Funder: Nvidia; FundRef: http://dx.doi.org/10.13039/100007065Funder: Wellcome Trust; FundRef: http://dx.doi.org/10.13039/100004440Funder: John Lucas Walker StudentshipFunder: Commonwealth Scholarship Commission; FundRef: http://dx.doi.org/10.13039/501100000867Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)−1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III–like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly

Ahmed, Md Firoz

Antrobus, Robin

Butt, Benjamin G

Crump, Colin M

Graham, Stephen C

Hill, Chris H

Houghton, Jack W

Ivanova, Lyudmila

Jeffries, Cy M

Owen, Danielle J

Svergun, Dmitri I

Welch, John J

English

Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)−1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III–like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly

Butt, Benjamin

Graham, Stephen

DESY

Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex

Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III-like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly

White Rose Research Online

Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III-like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.Remote synchrotron access was supported in part by the EU FP7 infrastructure grant BIOSTRUCT-X (Contract No. 283570) and access to P12 was supported by iNEXT funded by the Horizon 2020 programme of the European Commission (grant number 653706). A Titan V graphics card used for this research was donated by the NVIDIA Corporation. BGB is a Wellcome Trust PhD student, DJO was supported by a John Lucas Walker Studentship, and MFA was supported by Commonwealth Scholarship Commission PhD scholarship (BDCA801 2014-7). This work was supported by a Sir Henry Dale Fellowship (098406/Z/12/B), jointly funded by the Wellcome Trust and the Royal Society (to SCG)

Apollo (Cambridge)

Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex.

Figures and figure supplementsInsights into herpesvirus assembly from the structure of the pUL7:pUL51 complexBenjamin G Butt et alButt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 1 of 17RESEARCH ARTICLEFigure 1. HSV-1 pUL7:pUL51 forms a 1:2 heterotrimeric complex in solution. (A) SEC-MALS analysis ofrecombinant full-length pUL7:pUL51 complex. Weight-averaged molecular masses (colored solid lines) are shownacross the elution profiles (normalized differential refractive index, dRI, colored dashed lines) for samples injectedat 2.4, 4.9 and 9.7 mg/mL (blue, orange and purple, respectively). The expected molecular masses for 1:1, 1:2 and2:4 pUL7:pUL51 complexes are shown as dotted horizontal lines. (B) Averaged SAXS profiles through SEC elutionpeaks corresponding to 1:2 (blue) and 2:4 (red) complexes of pUL7:pUL51. Fits of representative GASBOR ab initiodummy-residue models to the scattering curves for each complex are shown in yellow. c2, fit quality. p, CorrelationMap (CorMap) P-value of systematic deviations between the model fit and scattering data (61). (C) RefinedFigure 1 continued on next pageButt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 2 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 1 continuedDAMMIN dummy-atom model reconstruction of the 2:4 pUL7:pUL51 complex, shown in two orthogonalorientations. (D) Representative GASBOR dummy-residue model of the 2:4 pUL7:pUL51 complex, shown in twoorthogonal orientations. This model comprises an anti-parallel dimer of heterotrimers, although we note thatparallel dimers are also consistent with the scattering data. (E) SEC-MALS of pUL7:pUL51(8-142) complex. Elutionprofiles and molecular masses are shown as in (A) for recombinant pUL7:pUL51(8–142) injected at 0.6, 1.1 and 3.9mg/mL (blue, orange and purple, respectively). (F) Schematic representation of pUL7 and pUL51. (G) AveragedSEC-SAXS profile through pUL7:pUL51(8–142) elution peak. Fit of a representative GASBOR ab initio dummy-residue model to the scattering curve is shown in yellow. (H) Refined DAMMIN dummy-atom model reconstructionof pUL7:pUL51(8–142) complex. (I) Representative GASBOR dummy-residue model of pUL7:pUL51(8-142). (J) Plotof the Guinier region (sRg < 1.3) for SAXS profiles shown in (B) and (G). The fit to the Guinier equation (yellow) islinear for each curve, as expected for aggregate-free systems. (K) p(r) vs r profiles for SAXS profiles shown in (B)and (G). (L) Dimensionless Kratky plot of SAXS profiles shown in (B) and (G). The expected maximum of the plotfor a compact globular domain that conforms to the Guinier approximation is shown (sRg = H3, (sRg)2I(s)/I0 = 3e 1,grey dotted lines).Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 3 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 1—figure supplement 1. HSV-1 pUL51 forms large soluble aggregates when purified in isolation. (A, B) SEC elution profiles of (A) His6-taggedwild-type pUL51 and (B) His6-tagged pUL51 where Cys9 was substituted with serine (C9S). Proteins were injected onto an S200 16/600 column (GEHealthcare) equilibrated in 20 mM Tris (pH 7.5), 200 mM NaCl, 1 mM DTT. Both proteins have extended elution profiles with peaks near the columnvoid volume (V0), consistent with their forming large soluble aggregates. Coomassie-stained SDS-PAGE analysis of eluted SEC fractions are shownbeneath each chromatogram. Note that there is a higher molecular weight band in (A), consistent with the presence of an SDS-resistant pUL51 dimer,despite the presence of 1 mM DTT in the SEC buffer and 2 mM DTT in the SDS-PAGE loading buffer. (C) Purified His6-tagged wild-type pUL51 wassubjected to SDS-PAGE either without additional treatment (lane 1) or following incubation with 50 mM b-mercaptoethanol (lane 2) or 50 mM DTT (lane3). Comparison with the His6-tagged pUL51 C9S mutant (lane 4) confirms that Cys9, the residue that becomes palmitoylated in mammalian cells(Nozawa et al., 2003), mediates disulfide bond mediated dimerization of recombinant wild-type pUL51. C9S substituted pUL51 (or truncations thereof)was thus used for all subsequent experiments with purified proteins.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 4 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 1—figure supplement 2. Predicted secondary structure of pUL7 and pUL51 homologues from representative human a-, b- and g-herpesviruses.Analyses of amino acid sequences were performed as described in Materials and Methods. Per-residue probabilities of forming a-helix (blue), b-sheet(purple) or coil (green) are shown, as is the probability of disorder (orange). Residues that are known (solid triangles) or predicted (empty triangles) tobe palmitoylated are marked: Cys9 of HSV-1 pUL51, Cys9 of VZV pORF7, Cys8 and Cys13 of HCMV pUL71, Cys10 and Cys11 of KSHV pORF55. Regionsof pUL7 and pUL51 a-helix and b-sheet observed in the pUL7:pUL51(8–142) core heterodimer structure are shown above the predictions as boxes. Thepredicted pUL7 and pUL51 secondary structural elements are largely conserved across herpesvirus families, although the first two helices of pUL7 areFigure 1—figure supplement 2 continued on next pageButt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 5 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 1—figure supplement 2 continuednot conserved in b-herpesviruses like HCMV. Additionally, the C-terminal regions of pUL51 homologues vary in length, although in all herpesvirusfamilies they are predicted to be largely unstructured.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 6 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 2. Structure of pUL7 in complex with pUL51. (A) Hetero-octamer of pUL7 and pUL51(8–142) observed in the crystallographic asymmetric unit.pUL7 and pUL51 are shown as green and cyan ribbons, respectively, in two orthogonal orientations. Inset shows residues arising from the pUL7 cloningtag (pink) that form an eight-stranded b-barrel with residues from pUL51. (B) Core heterodimer of pUL7 (residues 11–296) and pUL51 (residues 41–125).Selected secondary structure elements are labelled. (C) ‘Cut-through’ molecular surface representation of pUL7 (green) showing the intimate interactioninterface with the hydrophobic loop and helix a1 of pUL51 (cyan). pUL51 side chains are shown as sticks. (D) Molecular interactions between pUL51(cyan) and pUL7 (boxed residue names). Hydrophobic and hydrogen bond interactions are in black and red typeface, respectively. (E) Molecular surfacerepresentation of pUL7, colored by residue hydrophobicity from white (polar) to orange (hydrophobic). pUL51 is represented as a cyan ribbon withselected side chains shown.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 7 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 2—figure supplement 1. SEC-MALS of truncated pUL7:pUL51 complexes. (A, B) SEC elution profiles (differential refractive index, dashed lines)and weight-averaged molecular masses across the elution peaks (solid lines) are shown. (A) SEC-MALS of pUL7:pUL51(8–142) where pUL7 had beenpurified using an N-terminal GST tag that was subsequently removed using human rhinovirus 3C protease. Observed mass for the pUL7:pUL51(8–142)complex was 57.4 ± 2.2 kDa, compared with a theoretical mass of 62.7 kDa for a 1:2 heterotrimer. Samples were injected onto the column at 0.3 mg/mL(blue), 0.5 mg/mL (orange) and 1 mg/mL (purple). (B) SEC-MALS of pUL7:pUL51(41–142), injected onto the column at 0.3 mg/mL. The observed masswas 45.1 kDa, compared to a theoretical mass of 44.8 kDa for a 1:1 heterodimer (C) Coomassie-stained SDS-PAGE analysis of samples used for SEC-MALS analysis in (A), (B) and Figure 2. The GST purification tag was cleaved from all samples used for SEC-MALS, the pUL7 protein having beentagged at the N or C terminus during the initial purification steps as shown.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 8 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 2—figure supplement 2. The CUSTARD fold of pUL7. (A) Structure of pUL7:pUL51(8–142) core heterodimer is shown as ribbons, with pUL51colored white and pUL7 colored from blue (residue 11) to red (residue 296). Two orthogonal views are shown. (B) Schematic diagram of the topology ofpUL7. (C) HSV-1 pUL7 sequence, with secondary structure shown above. Residues that interact with pUL51 are highlighted in cyan.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 9 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 2—figure supplement 3. Cross-linking mass spectrometry analysis and pseudo-atomic modelling of the pUL7:pUL51(8–142) solutionheterotrimer. (A) Coomassie-stained SDS-PAGE analysis of purified pUL7:pUL51(8–142) following 30 min incubation at room temperature with varyingmolar excesses of the cross-linking agents DSSO (left) or DSBU (right). Theoretical migration of proteins corresponding to pUL7, pUL51(8–142), and 1:1,1:2, 2:2 or 2:4 complexes thereof, are indicated. (B) Cross-linking restraints used for pseudo-atomic modelling of the pUL7:pUL51(8–142) heterotrimer.Restraints used for all models (‘constant cross-links’) and permuted restraints (‘variable cross-links’) that were used for the five best-fit (lowest c2) modelsare shown. (C) The five best pseudo-atomic models (lowest c2) generated by fitting to the pUL7:pUL51(8–142) SAXS profile as described in Materialsand Methods using the restraints shown in (B). The core heterodimer of pUL7 (residues 11–234 and 253–296; green) and pUL51 (residues 41–89 and 96–125; #1, cyan) and the additional molecule of pUL51 (residues 41–89 and 96–125; #2, yellow) are shown as ribbons (right). Additional regions modelledusing I-TASSER or CORAL are shown as spheres. The fit of the computed scattering (yellow) to the pUL7:pUL51(8–142) SAXS profile (aqua) is shown foreach model (left), as are reduced c2 and CorMap P-values (Franke et al., 2015; Petoukhov et al., 2012).Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 10 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 3. Conservation of the pUL7:pUL51 interaction across herpesviruses. (A–D) HEK 293 T cells were co-transfected with GFP-tagged pUL7 homologues from human herpesviruses, or with GFP alone, and with mCherrytagged pUL51 homologues. Cells were lysed 24 hr post-transfection and incubated with anti-GFP (A, B, D) or anti-RFP (C) resin to capture protein complexes before being subjected to SDS-PAGE and immunoblotting using theantibodies shown. All immunoblots are representative of at least two independent experiments performed bydifferent scientists. (A) mCherry-tagged homologues of pUL51 are captured by GFP-pUL7 homologues, but not byGFP alone. (B) GFP-pUL7 co-precipitates with pUL51 (HSV-1) and pORF7 (VZV), but not with pUL71 (HCMV) orpORF55 (KSHV). (C) pUL51-mCherry co-precipitates with pUL7 but not with homologues from other herpesviruses.(D) The VZV pUL7 homologue pORF53 co-precipitates with VZV pORF7, but not with pUL51 homologues fromother herpesviruses. (E) Molecular surfaces of the pUL7 and pUL51 core heterodimer, colored by residueconservation across the a-herpesviruses. Residues that mediate the pUL7:pUL51 interaction are outlined.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 11 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 3—figure supplement 1. pUL51 does not co-precipitate pUL14 in uninfected cultured cells. (A) Core heterodimer of pUL7:pUL51 with residuesrequired for the reported pUL51 and pUL14 (Oda et al., 2016) highlighted in pink. Inset shows pUL7 and the first helix of pUL51 as surfaces, and sidechains of residues that were substituted with alanine in Oda et al., 2016 are shown as sticks. (B) HEK 293 T cells were co-transfected with myc-taggedpUL14 from HSV-1 along with GFP-pUL7 and pUL51-mCherry, as shown. Co-immunoprecipitation of myc-pUL14 with pUL51-mCherry is not observedeither in the presence or absence of pUL7. Image shown is representative of three independent experiments.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 12 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 4. Co-localization of the pUL7:pUL51 complex with trans-Golgi membranes is conserved across humanherpesviruses. HeLa cells were co-transfected with GFP-pUL7 and pUL51-mCherry, or with similarly-taggedhomologues from VZV, HCMV and KSHV. Cells were fixed 24 hr post transfection and immunostained using thetrans-Golgi marker protein TGN46 before imaging by confocal microscopy. Co-localization between the GFP,mCherry and far-red (TGN) fluorescence is observed in cells transfected with either HSV-1 pUL7:pUL51 or with thehomologous complexes from VZV, HCMV and KSHV. HSV-1 pUL7 and pUL51 also co-localize with striated cellperipheral structures (focal adhesions, see Figure 4—figure supplement 1 and Figure 4—figure supplement 2).Images are representative of experiments performed in three cell lines (TERT-immortalized human foreskinfibroblasts, U2-OS osteosarcoma cells and HeLa cells) by two independent scientists.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 13 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 4—figure supplement 1. The pUL7:pUL51 complex from HSV-1 co-localizes with focal adhesion marker paxillin, but homologues from otherhuman herpesviruses do not. HeLa cells were co-transfected with GFP-pUL7 and pUL51-mCherry, or with similarly-tagged homologues from VZV,HCMV and KSHV. Cells were fixed 24 hr post-transfection and immunostained for the focal adhesion marker protein paxillin before imaging by confocalmicroscopy. Co-localization between the GFP, mCherry and far-red (paxillin) fluorescence was only observed for cells transfected with HSV-1 pUL7-GFPand pUL51-mCherry. Images are representative of experiments performed in two cell lines (U2-OS osteosarcoma cells and HeLa cells).Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 14 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 4—figure supplement 2. The pUL7:pUL51 complex from HSV-1 co-localizes with focal adhesion marker zyxin but homologues from otherhuman herpesviruses do not. HeLa cells were co-transfected with GFP-pUL7 and pUL51-mCherry, or with similarly-tagged homologues from VZV,HCMV and KSHV. Cells were fixed 24 hr post-transfection and immunostained for the focal adhesion marker protein zyxin before imaging by confocalmicroscopy. Co-localization between the GFP, mCherry and far-red (zyxin) fluorescence was only observed for cells transfected with HSV-1 pUL7-GFPand pUL51-mCherry. Images are representative of experiments performed in three cell lines (TERT-immortalized human foreskin fibroblasts, U2-OSosteosarcoma cells and HeLa cells) by two independent scientists.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 15 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 5. Structural similarity of HSV-1 pUL51 to cellular ESCRT-III proteins. (A) pUL51 (cyan) is shown superposed on the helical hairpin (conservedhelices a1 and a2) of human CHMP4B (orange; PDB 4ABM) (Martinelli et al., 2012). (B and C) pUL51 (cyan) superposed on conserved helices a1 anda2 of CHMP4B homologues (B) yeast Snf7 (yellow; PDB 5FD7) (Tang et al., 2015) and (C) fly Shrub (violet; PDB 5J45) (McMillan et al., 2016). Notehelices a2 and a3 of the ESCRT-III core domains of that Snf7 and Shrub are elongated and continuous in polymeric forms of these proteins (Tang etal., 2015; McMillan et al., 2016). (D) Schematic representation of selected cellular ESCRT-III protein core domains and pUL51. Residues 1–150 ofcellular ESCRT-III proteins and 1–190 of pUL51 are depicted. Secondary structure of crystal structures shown in panels A–C are in solid lines (coil) andsolid boxes (helices). Predicted secondary structure (Petersen et al., 2009) outside these regions is shown as dotted lines (coil) and striped boxes(helices). The N-terminal region of pUL51 that forms a b-sheet with the pUL7 cloning tag, presumably an artefact of crystallization, and precedingresidues are shown in pink. The region of pUL51 used for electron microscopy analysis is shaded in grey. Myristoylation (CHMP6) or palmitoylation(pUL51) sites are indicated by green and purple sticks, respectively. (E–I) Negative stain transmission electron microscopy images of pUL51 filaments.Scale bars, 100 nm. (E–G) Representative images of pUL51 proto-filaments formed when 100 mM pUL51(1–170) in 20 mM tris pH 8.5 was incubated ongrids for 30 s before staining. (H, I) Representative images of pUL51 filaments formed when 10 mM pUL51(1–170) in 20 mM HEPES pH 7.5 was incubatedon grids for 1–2 min before staining. (J and K) The pUL7:pUL51 core heterodimer is shown superposed onto (J) two subunits of the putative Shrubhomopolymer (violet and pink; PDB 5J45) (McMillan et al., 2016), or (K) the complex of Shrub with the regulatory DM14-3 domain of Lgd (purple; PDB5VO5) (McMillan et al., 2017). pUL7 is shown as a green molecular surface. Spatial overlap between pUL7 and (J) the second subunit of Shrub, or (K)the Lgd DM14-3 domain, is denoted by asterisks.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 16 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular BiophysicsFigure 5—figure supplement 1. Purification of His-tagged pUL51(1-170). (A) Coomassie-stained SDS-PAGEanalysis of His-pUL51(1–170) purification from inclusion bodies, showing depletion of insoluble pUL51 from thebacterial cell lysate by centrifugation and the purified sample after refolding. (B) Circular dichroism spectrum ofHis-pUL51(1–170). The spectrum is consistent with the pUL51 N-terminal region having a predominantly a-helicalcomposition, as expected from the crystal structure and secondary structure predictions (Figure 2; Figure 1—figure supplement 2). Decomposition of the spectrum using CDSSTR (as implemented by DichroWeb) gives anoverall helical fraction of 0.8 (0.6 regular a-helix, 0.2 distorted a-helix) with a normalized root-mean-squaredeviation of 0.002 over 177 residues. MRE, mean residue ellipticity.Butt et al. eLife 2020;9:e53789. DOI: https://doi.org/10.7554/eLife.53789 17 of 17Research article Microbiology and Infectious Disease Structural Biology and Molecular Biophysics

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Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex

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