORF38-NLT) or pH 8.5 (pUL21 and pUL21-RVxF). Protein 127 \nwas eluted using wash buffer supplemented with additional imidazole (final concentration 250 128 \nmM) and proteins were subjected to size-exclusions chromatography using HiLoad 16/600 129 \nSuperdex 75 (PP1) or Superdex 200 (other) columns equilibrated in 20 mM Tris pH 8.5, 500 mM 130 \nNaCl, 1 mM DTT (for pUL21 and pUL21-RVxF) or 50 mM HEPES pH 8.0, 500 mM NaCl, 0.5 mM 131 \nTCEP (for PP1 and pORF38-NLT). Fractions were analysed by SDS-PAGE and those containing 132 \nthe desired protein were pooled, concentrated using centrifugal concentrators (Millipore) and 133 \nstored at 4°C (short-term) or snap-frozen in liquid nitrogen for long-term storage at -70°C. 134 \nIsothermal Titration Calorimetry (ITC) 135 \nITC experiments were performed using a MicroCal PEAQ-ITC automated calorimeter (Malvern 136 \nPanalytical). The solvent (ITC buffer) was 50 mM HEPES pH 8.0, 500 mM NaCl, 0.5 mM TCEP. 137 \nLyophilised peptides were dissolved into ITC buffer and their concentration was estimated from 138 \ndry mass. Proteins were diluted in ITC buffer and their concentration was estimated from the 139 \ntheoretical extinction coefficient at 280 nm (26). Titrations were conducted at 25°C using 12 x 140 \n3 µL injections with syringe and cell contents as listed in Table S1. For all reagents, control 141 \ntitrations (syringe into buffer or buffer into cell) showed very low non-specific heat evolution. 142 \nData were analysed using MicroCal PEAQ-ITC analysis software (Malvern Panalytical) and fitted 143 \nusing a one-site binding model.  144 \nFluorescence polarisation anisotropy 145 \nFluorescence anisotropy was measured using a SpectraMax i3 microplate reader (Molecular 146 \nDevices) with wavelengths 485 ± 20 nm (excitation) and 535 ± 25 nm (emission). Samples 147 \n(100 µL per well) were dispensed into low-bind black half-area 96-well microtitre plates 148 \n(Corning) and fluorescence anisotropy was recorded at 27°C.  Experiments were performed in 149 \n50 mM HEPES pH 8.0, 500 mM NaCl, 0.05% TWEEN-20 unless otherwise noted. For all 150 \ntitrations, fluorescent GADD34 RVxF+ϕϕ[xF] peptide was diluted from a 5 µM stock in DMSO 151 \ninto buffer for a final concentration of 4 nM (0.08% DMSO). To measure association, 2 nM 152 \npeptide was incubated with a serial dilution of PP1γ catalytic domain. For competition assays a 153 \nserial dilution of the competitor was mixed 1:1 with a pre-formed complex of 4 nM GADD34 154 \npeptide and 500 nM PP1γ catalytic domain, to yield a final concentrations of 2 nM GADD34 155 \npeptide, 250 nM PP1γ and competitor as indicated. The PP1γ dissociation constant (KD) was 156 \ncalculated by fitting the grating factor corrected anisotropy data to a one-site binding 157 \nequilibrium model in Prism version 7 (GraphPad). For competition experiments, IC50 values 158 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n5 \n \nwere calculated by fitting the grating factor corrected anisotropy data to a four-parameter 159 \ninhibitor concentration response curve using Prism version 7 (GraphPad). 160 \nMammalian cell culture 161 \nMycoplasma-free, human embryonic kidney 293 T (HEK 293T) cells (American Type Culture 162 \nCollection #CRL-3216) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 163 \nhigh glucose (Merck), supplemented with 10% (v/v) heat-inactivated fetal calf serum and 2 mM 164 \nL-glutamine (complete DMEM) in a humidified 5% CO2 atmosphere at 37 °C.  165 \nImmunoprecipitation of GFP-tagged bait protein from transfected cells  166 \nMonolayers of HEK 293T cells were transfected with 7.7 μg of DNA per 9 cm dish, using 167 \n18 μg/mL 25 kDa branched polyethylenimine (Merck) or using TransIt-LT1 (Mirus) in accordance 168 \nwith the manufacturer’s instructions. Cells were harvested 24 h post-transfection and 169 \nincubated for 30 min in ice-cold 10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Igepal 170 \nCA-630 (a.k.a. NP-40), 1% (v/v) EDTA-free protease inhibitor cocktail (Merck) and incubated 171 \n(4°C, 30 min) before clarification at 21,000×g for 10 min at 4 °C. Lysate protein concentrations 172 \nwere measured using the bicinchoninic acid (BCA) assay (Pierce) and normalised before affinity 173 \ncapture using GFP-Trap resin (ChromoTek) following the manufacturer’s protocol. Samples were 174 \neluted by heating the resin to 95°C for 5 min in 45 μL 2×SDS-PAGE loading buffer. Proteins were 175 \nseparated by SDS-PAGE and transferred to 0.45 μm nitrocellulose membranes (PerkinElmer or 176 \nCytiva). Membranes were stained with Ponceau S and imaged using a G:Box XX9 (Syngene) 177 \nbefore blocking in Tris-buffered saline with 0.1% TWEEN-20 (TBS-T) supplemented with 5% (w/v) 178 \nnon-fat milk powder. Immunoblotting was performed using antibodies diluted in blocking buffer 179 \nas listed below and immunoblots were imaged using an Odyssey CLx (LI-COR 180 \nBiosciences). Densitometry was performed using Image Studio Lite version 5.2 (LI-COR 181 \nBiosciences) and statistical tests were performed using Prism version 7 (GraphPad). 182 \nAntibodies  183 \nThe following primary antibodies and dilutions were used for immunoblotting: rabbit anti-CERT 184 \n1:10,000 (Abcam #Ab72536), mouse anti-PP1α 1:1000 (Santa Cruz #sc-271762), mouse anti-185 \nMyc 1:4000 (Millipore #05-724), rat anti-tubulin (clone YL1/2) hybridoma supernatant 1:40 (27). 186 \nSecondary antibodies from LI-COR Biosciences were diluted 1:10,000 as follows: IRDye 680RD 187 \ngoat anti-rat (#926-68029) and goat anti-mouse (#926-68020), or IRDye 800CW goat anti-rabbit 188 \n(#926-32221). 189 \nStructure prediction and analysis 190 \nStructures were predicted using a locally installed version of ColabFold version 1.5.3. Input 191 \nsequences were human PP1γ catalytic domain (residues 7-300, UniProt P36873) plus either 192 \nHSV-1 strain KOS pUL21 (residues 1-535, UniProt ID: F8RG07) or VZV strain Dumas pORF38 193 \n(residues 1-541, Uniprot P09289) in complex with PP1. Mean lipophilicity maps were calculated 194 \nusing ChimeraX (28) and molecular graphics were generated using an open-source build of 195 \nPyMOL (Schrödinger). 196 \nResults 197 \nThe TROPPO motif alone is insufficient to bind PP1 198 \nPrior studies of cellular regulatory proteins showed that peptides containing the RXvF and 199 \nϕϕ[xF] motifs are sufficient to bind the catalytic domain of PP1 (29). Using fluorescence 200 \nanisotropy, we confirmed that a peptide containing the RVxF and ϕϕ[xF] motifs of GADD34 201 \n(a.k.a. PP1 regulatory subunit 15A) binds purified PP1γ catalytic domain (residues 7–300) with 202 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n6 \n \n144 ± 84 nM affinity (mean ± SEM, n = three independent experiments each performed in 203 \ntechnical triplicate) (Fig. 1A), similar to previous isothermal titration calorimetry (ITC) affinity 204 \nmeasurements for the same peptide sequence (29). Competition fluorescence anisotropy 205 \nexperiments were performed, whereby peptides containing the TROPPO motifs of pUL21 or 206 \npORF38 were titrated into a pre-formed complex of PP1 plus the fluorescent GADD34 peptide. 207 \nNeither TROPPO-containing peptide was able to displace the GADD34 peptide, even when 208 \npresent at over 1000-fold molar excess (Fig. 1B). This suggested that either the TROPPO motifs 209 \ndo not bind PP1, or they bind a different site on the PP1 surface that does not overlap with the 210 \nRVxF binding groove. ITC was employed to distinguish between these possibilities. Surprisingly, 211 \ntitrating high concentrations (500–1000 µM) of pUL21 or pORF38 TROPPO-containing peptides 212 \nagainst the PP1 catalytic domain did not evolve heats above background levels (Fig. 1C,D, Table 213 \nS1), suggesting that neither peptide is sufficient to bind PP1. 214 \nThe C-terminal region of the pUL21 linker is dispensable for PP1 binding 215 \nThe N-terminal domain and linker region of pUL21 are necessary for immunoprecipitation (IP) of 216 \nPP1, with mutation of the TROPPO motif within the linker domain efficiently disrupting this 217 \nbinding (20). While the TROPPO motif is absolutely conserved across alphaherpesvirus pUL21 218 \nhomologues, the remainder of the pUL21 linker regions are only poorly conserved (Fig. 2A). 219 \nStrikingly, the region of the linker between the TROPPO motif and the C-terminal domain is 220 \nsignificantly longer in HSV-1 and HSV-2 pUL21 than in other herpesviruses such as VZV , the 221 \npUL21 homologue of which (pORF38) is also capable of binding PP1 (20).  222 \nTo probe the contribution of the linker region outside the TROPPO motif to PP1 binding, deletion 223 \nmutants of HSV-1 pUL21 were designed lacking linker residues that precede the TROPPO motif 224 \n(residues 216–237; pUL21ΔL), linker residues that follow the TROPPO motif (residues 257–276; 225 \npUL21ΔR), or both flanking regions (pUL21ΔLR). Wild-type and mutant pUL21, and the pUL21 226 \nhomologue ORF38 from VZV , were expressed as GFP fusion proteins and used for co-IP analysis 227 \nfollowing transient transfection in HEK293T cells (Fig. 2B). As previously reported, pUL21 and 228 \npORF38 efficiently precipitate endogenous PP1α and the interaction is disrupted by mutation of 229 \nthe TROPPO motif (pUL21-FV242AA and pORF38-FV255AA). Strikingly, removal of the N-230 \nterminal region of the pUL21 linker (pUL21ΔL or pUL21ΔLR) completely abolishes PP1α binding, 231 \nwhereas binding is retained when the C-terminal region of the pUL21 linker is removed 232 \n(pUL21ΔR). All pUL21 constructs retain CERT binding, consistent with localisation of CERT-233 \nbinding to the C-terminal domain of pUL21 (20, 23) and confirming the correct expression and 234 \nfolding of these mutants.  235 \nStructural model of the pUL21:PP1 and pORF38:PP1 complexes 236 \nExtensive attempts to co-crystallise WT or ΔR pUL21 with the catalytic subunit of PP1 proved 237 \nunsuccessful. Therefore, to investigate the relative contributions of the N-terminal domain, 238 \nlinker and TROPPO motif to PP1 binding, the structure of HSV-1 pUL21 (Fig. 3A) and VZV pORF38 239 \n(Fig. 3B) in complex with the catalytic domain of human PP1γ were predicted using AlphaFold2-240 \nMultimer (30) via the Colabfold pipeline (31). The models for pUL21 (Fig. 2A, pLDDT/ipTM = 241 \n86.0/0.936) and pORF38 (Fig. 2B, pLDDT/ipTM = 85.4/0.918) predict that the TROPPO motif and 242 \nseveral segments of the linker region preceding it associate with the N-terminal domain and 243 \nwith PP1. The per-residue confidence of the prediction (pLDDT) is high for residues of the 244 \nordered pUL21 and pORF38 N- and C-terminal domains and for the PP1 catalytic domain, plus 245 \nfor residues of the linker region that are predicted to interact with these domains (Fig. 3C). 246 \nAnalysis of the Predicted Aligned Error (PAE) for both models confirms that the orientation of the 247 \nN-terminal domain and linker region up to the TROPPO motif with respect to PP1 is confidently 248 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n7 \n \npredicted, while the relative orientations of linker region following the TROPPO motif and the C-249 \nterminal domains are not predicted with confidence (Fig. 3D). 250 \nPreviously, small angle X-ray scattering showed that pUL21 comprises two ordered domains 251 \nseparated by a highly flexible linker (20). Crystallographic structures showed the N-terminal 252 \ndomain of pUL21 (pUL21-N) to possess a distinctive α/β fold, with two anti-parallel β-sheets 253 \nthat pack against surface-exposed α-helices (the ‘inner sheet’ and ‘lower outer sheet’) plus a 254 \nthird β-sheet that packs against the inner sheet (the ‘upper outer sheet’) (18). Despite pUL21 255 \nresidues 1–216 being present in the crystallisation experiment and being conserved across 256 \nalphaherpesviruses, only residues 1–198 were resolved in electron density. In the predicted 257 \ncomplex, N-terminal domain residues 209–212 and residues 242–248 of the TROPPO motif are 258 \npredicted to become ordered, forming two strands of anti-parallel beta sheet that serve as a 259 \nbridge between the three-stranded pUL21-N upper outer sheet and the final β-sheet of the PP1 260 \ncatalytic domain (Fig. 3A). Residues 221-223 of the Linker (L) region are also predicted to 261 \nbecome ordered, forming a short β-strand that extends the pUL21-N inner sheet. We note that 262 \nthese newly-formed sheets are proximal to the amino terminus of pUL21, consistent with an 263 \ninability of N-terminally GFP-tagged pUL21-N-plus-linker to bind PP1 (20). 264 \nAll residues of the pUL21 TROPPO motif (239VSFVQVKHI248) are predicted to be in close 265 \nassociation with PP1 (Fig. 3E). Surprisingly, the TROPPO motif is predicted to bind at the same 266 \nsurface hydrophobic groove as cellular RVxF-containing peptides, adopting a similar loop-plus-267 \nsheet conformation as GADD34 residues 553–568 (29). The first residue of the TROPPO and the 268 \nthree preceding (236RATV239) of pUL21 align structurally with residues 555KVRF558 of the GADD34 269 \nRVxF motif (Fig. 3F), with pUL21 A237 and V239 predicted to bind the PP1 hydrophobic pockets 270 \noccupied by GADD34 V556 and F558 (Fig. 3G). Most cellular regulatory proteins that bind PP1 271 \npossess a second PP1-binding SLiM, ϕϕ[xF], that is C-terminal to the RVxF (16). In the predicted 272 \ncomplex, residues 244VKHI247 of the pUL21 TROPPO adopt a similar conformation to the GADD34 273 \nϕϕ[xF] residues 564VHFL567 (Fig. 3G). 274 \nWe previously showed that pUL21 mutations F242E or V243D are sufficient to abolish PP1 275 \nbinding (23). The severity of the V243D mutation is readily explained by the complex prediction, 276 \nthis residue is predicted to interact with hydrophobic residues on the surface of PP1 (Fig. S1) 277 \nand there is a valine at the equivalent position in GADD34 (Fig. 3G). The severity of the F242E 278 \nmutation is less obvious when considering only the TROPPO motif, as the side chain is 279 \npredicted to point away from PP1 (Fig. 3G). However, it becomes clearer when one considers 280 \nthe context of pUL21-N and the residues of the linker that are predicted to become ordered. The 281 \nside chain of F242 is predicted to be largely buried, interacting with hydrophobic side chains of 282 \npUL21 A48 and F50, plus the hydrophobic face of the R24 guanidyl group (Fig. 3H). Mutation to a 283 \ncharged residue would be predicted to disrupt this packing. The TROPPO motif of pORF38 forms 284 \nvery similar interactions with both the pORF38 N-terminal domain and PP1 in the model of the 285 \npORF38:PP1 complex (Fig. S1). Loss of hydrophobic interactions would thus similarly explain 286 \nwhy the FV255AA mutations abolished binding to PP1 (Fig. 2B) (20). 287 \nSite-d irected mutagenesis confirms the predictive power of the pUL21:PP1 model 288 \nWhile the models of pUL21 and pORF38 in complex with PP1 were compellingly consistent with 289 \nboth each other and with previous results, structurally informed mutagenesis is required to 290 \nrigorously test their predictive power. To this end, single amino acids mutations were introduced 291 \nin both PP1 and pUL21, and their ability to interact in cells was probed by c o I P. 292 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n8 \n \nFor PP1, we utilised three published mutations that have all been show to severely decrease 293 \nPP1 regulatory protein binding: F257A (32), which is at the RVxF hydrophobic pocket into which 294 \nthe side chain of pUL21 V239 is predicted to bind (Fig. 4A); and L289R or C291R (33), which are 295 \non the final β-sheet of the PP1 catalytic domain and contribute to the RVxF hydrophobic surface 296 \nwith which pUL21 A237 is predicted to associate (Fig. 4A). HEK293T cells were co-transfected 297 \nwith pUL21-GFP and WT or mutant Myc-PP1, and lysates were subjected to GFP affinity capture 298 \nbefore SDS-PAGE and immunoblotting. The coIP of all three mutants was profoundly less 299 \nefficient than for WT Myc-PP1 (Fig. 4B). However, we consistently observed lower abundance of 300 \nthese mutants in the cell lysates used for IP analysis, suggesting lower expression or increased 301 \nturnover in transfected cells. To account for this, the amount of input and bound Myc-PP1 was 302 \nquantified and the ratio of bound:input was calculated (Fig. 4C), confirming that all three 303 \nmutations significantly disrupt binding to pUL21-GFP. 304 \nFor pUL21, a panel of mutations were designed to probe the predicted interactions of residues 305 \nat the interface with both PP1 and pUL21-N. Five mutations were designed to disrupt the 306 \ninteraction: Q174R, which would replace a buried residue on pUL21-N sheet β14 that contacts 307 \nQ244 of the TROPPO motif (Fig. 4D); T209R, which would replace a small buried side chain that 308 \ncontacts pUL21-N with a large charged side chain (Fig. 4D); VV211AA, where two hydrophobic 309 \nside chains (including one that is buried) in the sheet that bridges the TROPPO motif and pUL21-310 \nN β1 are removed (Fig. 4D); A218E, where a small hydrophobic residue that interacts with a 311 \nsurface hydrophobic patch on PP1 is replaced with a charged residue (Fig. 4D); and R250E, 312 \nwhere the charge of a side chain that interacts with the PP1 D277 side chain is inverted (Fig. 4D). 313 \nThree mutations were designed to enhance PP1 binding: A237V , substituting alanine for the 314 \ncanonical RVxF valine residue (Fig. 4A); V239F , substituting valine for the canonical RVxF 315 \nphenylalanine residue (Fig. 4A); and H247F , substituting for the equivalent residue in GADD34 316 \nwhere the larger hydrophobic side chain could make more extensive interactions with the aryl 317 \nportion of the PP1 K297 side chain (Fig. 4D). A control mutation A232K was also introduced, as 318 \nsubstitution of a small hydrophobic side chain for a large charged one on a mobile surface-319 \nexposed loop (Fig. 4A) would be predicted to leave PP1 binding unaltered. 320 \nHEK293T cells were transfected with WT or mutant pUL21-GFP and the coIP of endogenous 321 \nPP1α was monitored by immunoblotting (Fig. 4E). Pleasingly, all designed mutations had the 322 \npredicted effect: PP1α coIP was abolished by the Q174R, T209A, VV211AA and A218E 323 \nsubstitutions, in addition to the control FV242AA mutation, and the R250E substitutions 324 \ndramatically reduced binding. All proteins were present at similar abundance in the input 325 \nsamples and all retained binding to CERT, confirming their correct folding. As predicted, the 326 \nsurface-loop substitution A232K did not dramatically alter PP1α coIP . The A237V, V239F and 327 \nH247F mutants all co-precipitated more PP1α than did WT pUL21-GFP , suggesting that all three 328 \nhave higher affinity for the PP1 catalytic subunit. Taken together, these mutagenesis results 329 \nstrongly support the predicted structure of the pUL21:PP1 complex. 330 \nAlphaherpesvirus proteins compete directly with cellular regulatory proteins for PP1 binding 331 \nSite-directed mutagenesis confirmed the predictive power of the pUL21:PP1 complex, but the 332 \naffinity of the interaction between PP1 and alphaherpesvirus pUL21 homologues remained 333 \nunknown. Attempts to perform ITC using recombinant full-length pUL21 and PP1 catalytic 334 \ndomain were unsuccessful as neither protein could be concentrated sufficiently to serve as 335 \ntitrant in the syringe. Similarly, attempts to purify full-length pORF38 following recombinant 336 \nexpression in E. coli were frustrated by low protein expression and solubility. As the R region of 337 \nthe linker and C-terminal domain are dispensable for PP1 binding, we designed expression 338 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n9 \n \nconstructs encoding the pUL21 and pORF38 N-terminal domains plus the Linker region up to 339 \nthe end of the TROPPO motif (the NLT region) with a C-terminal hexahistidine tag (Fig. 5A). The 340 \npORF38-NLT construct was readily expressed and purified to concentrations suitable for ITC 341 \n(Fig. 5B). ITC titration of pORF38-NLT into PP1 showed that the proteins form a 1:1 complex with 342 \naffinity of 497 ± 143 nM (mean ± SEM, three independent experiments) (Fig. 5C and Table S1). 343 \nThis is substantially weaker binding than observed for the GADD34 peptide binding PP1 via 344 \nfluorescence anisotropy (Fig. 1A) or ITC (29). 345 \nTo definitively test whether pORF38 binds the same hydrophobic groove as cellular RVxF motifs, 346 \ncompetition fluorescence anisotropy was performed via titration of pORF38-NLT into a pre-347 \nformed complex of PP1 plus the fluorescent GADD34 peptide. The liberation of GADD34 peptide 348 \nat increasing concentrations of pORF38-NLT, as evidenced by a decrease in fluorescence 349 \nanisotropy, confirmed that pORF38 competes with the RVxF peptide for PP1 binding (Fig. 5D), 350 \nwith an IC50 of 417 ± 11 nM (mean ± SEM, three independent experiments each performed in 351 \ntechnical triplicate). 352 \nSimilar competition fluorescence anisotropy experiments with purified full-length pUL21 (Fig. 353 \n5E) were performed in buffer optimised to enhance solubility of the protein (Tris pH 8.5 in place 354 \nof HEPES pH 8). Under these conditions PP1 binds the GADD34 peptide with slightly higher 355 \naffinity (55 ± 21 nM, mean ± SEM of three independent experiments performed in technical 356 \ntriplicate; Fig. S2). Full-length pUL21 is unable to out-compete GADD34 for binding to PP1 (Fig. 357 \n5F), suggesting that the affinity of PP1 for full-length pUL21 is lower than for pORF38 – 358 \nconsistent with the observation that PP1α is consistently co-immunoprecipitated more 359 \nefficiently by pORF38-GFP than by pUL21-GFP (Fig. 2B and Fig. 3H of (20)). To test whether 360 \noptimisation of the TROPPO sequence to better match a canonical RVxF motif could enhance 361 \nbinding, we generated, expressed and purified a pUL21-H6 mutant where residues A237 and 362 \nV239 had been substituted for valine and phenylalanine, respectively (pUL21-RVxF , Fig. 5E). 363 \nFluorescence anisotropy confirms that pUL21-RVxF competes with the GADD34 peptide for 364 \nbinding to the PP1 hydrophobic groove with an IC50 of 337 ± 31 nM (mean ± SEM, three 365 \nindependent experiments each performed in technical triplicate). 366 \nDiscussion 367 \npUL21 is required for efficient replication and spread of HSV-1 (20, 34–36). We previously 368 \nidentified HSV-1 pUL21 as a PP1 regulatory protein and that it contained a novel SLiM required 369 \nfor PP1 binding, the TROPPO motif (20). The absence of identifiable sequence homology with 370 \nother known PP1-associated SLiMs (10) raised the tantalising possibility that the TROPPO motif 371 \nbinds PP1 via a novel surface that could be targeted for the development of antiviral 372 \ncompounds. Combining structure prediction, site-directed mutagenesis, immunoprecipitation 373 \nand biophysical characterisation, we now show that the TROPPO motif binds the same 374 \nhydrophobic surface of PP1 as cellular RVxF and ϕϕ[xF] SLiMs. Competition fluorescence 375 \nanisotropy confirms that TROPPO-containing proteins compete directly with cellular RVxF and 376 \nϕϕ[xF] motifs for binding to PP1 (Fig. 5).  377 \nPP1 catalytic domains are unlikely to exist in their apo form within cells (10) and, as such, 378 \npUL21 and pORF38 must compete with cellular regulatory proteins to access PP1 in infected 379 \ncells. There are over 200 mammalian PP1 regulatory proteins (9, 12) and their affinities for PP1 380 \nrange from ~10 nM (15, 37) to ~1 µM (38). Given the competition for binding, it is puzzling that 381 \npORF38 has evolved to bind PP1 with only modest affinity (~500 nM) (Fig. 5C). The PP1-binding 382 \nof pUL21 must be even lower, as unlike pOPF38-NLT wild-type pUL21 cannot displace a 383 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n10 \n \nGADD34 RVxF- and ϕϕ[xF]-containing peptide from PP1. One striking feature of the predicted 384 \npUL21:PP1 complex is that the TROPPO motif containing the ϕϕ[xF] motif lies sandwiched 385 \nbetween PP1 and the pUL21-N, forming part of an extended β-sheet that bridges core β-sheets 386 \nacross the two proteins (Fig. 2A). While the structure of regulatory protein spinophilin (15) 387 \nshowed a sheet-turn-sheet binding PP1 at the ϕϕ[xF] interacting region, adding two strands to 388 \nthe PP1 core β-sheet, we are unaware of any examples where the RVxF and ϕϕ[xF] form a 389 \nstructured bridge between PP1 and another folded domain. The formation of this extended 390 \nsheet, with concomitant burial of hydrophobic residues like F242 into a newly-extended 391 \nhydrophobic core of pUL21-N (Fig. 3H), would stabilise the interaction between pUL21 and PP1 392 \nonce formed. Such cooperativity would restrain the TROPPO motif in the correct conformation 393 \nfor PP1 binding despite the modest affinity (39). 394 \nPublished RVxF consensus sequences (13, 40, 41) invariably have a valine or isoleucine and 395 \nphenylalanine or tryptophan in the second and fourth position, respectively, whereas in pUL21 396 \nand pORF38 the equivalent residues are alanine and valine (Fig. 3). It is not clear that there are 397 \nstructural constraints preventing the virus from evolving an optimal RVxF motif since, like most 398 \nregulatory protein regions that bind PP1 (12), the linker region of pUL21 is intrinsically 399 \ndisordered (20). Mutation of A237 to valine or V239 to phenylalanine enhances the coIP of PP1α 400 \nby pUL21 in transfected cells (Fig. 4E) and the A237V+V239R double substitution gives pUL21-401 \nRVxF the ability to compete with the GADD34 peptide for PP1 binding (Fig. 5F). HSV-1 encodes a 402 \nsecond PP1 regulatory protein, ICP34.5, that has high sequence homology to GADD34 and 403 \nbinds PP1 via a canonical RVxF motif (17, 42). There is thus nothing to prevent HSV-1 encoding a 404 \nprotein with a bona fide RVxF motif, and we hypothesise that selective pressure related to the 405 \nmolecular function of pUL21 has driven it to retain a suboptimal RVxF motif. Given the high 406 \ndegree of TROPPO sequence conservation across pUL21 homologues and corresponding 407 \nabsence of canonical RVxF motifs, we conclude that this evolutionary constraint must be 408 \ncommon to all alphaherpesviruses. 409 \nWhat might drive these viruses to maintain a low-affinity PP1 recruitment domain? One clue 410 \ncomes from in vitro evolution experiments, where serial passage of HSV-1 encoding pUL21 411 \nmutated to prevent PP1 binding led to compensatory mutations in the virus-encoded kinase 412 \npUS3 (20). These two proteins have overlapping substrates, with pUL21-mediated 413 \ndephosphorylation directly antagonising pUS3 kinase activity. A balance of activity is clearly 414 \ncritical for the virus: abolishing expression of either pUS3 or pUL21 severely diminishes virus 415 \nreplication in cultured keratinocytes, as does removal of pUL21 PP1 binding, but passage of the 416 \nPP1-binding pUL21 mutants led to rapid accumulation of mutations that lowered pUS3 kinase 417 \nactivity and restored virus fitness. The peak of pUL21 expression is at late times (18 hours) post-418 \ninfection in cultured keratinocytes, whereas pUS3 abundance peaks at 6 hours post-infection 419 \nand then declines slightly (43). We hypothesise that the PP1-binding motif of pUL21 is 420 \nspecifically tuned to prevent effective PP1 binding at early times post-infection, when pUS3 421 \nactivity is presumably pro-viral. Instead, the presence of a suboptimal PP1 interaction motif 422 \nwould allow pUL21 to effectively compete for PP1 binding only at late times post infection and 423 \nin specific subcellular locations, for example the nuclear envelope, where the protein is highly 424 \nabundant (20). This fine-tuning of activity would be especially important for a multi-functional 425 \nprotein like pUL21, with roles cell-to-cell spread (20, 34, 36, 44–46), preventing futile genome 426 \nrelease by nascent viral capsids (35, 47) and virus assembly (45, 46, 48, 49), in addition to its 427 \nrole in regulating viral nuclear egress (44, 45, 50–52). High affinity binding to PP1 could recruit 428 \nphosphatase activity to the wrong cellular or viral complexes, or it could inappropriately 429 \nsequester PP1 and thereby prevent other PP1-mediated dephosphorylation events within the 430 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n11 \n \ncell that promote virus replication. Similar fine-tuning of PP1 recruitment has been observed for 431 \nHIV , where mutation of the suboptimal QVCF motif to RVCF enhanced PP1 binding but reduced 432 \nthe ability of Tat to stimulate HIV transcription (5). 433 \nIn conclusion, we show that the alphaherpesvirus pUL21 homologues bind the same PP1 434 \nhydrophobic surface groove as cellular RVxF and ϕϕ[xF] motifs. Extensive site-directed 435 \nmutagenesis supports a predicted structure of the pUL21:PP1 complex where intrinsically 436 \ndisordered regions of the pUL21 linker fold into an extended β-sheet that bridges the cores of 437 \nPP1 and the pUL21 N-terminal domain. The conservation of suboptimal binding residues within 438 \nthe region structurally equivalent to the cellular RVxF implies that the viruses have deliberately 439 \nregulated PP1 binding affinity to support optimal virus replication. This highlights the careful 440 \nregulation of kinase and phosphatase activity required to support efficient virus replication and 441 \nspread. Our work also highlights how PP1 can be recruited by proteins lacking any identifiable 442 \nPP1-binding SLiM, suggesting that the pool of viral and cellular PP1 regulatory proteins may be 443 \nlarger than previously imagined.  444 \nAcknowledgements 445 \nWe thank Owen Tutt (University of Cambridge) for technical assistance. For the purpose of open 446 \naccess, the authors have applied a Creative Commons Attribution (CC BY) licence to any Author 447 \nAccepted Manuscript version arising from this submission. 448 \nConflicts of interest 449 \nThe author(s) declare that there are no conflicts of interest. 450 \nFunding information 451 \nThis work was funded by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and 452 \nthe Royal Society (098406/Z/12/B) to SCG. HM is funded by the University of Cambridge School 453 \nof Clinical Medicine Doctoral Training Programme in Medical Research. DSC is funded by a 454 \nBritish Skin Foundation PhD studentship (028/S/22). THB was funded by a University of 455 \nCambridge Department of Pathology PhD studentship. JED is a Wellcome Trust Senior Research 456 \nFellow (219447/Z/19/Z). 457 \nData availability 458 \nThe models of pUL21 and pORF38 in complex with PP1 have been deposited to the University of 459 \nCambridge Data Repository (DOI). The authors confirm that all other data supporting the 460 \nfindings of this study are available within the article and/or its supplementary materials. 461 \n  462 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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(2019) Affinity and specificity of motif-based protein-protein 580 \ninteractions. Curr Opin Struct Biol. 54, 26–33 581 \n40.  Meiselbach, H., Sticht, H., and Enz, R. (2006) Structural analysis of the protein 582 \nphosphatase 1 docking motif: molecular description of binding specificities identifies 583 \ninteracting proteins. Chem Biol. 13, 49–59 584 \n41.  Wakula, P ., Beullens, M., Ceulemans, H., Stalmans, W., and Bollen, M. (2003) Degeneracy 585 \nand function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1. 586 \nJ Biol Chem. 278, 18817–18823 587 \n42.  Chou, J., and Roizman, B. (1994) Herpes simplex virus 1 gamma(1)34.5 gene function, 588 \nwhich blocks the host response to infection, maps in the homologous domain of the genes 589 \nexpressed during growth arrest and DNA damage. Proc Natl Acad Sci U S A. 91, 5247–5251 590 \n43.  Soh, T. K., Davies, C. T. R., Muenzner, J., Hunter, L. M., Barrow, H. G., Connor, V ., Bouton, C. 591 \nR., Smith, C., Emmott, E., Antrobus, R., Graham, S. C., Weekes, M. P ., and Crump, C. M. 592 \n(2020) Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-593 \nSurface Remodeling via pUL56-Mediated GOPC Degradation. Cell Rep. 33, 108235 594 \n44.  Le Sage, V ., Jung, M., Alter, J. D., Wills, E. G., Johnston, S. M., Kawaguchi, Y ., Baines, J. D., 595 \nand Banfield, B. W. (2013) The herpes simplex virus 2 UL21 protein is essential for virus 596 \npropagation. J Virol. 87, 5904–5915 597 \n45.  Roddy, K., Grzesik, P ., Smith, B. J., Ko, N., Vashee, S., and Desai, P . J. (2025) The loss of both 598 \npUL16 and pUL21 in HSV-1-infected cells alters capsid-tegument composition, nuclear 599 \nmembrane architecture, cytoplasmic maturation and cell-to-cell spread. J Gen Virol. 106, 600 \n002083 601 \n46.  Finnen, R. L., Muradov, J. H., Le Sage, V ., and Banfield, B. W. (2024) Disruption of herpes 602 \nsimplex virus type 2 pUL21 phosphorylation impairs secondary envelopment of 603 \ncytoplasmic nucleocapsids. J Virol. 98, e0065624 604 \n47.  Thomas, E. C. M., Bossert, M., and Banfield, B. W. (2022) The herpes simplex virus 605 \ntegument protein pUL21 is required for viral genome retention within capsids. PLoS 606 \nPathog. 18, e1010969 607 \n48.  Han, J., Chadha, P ., Starkey, J. L., and Wills, J. W. (2012) Function of glycoprotein E of 608 \nherpes simplex virus requires coordinated assembly of three tegument proteins on its 609 \ncytoplasmic tail. Proc Natl Acad Sci U S A. 109, 19798–803 610 \n49.  Ahmad, I., and Wilson, D. W. (2020) HSV-1 Cytoplasmic Envelopment and Egress. Int J Mol 611 \nSci. 21, E5969 612 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n15 \n \n50.  Gao, J., Finnen, R. L., Sherry, M. R., Le Sage, V ., and Banfield, B. W. (2020) Differentiating 613 \nthe Roles of UL16, UL21, and Us3 in the Nuclear Egress of Herpes Simplex Virus Capsids. J 614 \nVirol. 94, 00738–20 615 \n51.  Muradov, J. H., Finnen, R. L., Gulak, M. A., Hay, T. J. M., and Banfield, B. W. (2021) pUL21 616 \nregulation of pUs3 kinase activity influences the nature of nuclear envelope deformation 617 \nby the HSV-2 nuclear egress complex. PLoS Pathog. 17, e1009679 618 \n52.  Nahas, K. L., Connor, V ., Wijesinghe, K. J., Barrow, H. G., Dobbie, I. M., Harkiolaki, M., 619 \nGraham, S. C., and Crump, C. M. (2025) Applying 3D correlative structured illumination 620 \nmicroscopy and X-ray tomography to characterise herpes simplex virus-1 morphogenesis. 621 \n10.7554/eLife.105209.1 622 \n 623 \n  624 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n16 \n \nFigure 1. Alphaherpesvirus TROPPO peptides do not bind PP1. 625 \nA Fluorescence anisotropy of PP1γ catalytic domain (residues 7–300) binding 2 nM fluorescently 626 \nlabelled peptide containing the GADD34 RVxF and ϕϕ[xF] motifs (residues 552–568). Mean ± SD 627 \nis shown for technical triplicate measurements. Affinity (KD) is average of three independent 628 \nexperiments. B Competition fluorescence anisotropy of peptides containing the 629 \nalphaperhesvirus TROPPO motif . Pre-formed complexes of 2 nM fluorescent GADD34 peptide 630 \nwith 250 nM PP1 were incubated with increasing concentrations of TROPPO-containing 631 \npeptides from pUL21 (residues 234–250; left) and pORF38 (residues 246–263; right). Neither 632 \npeptide successfully displaced GADD34 RVxF . Mean ± SD is shown for technical triplicate 633 \nmeasurements. C, D Isothermal titration calorimetry of PP1γ catalytic domain with TROPPO 634 \ncontaining peptides from C pUL21 and D pORF38. For each, baseline-corrected differential 635 \npower (DP) versus time (left) and integrated change in enthalpy (ΔH) versus molar ratio (right) 636 \nare shown. Figures are representative of three (C) or two (D) independent experiments. 637 \n  638 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n17 \n \nFigure 2. Contribution of the pUL21 linker region to PP1 binding. 639 \nA Alignment of alphaherpesvirus pUL21 homologue sequences spanning the linker between 640 \npUL21 N- and C-terminal domains, plus the N-terminal domain residues that were disordered in 641 \nthe pUL21-N crystal structure (18). The following sequences were aligned using ClustalW 642 \n(Abbreviation and Uniprot ID are shown in parentheses): HSV-1 (HSV1, P10205), HSV-2 (HSV2, 643 \nG9I242), cercopithecine herpesvirus 2 (CHV-2, Q5Y0T2), saimiriine herpesvirus 1 (SHV-1, 644 \nE2IUE9), bovine alphaherpesvirus 1 (BHV-1, Q65563), leporid alphaherpesvirus 4 (LHV-4, 645 \nJ9QYM9), feline herpesvirus 1 (FHV-1, D1FXW1), equine herpesvirus 1 (EHV-1, P28972), 646 \npseudorabies virus (PRV , Q04532), anatid herpesvirus 1 (AHV-1, A4GRJ2), varicella-zoster virus 647 \n(VZV , Q6QCT9), turkey herpesvirus (MHV-1, Q9DPR5). The regions of the linker preceding (L 648 \nregion, residues 216–237, grey) or following (R region, residues 257–276, grey) the TROPPO motif 649 \n(pink) are highlighted. B HEK293T cells were transfected with plasmids expressing wild-type 650 \n(WT) or mutated HSV-1 pUL21-GFP or VZV pORF38-GFP . At 24 hours post-transfection the cells 651 \nwere lysed, subjected to immunoprecipitation using a GFP affinity resin, and captured proteins 652 \nwere subjected to SDS-PAGE and immunoblotting using the listed antibodies. Ponceau S (PonS) 653 \nstaining of the nitrocellulose membrane before blocking is shown, confirming efficient capture 654 \nof GFP-tagged proteins. Figure is representative of two independent experiments.  655 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n18 \n \nFigure 3. HSV-1 pUL21 and VZV pORF38 are predicted to bind the ‘RVxF’ hydrophobic groove 656 \nof PP1. 657 \nA,B AlphaFold2-Multimer models of human PP1γ catalytic domain (residues 7–300; yellow 658 \nribbons) in complex with A HSV-1 pUL21 (purple ribbons) or B VZV pORF38 (rose blush ribbons), 659 \nwith N- and C-terminal domains in lighter and darker shades, respectively. For each model, 660 \nregions of the linker between the N- and C-terminal domains are coloured grey and the TROPPO 661 \nmotif is in bright pink. In A the region of the conserved N-terminal domain missing from the 662 \ncrystal structure (18) is light pink and inset shows the packing of predicted β-sheets at the 663 \npUL21-N:PP1 interface. C Per-residue predicted local distance difference test (pLDDT) of the 664 \npUL21:PP1 (top) and pORF38:PP1 (bottom) models, where values above 70 represent residues 665 \npredicted with high local confidence. Domains are coloured as in (A, B). D Predicted aligned 666 \nerror of pUL21:PP1 (left) and pORF38:PP1 (right) models. Regions of the matrix where the 667 \nrelative orientations of residues are predicted with high confidence are shown in green. E 668 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n19 \n \nSuperposition of the pUL21:PP1 predicted structure on the crystal structure of PP1α in complex 669 \nwith the GADD34 RVxF+ϕϕ[xF] motif (PDB 4XPN) (29). PP1 is shown as a molecular surface 670 \ncoloured by mean lipophilicity, from orange (hydrophobic) to cyan (polar), and PP1-binding 671 \nmotifs are shown as ribbons. F Structure-based alignment of the pUL21 and GADD34 PP1-672 \nbinding sequences, with SLiMs highlighted. G Predicted molecular interactions at the RVxF 673 \nbinding groove of PP1. Selected sticks are shown with carbon atoms green (GADD34) or 674 \ncoloured as in (A) (PP1 and pUL21). H Predicted interactions between pUL21-N and residues of 675 \nthe TROPPO motif. Selected sticks are shown with carbon atoms coloured as in (A). 676 \n  677 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n20 \n \n 678 \nFigure 4. Site-directed mutagenesis supports pUL21 binding the hydrophobic RVxF groove 679 \nof PP1. 680 \nA Model of the pUL21:PP1 complex, highlighting predicted interactions with PP1 residues 681 \nimportant for binding regulatory subunits containing RVxF motifs. Structures are shown as 682 \nribbons for PP1 (yellow), the pUL21 linker (grey), TROPPO (bright pink), and residues of pUL21-N 683 \nmissing from the crystal structure (light pink) (18). Selected residues shown as sticks. B 684 \nHEK293T cells were co-transfected with plasmids expressing pUL21-GFP and wild-type (WT) or 685 \nmutated Myc-tagged human PP1α. At 24 hours post-transfection the cells were lysed, subjected 686 \nto immunoprecipitation using a GFP affinity resin, and captured proteins were subjected to 687 \nSDS-PAGE and immunoblotting using the listed antibodies. Ponceau S (PonS) staining of the 688 \nnitrocellulose membrane before blocking is shown, confirming efficient capture of GFP-tagged 689 \nproteins. Figure is representative of at least three independent experiments (see below). C 690 \nQuantitation of Myc-PP1 co-immunoprecipitation by pUL21-G F P. Ratio of bound to input signals 691 \nas determined by densitometry is shown as individual points and mean ± SD from seven (WT), 692 \nfive (F257A and L289R) or three (C291R) independent experiments. One-way ANOVA with Holm-693 \nSidak’s multiple comparisons test was used for the statistical analysis (***p < 0.001). D Model 694 \nof the pUL21:PP1 complex, highlighting pUL21 residues at the interfaces with PP1 and pUL21-N. 695 \nRibbons are shown with selected residues represented as sticks, coloured as in (A). E HEK293T 696 \ncells were transfected with plasmids expressing wild-type (WT) or mutated HSV-1 pUL21-GFP . 697 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n21 \n \nAt 24 hours post-transfection the cells were lysed, subjected to immunoprecipitation using a 698 \nGFP affinity resin, and captured proteins were subjected to SDS-PAGE and immunoblotting 699 \nusing the listed antibodies. Ponceau S (PonS) staining of the nitrocellulose membrane before 700 \nblocking is shown, confirming efficient capture of GFP-tagged proteins. Figure is representative 701 \nof two independent experiments. 702 \n  703 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n22 \n \n 704 \nFigure 5. Alphaherpesvirus pUL21 homologues compete with cellular RVxF motifs for 705 \nbinding PP1. 706 \nA Schematic of pORF38-NLT, which encodes the N-terminal domain plus Linker region to the 707 \nend of the TROPPO motif. B Coomassie-stained SDS-PAGE of purified C-terminally 708 \nhexahistidine-tagged pORF38-NLT. C ITC of PP1γ catalytic domain with pORF38-NLT. Baseline-709 \ncorrected differential power (DP) versus time (left) and integrated change in enthalpy (ΔH) 710 \nversus molar ratio (right) is shown. Figure is representative of three independent experiments 711 \n(Table S1), and average values for affinity (KD), stoichiometry (N) and ΔH are shown. D 712 \nCompetition fluorescence anisotropy confirms that pORF38-NLT competes with RVxF+ϕϕ[xF] 713 \npeptides for PP1 binding. A pre-formed complex of 2 nM fluorescent GADD34 RVxF+ϕϕ[xF] 714 \npeptide with 250 nM PP1 was incubated with increasing concentrations of pORF38-NLT, 715 \nresulting in displacement of the GADD34 peptide. Mean ± SD is shown for technical triplicate 716 \nmeasurements. Average IC50 from three independent experiments is shown. E Coomassie-717 \nstained SDS-PAGE of purified C-terminally hexahistidine-tagged pUL21 WT and A237V+V239F 718 \n(RVxF) mutant. F Competition fluorescence anisotropy of WT (purple) and RVxF (blue) pUL21. A 719 \npre-formed complex of fluorescent 2 nM GADD34 peptide with 250 nM PP1 was incubated with 720 \nincreasing concentrations of pUL21. Mean ± SD is shown for technical triplicate measurements. 721 \nAverage IC50 from three independent experiments is shown. 722 \n  723 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n23 \n \n 724 \nFigure S1. Comparison of (predicted) peptide conformations at the PP1 hydrophobic 725 \ngroove. 726 \nA–C PP1 is shown as a molecular surface coloured by mean lipophilicity, from orange 727 \n(hydrophobic) to cyan (polar). A Structure of the crystal structure of PP1α catalytic domain in 728 \ncomplex with the GADD34 RVxF+ϕϕ[xF] motif (PDB 4XPN) (29). The GADD34 peptide is shown 729 \nas sticks with green carbon atoms. B AlphaFold2-Multimer model of PP1γ catalytic domain in 730 \ncomplex with HSV-1 pUL21. The TROPPO motif (pink carbon atoms) and flanking residues (grey 731 \nalpha carbons) are shown as sticks. C AlphaFold2-Multimer model of PP1γ catalytic domain in 732 \ncomplex with VZV pORF38. The TROPPO motif (pink carbon atoms) and flanking residues (grey 733 \nalpha carbons) are shown as sticks. 734 \n  735 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint \n\n24 \n \n 736 \nFigure S2. Association of GADD34 peptide and PP1 in Tris pH 8.5 buffer. 737 \nFluorescence anisotropy of PP1γ catalytic domain (residues 7–300) binding 2 nM fluorescently 738 \nlabelled peptide containing the GADD34 RVxF and ϕϕ[xF] motifs (residues 552–568). Mean ± SD 739 \nis shown for technical triplicate measurements. Affinity (KD) is average of three independent 740 \nexperiments. 741 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 25, 2025. ; https://doi.org/10.1101/2025.07.22.666160doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}