Abstract
Human cytomegalovirus can cause severe birth defects upon infection in pregnant women and complications in immunocompromised patients. A major challenge for vaccine design is our incomplete understanding of the diverse protein complexes this virus uses to infect cells. In Herpesviridae, glycoproteins H and L (gH and gL) form complexes with other viral proteins that bind receptors to mediate cell-type-specific entry. Here we identify a distinct gH complex that is abundant on human cytomegalovirus virions and enhances infection of endothelial cells. In this complex, gH associates with UL116 and UL141 (an immunoevasin previously known to function intracellularly) but not with gL. We term this the gH-associated tropism and entry (GATE) complex and provide the cryo-electron microscopy structure at ~3.5 Å. The structure shows gH-only scaffolding, UL141-mediated dimerization and a heavily glycosylated UL116 cap. These findings identify a third virion surface complex that promotes cell entry and may represent a new target for vaccines or antiviral therapies.
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Data availability
All data needed to evaluate the conclusions are present in the article and/or the extended data. Requests for resources and reagents should be directed to and will be fulfilled by corresponding authors. Structural models of the HCMV GATE complex have been deposited in the Protein Data Bank (www.rcsb.org) under accession numbers 9DIX (GATE complex) and 9DIY (focused gH–UL116). Cryo-EM maps are deposited in the EM Database (https://www.emdataresource.org/) with the following IDs: EMD-46920 (GATE complex) and EMD-46921 (focused gH–UL116). Source data are provided with this paper.
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Acknowledgements
We thank R. Diaz Avalos and the LJI CryoEM centre for assistance with data collection. Equipment of the LJI cryoEM core was supported by NIH U19109762-S1, the GHR Foundation and private donations. We thank S. Schendel (LJI) for expert assistance with scientific language editing. We thank the LSUHS Research Core Facility (RRID: SCR_024775) for assistance with confocal microscopy imaging and qPCR. We thank J. von Einem (Universitätsklinikum Ulm, Ulm, Germany) and G. Li (Johnson & Johnson, Rockville, MD) for advice on glycerol tartrate gradient purification of virions. We thank W. J. Britt (University of Alabama, Birmingham, AL, USA), G.W.G. Wilkinson (Cardiff University, UK) and T. Shenk (Princeton University, Princeton, NJ, USA) for generously sharing antibodies. This work was supported by NIH grants R01AI116851 to J.P.K., R01AI139749 and R01AI101423 to C.A.B., T32HL155022 to L.A.H., P20GM134974 (a COBRE award at LSUHS), an ARPA-H APECx contract 1AY1AX000055 (E.O.S., C.A.B. and J.P.K.), institutional funds from the La Jolla Institute for Immunology (E.O.S. and C.A.B.), an LSUHS Chancellor’s Aim High award (J.P.K.) and a Malcolm Feist Cardiovascular Fellowship (L.A.H.). The contents of this study do not necessarily represent the official views of, nor an endorsement by, the NIH, ARPA-H, HHS or the US Government.
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Contributions
J.P.K., C.A.B. and E.O.S. conceived the idea and developed and supervised the project. M.N.A.S. constructed recombinant HCMVs and carried out IP experiments. L.A.H. carried out absolute infectivity and percentage of infection assays, glycerol–sodium-tartrate purification of virions, western blotting, confocal microscopy imaging and analysis, viral growth curves and plaque size measurements and designed tropism studies. M.J.N. designed and constructed expression plasmids, purified proteins, carried out cryo-EM and negative stain electron microscopy (nsEM) studies, and solved the GATE complex structure. J.Y. assisted with cloning and GATE complex purification. S.B. and K.Y. performed various binding and biochemical studies with the purified GATE. M.M. performed native PAGE and nsEM of the trimeric form of the GATE complex. M.J.N., L.A.H., E.O.S., J.P.K. and C.A.B. wrote the paper and contributed to paper revision and editing.
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M.J.N., M.N.A.S., E.O.S., C.A.B. and J.P.K. are listed as coinventors on US, European and international patent applications related to the identification of GATE-3, US20230293673A1, EP4192494A4 and WO2022032177A1. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 UL141 localizes at the cVAC and is incorporated into HCMV virions.
a, Infected cell lysates and purified virions from HCMV strains TB40/E (141-) and TR3 (141 + ) were compared for the indicated viral glycoproteins. These are representative blots of two independent experiments. b, Immunofluorescent staining of fibroblasts infected with TB40/E viruses that are UL141-null (TB40141-) or express FLAG-tagged UL141 (TB40141F) at 3 dpi (MOI 1 TCID50). Cells were stained with rabbit-derived anti-UL141 (magenta) and mouse-derived anti-gB (green), to identify the cytoplasmic viral assembly compartment (cVAC) (white arrowheads). HCMV encodes Fc-gamma receptors that localize at the cVAC and bind to human and rabbit antibodies, but not mouse antibodies. For all confocal microscopy, recombinant human IgG Fc is used to block viral Fc receptors during the assessment of UL141 localization to the cVAC. In addition to using human Fc, we also use (c) mouse anti-FLAG antibody to detect UL141 (green) and rabbit-derived calnexin (CNX) antibody to identify the endoplasmic reticulum (magenta) in TB40141F infected fibroblasts. Scale bars denote 25 μm. These are representative micrographs of three independent experiments yielding comparable outcomes.
Extended Data Fig. 2 Purification and oligomeric state of the HCMV gH/UL116/UL141 GATE.
a, Schematic representation of the expression and purification process for the HCMV gH/UL116/UL141 GATE complex. b, Size exclusion chromatography profile of the gH/UL116/UL141 GATE complex. Fractions were analyzed by SDS-PAGE under non-reducing conditions, with the fraction indicated by blue asterisks used for cryo-EM studies. c, Western blot analysis of fraction 8, probed with anti-His, anti-gH, and anti-strep antibodies to detect UL141, gH, and UL116, respectively. d, Fractions from size exclusion chromatography were analyzed to determine the oligomeric distribution of the gH/UL116/UL141 GATE complex. Molecular weight markers (in kDa) are indicated on the left. Two predominant species, ~720 kDa and ~480 kDa, were observed, suggesting different oligomeric states. Fractions containing these species are marked with asterisks. e, Representative 2D class averages from negative stain electron microscopy of the size exclusion chromatography fractions. Particles from the ~720 kDa and ~480 kDa fractions exhibit distinct structural features, resembling either a “plus sign” or an “H” shape, suggesting that the gH/UL116/UL141 GATE complex can exist both as a heterotrimer and as a dimer of heterotrimers (hexamer). These data are representative of five independent experiments.
Extended Data Fig. 3 Cryo-electron microscopy processing of the HCMV gH/UL116/UL141 GATE complex.
a, Overview of the representative cryo-EM data processing workflow for the gH/UL116/UL141 GATE complex. b, (Left) Front and back views of the cryo-EM map of the locally refined and symmetry-expanded gH/UL116 region. (Right) Corresponding front and back views of the atomic model, shown as a ribbon diagram, highlighting the gH–UL116 interaction. gH is depicted in grey, UL116 in purple, and UL141 in teal. Resolved N-linked glycans, identified from focused refinements, are highlighted in yellow.
Extended Data Fig. 4 Cryo-EM structure validation.
a, Gold-standard Fourier shell correlation (FSC) curves for the refinements of the HCMV gH/UL116/UL141 dimer (left) and the symmetry-expanded focused local refinement of the gH-UL116 interface (right). b, Conical FSC (cFSC) analysis of the half maps. The blue cFSC summary plot displays the mean, minimum, maximum, and standard deviation of correlations at each spatial frequency. The green histogram shows the distribution of 0.143 threshold crossings, corresponding to the spread of resolution values across different directions. c, Euler angle distribution plot of the particles used in the final 3D reconstructions, demonstrating complete coverage of projections as generated in CryoSPARC. d, Final reconstructions filtered and colored by local resolution, as estimated in CryoSPARC.
Extended Data Fig. 5 Cryo-EM structure validation and model quality assessment.
a, Map versus model FSC curves calculated with and without masking, using the Phenix package. Curves are shown for the HCMV gH/UL116/UL141 GATE dimer (left) and the symmetry-expanded focused local refinement of the gH-UL116 interface (right). b, Cryo-EM maps for fragments of gH (left), UL116 (middle), and UL141 (right) from the gH/UL116/UL141 dimer, demonstrating the quality of the map. The cryo-EM map is displayed as a mesh. c, Cryo-EM maps for a fragment of gH and UL116 from the symmetry-expanded focused refinement of the gH-UL116 interface, illustrating the quality of the cryo-EM map. The map is shown as a mesh.
Extended Data Fig. 6 Electrostatic surface potential and glycosylation of the HCMV gH/UL116/UL141 GATE.
a, Electrostatic surface potential of the HCMV GATE complex displayed on a space-filling model, with positively charged regions in blue and negatively charged regions in red. The negatively charged cleft is outlined. Electrostatic potential maps were generated using the PDB2PQR and APBS software. b, Side and top views of the glycosylation site distribution on the HCMV gH/UL116/UL141 GATE complex. c, Inset showing the glycosylation site distribution at the gH-UL116 interaction site, as resolved in the symmetry-expanded focused refinement of the gH-UL116 interface.
Extended Data Fig. 7 Structural comparison of gH from the HCMV GATE, trimer, and pentamer.
a, Structural representation and ___domain organization of gH in the HCMV GATE (left), trimer (middle), and pentamer (right). The gH domains I–IV are colored yellow, orange, red, and purple, respectively. In the GATE, the gH DI ___domain undergoes a substantial rotational shift relative to the trimer and pentamer, transforming the gH subunit from a straight rod in the trimer and pentamer to a crescent shape in the GATE structure. b, Structural alignment of individual gH domains comparing the GATE with the trimer (top) and the pentamer (bottom). The structures were aligned using the indicated number of Cα atoms from the respective PDB files, and the alignment was quantified by the indicated r.m.s.d. values.
Extended Data Fig. 8 gH and TRAIL-R2 share a similar binding site on UL141.
a, Structural comparison of UL141 in the GATE complex with unbound UL141 (left) and UL141 bound to TRAIL-R2 (right). Structures were aligned as dimers using all Cα atoms in the respective PDB files. The alignment is quantified by the indicated root-mean-square deviation (r.m.s.d.) values. b, The extracellular ___domain of UL141 (teal) is shown within the gH/UL116/UL141 GATE complex, with gH in grey and UL116 in purple. Glycans are depicted as yellow sticks. Distance measurements indicate the contribution of the gH stalk (37.4 Å), the distance from the top of the stalk to UL141 (38 Å), and the estimated total distance of UL141 from the viral membrane (~75.4 Å). Given that the final 25 C-terminal residues of UL141 are unresolved, they could extend ~85 Å if fully disordered. This suggests that UL141 is membrane-anchored, possibly positioned even closer due to the twisted conformation of the complex. c and d, Structural models of (c) UL141 in the GATE complex and (d) UL141 bound to TRAIL-R2 illustrate that gH and TRAIL-R2 occupy overlapping binding sites on UL141. The buried surface area for each interaction is indicated. UL141 is shown as a cartoon representation, while gH, UL116, and TRAIL-R2 are depicted as surface models. In the GATE, several regions of UL141 that were disordered in the unbound and TRAIL-R2-bound structures become well-ordered (highlighted in yellow). e, Surface representation of a UL141 monomer, with the TRAIL-R2 binding footprint highlighted in pink, the gH binding footprint in grey, and their overlapping region in orange. The buried surface area of the overlap is quantified, representing ~25% of the TRAIL-R2 binding site.
Extended Data Fig. 9 UL141 promotes endothelial cell tropism independently of the pentamer complex.
a, Representative images of UL141-dependent spread in endothelial cells. Fibroblasts and endothelial cells were infected with 50 or 100 genome equivalents/cell, respectively, of 141- or 141 + TB40/E viruses produced by fibroblasts. Cells were stained for IE1 (green) and Hoechst (blue) at the indicated days post-infection to monitor viral spread. Scale bars denote 800 μm. b, Low MOI (0.01 TCID50) viral growth kinetics in HUVEC infected with 141- or 141+ viruses up to 14 dpi (n = 3). Data were logarithmically transformed to fit a Gaussian distribution prior to calculating statistical significance via 2-way ANOVA (two-tailed). ***P = .0009 for 12 dpi data points. c, Non-reducing SDS-PAGE of HUVEC cell lysates and HUVEC-derived virions. Cells were infected with 141- and 141+ viruses to measure virion incorporation of known HCMV entry complexes, trimer (gH/gL/gO) and pentamer (gH/gL/128). Lysates were immunoblotted for gL to identify covalently-linked entry complexes, major capsid protein (MCP) to measure virion abundance, and UL148 to assess the purity of the virion preparations. d, Quantification of band intensities for gH/gL/gO and gH/gL/128 abundance in HUVEC-derived virions from two independent experiments. Band intensities of 141- and 141+ virions were normalized to MCP. Error bars represent ± SEM.
Extended Data Fig. 10 UL141 enhances the infectivity of the pentamer-null AD169 strain in epithelial cells.
a, Epithelial cells (ARPE-19) were incubated with 50 genome equivalents/cell of UL141-null (141-) or UL141-repaired (141 + ) TB40/E that either express UL116 or are UL116-deficient (Δ116). Cells were stained for IE1 to measure the percentage of infected cells. Significance was determined by a two-tailed ratio paired t-test, as all conditions for each biological replicate were assessed in parallel (141- and 141 + , n = 5; Δ116141- and Δ116141 + , n = 3). Error bars represent ± SEM. **P = .0086. Scale bars represent 400 μm. b, Schematic of the pentamer-null AD169 viruses used in c-f. c, ARPE-19 were infected with AD169 or UL141-restored AD169 (AD169141+) at MOI 0.1 TCID50. Cells were stained for IE1 at 5 and 10 days post-infection (dpi) to measure the size of foci, or plaques, as IE1+ nuclei/plaque. Each point in the bar graph represents a biological replicate (5dpi, n = 6; 10 dpi, n = 7) *P = .0406. Error bars represent ± SEM. d, Mean plaque sizes for each 10 dpi biological replicate shown in (c). Error bars represent ± SEM. Enumerated plaques are reported in Supplementary Table 2. e, QQ plots displaying the lognormality of raw plaque size data. After log10 transformation, data fit a Gaussian distribution and were used to calculate statistical significance via two-tailed analyses: (c) Welch’s t-test or (d) 2-way ANOVA. f, Representative image of AD169 versus AD169141+ plaques in ARPE-19 cells at 10 dpi. Cells were stained for IE1 (green). Scale bars represent 100 μm.
Supplementary information
Source data
Source Data Fig. 1
a, Unprocessed western blots for Fig. 1b. b, Unprocessed western blots for Fig. 1c. c,d, Unprocessed western blots for Fig. 1e (FLAG IP) and Fig. 1f (myc IP).
Source Data Fig. 4
Unprocessed western blots for Fig. 4d.
Source Data Fig. 4
Mean absolute infectivity with s.e.m. values and statistics for Fig. 4b. Average percentage of infection of fibroblasts and endothelial cells infected with UL116-null TB40/E (Fig. 4c). Intensity profiles (with the cVAC highlighted in grey), Pearson’s correlations, simple linear regressions and unpaired t-test results with Welch’s corrections for Fig. 4f–h,j–l.
Source Data Fig. 5
Average percentage of infection and ratio paired t-test statistics for fibroblast-derived viruses (Fig. 5b) and endothelial cell-derived viruses (Fig. 5c).
Source Data Extended Data Fig. 1/Table 1
Unprocessed western blots.
Source Data Extended Data Fig. 9/Table 9
Unprocessed blots (Fig. 9c).
Source Data Extended Data Fig. 9/Table 9
Viral titres (TCID50 per millilitre) for MOI 0.1 TCID50 growth curves and two-way ANOVA statistics shown in Extended Data Fig. 9b. Calculated band intensities of major capsid protein (MCP), gH–gL–UL128 and gH–gL–gO in UL141-null (141−) and UL141-repaired (141+) virions (Fig. 9d).
Source Data Extended Data Fig. 10/Table 10
Mean percentage of infection and statistical source data (Extended Data Fig. 10a). Mean plaque size and log10-transformed plaque size data (Extended Data Fig. 10c). IE1+ nuclei recorded for all foci at 10 dpi for each independent experiment (Extended Data Fig. 10d).
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Norris, M.J., Henderson, L.A., Siddiquey, M.N.A. et al. The GATE glycoprotein complex enhances human cytomegalovirus entry in endothelial cells. Nat Microbiol (2025). https://doi.org/10.1038/s41564-025-02025-4
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DOI: https://doi.org/10.1038/s41564-025-02025-4
