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Journal of Virology logoLink to Journal of Virology
. 2014 Dec 24;89(5):2979–2984. doi: 10.1128/JVI.03516-14

The Unusual Fold of Herpes Simplex Virus 1 UL21, a Multifunctional Tegument Protein

Claire M Metrick a, Pooja Chadha b, Ekaterina E Heldwein a,
Editor: R M Sandri-Goldin
PMCID: PMC4325721  PMID: 25540382

Abstract

UL21 is a conserved protein in the tegument of alphaherpesviruses and has multiple important albeit poorly understood functions in viral replication and pathogenesis. To provide a roadmap for exploration of the multiple roles of UL21, we determined the crystal structure of its conserved N-terminal domain from herpes simplex virus 1 to 2.0-Å resolution, which revealed a novel sail-like protein fold. Evolutionarily conserved surface patches highlight residues of potential importance for future targeting by mutagenesis.

TEXT

Herpesviruses are double-stranded DNA (dsDNA), enveloped viruses that cause lifelong latent infections and ailments ranging in severity from skin lesions to blindness, encephalitis, and cancer (1, 2). A unique feature of all herpesviruses is a multiprotein tegument layer between the capsid and the envelope. Besides being necessary for viral assembly, tegument proteins also play critical roles at early stages of the viral replication cycle, in which some are released to regulate expression of viral (3) or cellular genes (4) while others remain bound to the capsid and mediate its trafficking (57). The involvement of tegument proteins at multiple stages during viral replication makes them attractive antiviral targets, yet few have been characterized in detail.

Herpes simplex virus 1 (HSV-1) UL21 is a 535-amino-acid tegument protein that is conserved within the Alphaherpesvirus subfamily (8) and may have analogs among other herpesviruses. UL21 is important for replication in culture because UL21-null mutants of HSV-1 and pseudorabies virus (PRV) show reductions in titer and small plaques (9, 10), whereas HSV-2 cannot replicate without UL21 (8). A lack of UL21 also leads to defects in pathogenesis of PRV in mice and pigs (1114). UL21 is involved in secondary envelopment (15) and cell-cell spread of mature virions (16) through binding of the tegument proteins UL16 (15, 17) and UL11 (16, 17). The UL21-UL16-UL11 heterotrimer binds the cytoplasmic domain of glycoprotein E (gE), a viral glycoprotein required for viral cell spread (18, 19) and cell-cell fusion of infected cells (20), and regulates these functions (16). UL21 may also have a role in cytosolic capsid transport through association with microtubules (21). In the absence of UL21, expression of viral genes is delayed, possibly due to lower mRNA levels (8, 10). Finally, capsids of UL21-null HSV-2 are unable to undergo nuclear egress (8), suggesting a potential nuclear function for UL21, which localizes not only to the cytoplasm but also to the nucleus (17, 21), specifically the nuclear rim (8, 9, 16).

Although UL21 clearly plays multiple roles in the viral replication cycle, little is known about it due to the lack of sequence homology to other proteins and structural information. Parsing out these functions requires a roadmap in the form of a high-resolution three-dimensional structure. Toward this goal, we determined the crystal structure of the conserved N-terminal domain of UL21 (residues 1 to 216), which we termed UL21N. The crystal structure revealed a novel sail-like fold and evolutionarily conserved features and provides an important framework for elucidating the multiple roles of UL21 in the viral replication cycle and pathogenesis.

Full-length HSV-1 strain 17 UL21 expressed in Escherichia coli underwent spontaneous proteolysis, generating a stable N-terminal fragment (UL21N, residues 1 to 216). The corresponding fragment was amplified from the full-length gene and subcloned into the pET24a vector preceded by an N-terminal StrepII tag and a Gly-Ser linker. UL21N was expressed in Rosetta E. coli (Novagen) using overnight induction with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16°C. Cell pellets from 1 liter of culture were resuspended in buffer A [50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 100 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP)] with cOmplete protease inhibitor cocktail (Roche) and egg white avidin (Sigma) and lysed using a French press. UL21N was purified from the clarified lysate with StrepTactin Sepharose resin (GE Healthcare) and size exclusion chromatography (Superdex 200, buffer A) and stored with 1× Halt protease inhibitor cocktail (Pierce).

Crystals were grown by vapor diffusion in hanging drops {2 μl protein at ∼5 mg/ml and 2 μl crystallization solution [0.8 to 1.2 M ammonium sulfate, 100 mM HEPES (pH 7.5), 7 to 10% 2-methyl-2,4-pentanediol (MPD)]} at room temperature and flash frozen in crystallization solution with 15% MPD for data collection. Hg derivative crystals were prepared from native crystals by soaking them in crystallization solution with 1.7 mM thimerosal. X-ray diffraction data were collected at 100 K on beamline X25 at the National Synchrotron Light Source and processed with the HKL2000 software package (22) (Table 1). The experimental phases were obtained using single anomalous dispersion (SAD) data collected for the thimerosal derivative crystals as implemented in the autoSHARP program (23). The model was built manually into the experimental SAD electron density using Coot (24) and improved over several cycles of phase combination and density modification using SHARP (23). The model was refined against native data in the phenix.refine structure refinement program (25) (Table 1), with 5% of the reflections set aside as a reference, and rebuilt in Coot (24). The final Rwork is 0.166, and Rfree is 22.1% (Rwork and Rfree are defined as ∑||Fobs| − |Fcalc||/∑||Fobs| for the reflections in the working or the test set, respectively). There is one UL21N molecule in the asymmetric unit, and the model contains residues 1 to 198 of UL21, the N-terminal StrepII affinity tag, and the Gly-Ser linker. Residues 38 to 46, 76 to 87, and 199 to 216 were not resolved despite being present in the crystals. S0 is the first residue of the β1 strand, and residues QEF of the StrepII tag form a 310 helix.

TABLE 1.

Data collection and refinement statistics

Parameter Value for:
Native crystal Thimerosal-soaked crystal
Data collectiona
    Space group P6322 P6322
    Cell dimensions
        a, b, c (Å) 108.35, 108.35, 65.24 109.48, 109.48, 65.29
        α, β, γ (°) 90, 90, 120 90, 90, 120
    Resolution (Å) 41.68-2.05 (2.09-2.05) 47.41-2.80 (2.90-2.80)
    Rsym or Rmerge 0.061 (0.475) 0.111 (0.651)
    II 23.26 (2.52) 23.96 (5.78)
    Completeness (%) 99.78 (99.58) 100.00 (100.00)
    Redundancy 5.3 (5.5) 18.4 (19.1)
Refinement
    Resolution (Å) 30.81-2.05
    No. of reflections (free) 14,225 (713)
    Rwork/Rfreeb 0.166/0.221
    No. of atoms
        Protein 1,643
        Water 66
    B-factors
        Protein 51.80
        Water 49.40
    RMSc deviations
        Bond lengths (Å) 0.007
        Bond angles (°) 1.08
    Ramachandran plotd
        Favored (%) 98.49
        Allowed (%) 1.51
        Outliers (%) 0.0
a

Values in parentheses are for the highest-resolution shell.

b

Rwork and Rfree are defined as ∑||Fobs| − |Fcalc||/∑||Fobs| for the reflections in the working or the test set, respectively.

c

RMS, root mean square.

d

As determined using Molprobity (molprobity.biochem.duke.edu) (34).

UL21N adopts a single-domain structure of an unusual α/β fold that resembles a wind-filled sail with dimensions of 50 by 30 by 30Å. A DALI search (26) revealed no appreciable overall structural similarity to other proteins. UL21N is composed of two clearly defined “halves,” segregated by the following secondary structure: the oblong β bouquet, which is composed of three antiparallel β sheets (β1 to β14), and the α crescent, which is composed of four α-helices (α1 to α4) and two 310 helices (η1 and η2) that are arranged along one face of the molecule (Fig. 1). Short 310 helix η2 and α-helix α4 look like one long helix broken by completely conserved G190 (Fig. 2). The β bouquet consists of three antiparallel β sheets oriented at an angle to the longest axis of UL21N, creating the appearance of a bouquet arranged with the following topology: an inner seven-stranded β sheet [β2-β3-β4-β5-(β7)-β6-β14], a three-stranded upper outer β sheet (β1-β11-β10), and a four-stranded lower outer β sheet (β8-β9-β12-β13) (Fig. 1). Strand β5 is shorter than the neighboring strand β6 and is effectively extended by the strand β7 such that both β5 and β7 form hydrogen bonds along the same margin of strand β6 (Fig. 1A and C). The secondary structure of UL21N matches predictions (27), except that neither strands β1 and β8 nor 310 helices η1 and η2 were predicted, long strand β14 was predicted to be a helix, and helix α2 was predicted to be a strand.

FIG 1.

FIG 1

UL21N structure. The crystal structure of UL21N is shown in color based on secondary structure (A) and in rainbow coloring (B), from blue (N terminus) to red (C terminus). The StrepII tag is shown in gray. Residues unresolved in the structure are shown as dashed lines. (C) Topology diagram, colored as described for panel A. Secondary structure elements are numbered sequentially, and their amino acid boundaries are given. Helices are show as cylinders, strands as arrows, loops as solid lines, and unresolved loops as dashed lines. All structures were made using Pymol (http://www.pymol.org).

FIG 2.

FIG 2

Multiple-sequence alignment of UL21 homologs from 16 alphaherpesviruses. Sequence alignment was generated and analyzed using ClustalW (32) and ESPRIPT (33) using sequences from GenBank accession numbers ACM62243, AEV91359, AAU88086, ADO13807, AAK69349, AFR32463, ACT88337, AAT67298, AAA47475, ABO26208, AAT07796, AAR86141, CAA88112, AEI00223, AAF66756, and AAG45758. Only the alignment of residues corresponding to residues 1 to 216 of HSV UL21N is shown. The secondary structure of HSV-1 UL21 is shown above the aligned sequences. Similar residues are shown in red text. Identical residues are boxed in red with white text, and those exposed on the surface of UL21N are marked by asterisks. Gray asterisks identify conserved residues that are surface exposed in the model but are likely obscured in the protein by the unresolved loop containing residues 76 to 87.

UL21 is 5.2% identical among 16 alphaherpesviruses, and 21 of 28 identical residues are within UL21N (Fig. 2). Twelve of these are surface exposed (Fig. 2 and 3A) but do not form obvious clusters, which prevented clear assignment of potentially important sites on the surface of UL21N based on sequence identity alone. Evolutionary trace analysis (ETA) (28) was performed on the same sequence alignment. ETA generates a phylogenetic tree from a sequence alignment of homologous proteins. At each partition, or branch point, closely related sequences are grouped into classes, trace residues are defined as conserved within specific classes, and these residues are assigned importance scores based on the partition at which they appear. Clustering of important trace residues on the protein surface may indicate regions of functional importance (28). ETA has been used to detect functional sites in a number of proteins (20, 29), including PRV UL37 (31). ETA of UL21N yielded several surface clusters of trace residues of increasing importance (Fig. 3B).

FIG 3.

FIG 3

Analysis of conservation and charge on the surface of UL21N. A dotted line marks a cavity likely obscured by unresolved residues 76 to 87. (A) Completely conserved residues on the surface of UL21N are shown in magenta. Four orientations based on 90° rotations around the vertical axis are shown. (B) Class-specific residues identified by universal evolutionary trace analysis (http://mammoth.bcm.tmc.edu/uet/) are highlighted on the surface of UL21N. The 25% of residues with the highest importance scores are shown. (C) An electrostatic surface potential map of UL21N was generated using the PBEQ Solver function in the Charmm program (http://www.charmm-gui.org/?doc=input/pbeqsolver). (D) Potential functional regions assigned on the surface of UL21N. Potential functional regions are composed of the following residues: region 1 (red), D13, D105, D111, and E113; region 2 (orange), M1, E2, R55, N156, Y163, P165, F170, and L172; region 3 (yellow), Y5, Y17; region 4 (green), Y67, R69, S70, E71, D116, E117, and E121.

Analysis of the electrostatic potential on the surface of UL21N revealed two large negatively charged patches, consistent with its calculated isoelectric point of 5.2, one of which coincides with a shallow depression and the other of which wraps around the nearby side (Fig. 3C). A number of the residues within these charged patches were also identified as important trace residues in ETA. In combination, conservation and charge patterns pinpoint several surface regions of potential functional importance (Fig. 3D), which may participate in binding the known partner UL16, in interacting with UL21C, or in binding to as yet unknown ligands. Region 1 (D13, D105, D111, E113) sits on one flat face of UL21N and is comprised of four aspartate and glutamate residues, two of which are also identified in ETA. Region 2 (M1, E2, R55, N156, Y163, P165, F170, L172) extends to three sides of the protein and contains identical and important ETA trace residues. Region 3 (Y5, Y17) comprises two conserved, surface-accessible tyrosine residues, and the large region 4 (Y67, R69, S70, E71, D116, E117, E121) wraps around one short side and contains residues identified in electrostatic and ETA analyses. These regions provide a more educated starting point for mutational analysis in the context of protein biochemistry and viral infection.

Although UL21 plays multiple roles in the viral replication cycle, including nuclear egress, cytoplasmic capsid trafficking, budding, and cell-cell spread, the molecular mechanisms by which UL21 enables these and other processes remain poorly understood. The novel sail-shaped structure of UL21N provides a three-dimensional template for targeted mutational analysis and enables structure-guided functional exploration of the multiple roles of UL21 in the replication and pathogenesis of alphaherpesviruses.

Protein structure accession number.

Atomic coordinates and structure factors for the UL21N structure have been deposited in the RCSB Protein Data Bank under accession number 4U4H.

ACKNOWLEDGMENTS

We thank John Wills for helpful discussions related to this project. We also thank A. Héroux (National Synchrotron Light Source) for collecting and processing X-ray diffraction data.

This work was funded by the Burroughs Wellcome Fund Award for Investigators in Pathogenesis (E.E.H.). P.C. was supported by NIH grant AI071286 to John W. Wills. Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under contract no. DE-AC02-98CH10886.

C.M.M. performed all of the experiments described in this work. P.C. performed early work important for construct design and generated the full-length StrepII-UL21 construct. C.M.M. and E.E.H. designed research, analyzed data, and wrote the article.

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