Have NEC Coat, Will Travel: Structural Basis of Membrane Budding During Nuclear Egress in Herpesviruses

Abstract

Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. Furthermore, unlike other DNA viruses that replicate in the nucleus, herpesviruses do not exit it by passing through the nuclear pores or by rupturing the nuclear envelope. Instead, herpesviruses have a complex mechanism of nuclear escape whereby nascent capsids bud at the inner nuclear membrane to form perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytosol. This makes them some of the very few known viruses that bud into the nuclear envelope. The envelope acquired during nuclear budding does not end up in the mature viral particle but instead allows the capsid to translocate from the nucleus into the cytosol. The viral nuclear egress complex (NEC) is a critical player in the nuclear egress, yet its function and mechanism have remained enigmatic. Recent studies have demonstrated that the NEC buds membranes without the help of other proteins by forming a honeycomb coat, which established the NEC as the first virally encoded budding machine that operates at the nuclear, as opposed to cytoplasmic, membrane. This review discusses our current understanding of the NEC budding mechanism, with the emphasis on studies that illuminated the structure of the NEC coat and its role in capsid budding during herpesvirus nuclear escape.

Keywords: Budding; Coat; Deenvelopment; Envelopment; Herpesviruses; Hexagonal; Honeycomb; Lattice; NEC; Nuclear egress; Scission; Structure; UL31; UL34.

Fig. 1

Herpesvirus egress. Herpesviruses assemble their capsids and package their DNA genome in the nucleus. Nucleocapsids bud at the inner nuclear membrane (INM), with the help of the nuclear egress complex (NEC), to form the perinuclear viral particles, which fuse with the outer nuclear membrane (ONM). As the result, the capsids are released into the cytoplasm where they undergo further maturation steps, e.g., assembly of a tegument layer around the capsids. During the second budding event, at the cytoplasmic membranes derived from the Trans-Golgi Network or early endosomes, the capsids acquire their final lipid envelope, which contains glycoproteins required for cell entry. The mature capsids are released from the cell through the secretory pathway. Reprinted from Bigalke, J.M., Heldwein, E.E., 2016. Nuclear exodus: herpesviruses lead the way. Annu. Rev. Virol., 3.

Fig. 2

Sequence alignment of UL31 and UL34 proteins from PRV, HSV-1, and HCMV. Secondary structure for PRV and HSV-1 proteins is indicated above the sequences and for HCMV proteins, below the sequence alignment. HCMV bears additional residues at the C-termini of UL31 and UL34, which were absent from the crystallized constructs. Sequence alignment was done with Clustal Omega (Sievers and Higgins, 2014) and visualized with ESPript 3 (Robert and Gouet, 2014).

Fig. 3

Comparison of HSV-1, PRV, and HCMV NEC crystal structures. All structures are shown in the same orientation. UL31 and UL34 form an elongated complex, with UL31 wrapping its N-terminal hook around UL34. The two molecules interact extensively, which implies high binding affinity. The membrane-proximal end is located at the bottom of the heterodimer in this orientation. The regions important for membrane interaction are missing from the structure and are indicated schematically, along with the membrane, only for HSV-1 but are expected to have a similar location in PRV and HCMV NEC. The C-terminal helix (α4) in HSV-1 UL34 was not resolved in the crystal structure. Overall, NEC structures from three different viruses are very similar, but the relative orientations of UL31 and UL34 are slightly different in HCMV (PDB ID: 4ZXS, 4Z3U, and 5DOB).

Fig. 4

Detailed analysis of the UL31–UL34 interaction. The bar diagram of the crystallized constructs (numbered) and the regions of interactions separated into two interfaces: 1 (blue) and 2 (red). Interface 1 includes the UL31 (UL53) N-terminal hook and multiple regions throughout UL34 (UL50). Interface 2 is restricted to several residues within the C-terminal half of UL34 (UL50) and residues within the globular core in UL31 (UL53). Brackets indicate the binding sites within UL31 and UL34 predicted on the basis of deletion mutagenesis, prior to crystal structures (bar diagram). UL31 and UL34 from HSV-1, PRV, and CMV were superimposed to visualize the similarities in folds (PDB codes 4ZXS, 4Z3U, 5DOB). All UL31 (UL53) molecules contain a zinc-binding motif, with zinc coordinated by three strictly conserved cysteines and one histidine. This element is likely to be important for structural integrity of the complex. Interface 1 and 2 differ between the viruses with regard to salt bridges and hydrogen bonds, but one salt bridge at interface 1 is conserved in all three structures (inlet bottom right) and may be important for complex formation throughout all herpesviruses.

Fig. 5

Comparison of hexagonal lattice in HSV-1 (two crystal lattices), HCMV (crystal lattice), and PRV (model derived from cryoET data). (A) For each lattice, three connected hexameric rings are shown side by side in a top view, perpendicular to the sixfold symmetry axis, and a side view. One NEC heterodimer is highlighted in every lattice. The two-, three-, and sixfold axes in each lattice are indicated by lense, triangle, and star symbols, respectively. Representative dimer, trimer, and hexamer are indicated by dashed lines in the HSV-1 C/D lattice. The hexameric rings are very similar in both HSV-1 lattices and the HCMV lattice but differ in the PRV lattice model. HSV-1 A/B is rings are turned toward each other in a 10.5 degree angle compared to the C/D lattice. The PRV lattice model is the only curved lattice in the side view while the rest of the lattices are planar. All crystal lattices are ∼78 Å thick. For HSV-1 NEC (B) and PRV NEC (C), cryoET lattices are shown for comparison. The lattices are thicker than in the crystal structures due to the presence of the membrane-proximal regions, absent from all crystallized NEC constructs. The diameter of the hexameric ring in the PRV cryoET lattice is slightly smaller than other ring diameters due to the curvature of the lattice and the positioning of the slice (purple dashed line in side view). HSV-1 NEC cryoET image in (B) is reprinted from Bigalke, J.M., Heldwein, E.E., 2015b. Structural basis of membrane budding by the nuclear egress complex of herpesviruses. EMBO J., 34, 2921–2936. PRV NEC cryoET image in (C) is reprinted from Hagen, C., Dent, K.C., Zeev-Ben-Mordehai, T, Grange, M., Bosse, J.B., Whittle, C., Klupp, B.G., Siebert, C.A., Vasishtan, D., Bauerlein, F.J., Cheleski, J., Werner, S., Guttmann, P., Rehbein, S., Henzler, K., Demmerle, J., Adler, B., Koszinowski, U., Schermelleh, L., Schneider, G., Enquist, L.W., Plitzko, J.M., Mettenleiter, T.C., Grunewald, K., 2015. Structural basis of vesicle formation at the inner nuclear membrane. Cell, 163, 1692–1701. http://dx.doi.org/10.1016/j.cell.2015.11.029, under Creative Commons Attribution License, https://creativecommons.org/licenses/by/4.0/.

Fig. 6

Detailed comparison of hexameric and interhexameric interfaces in HSV-1 A/B, C/D, and HCMV crystal lattices, and the PRV lattice model. (A) Interfaces are colored hotpink (hexameric), dark blue (dimeric), and teal (trimeric) to match the representative hexamer, dimer, and trimer in Fig. 5A. Bar diagrams (B) are colored according to the same color scheme. Hexameric interfaces are very similar in both HSV-1 and the HCMV lattices. In PRV, this interface is shifted toward the center of the molecule in this orientation. These differences are also apparent in the bar diagram. HSV-1 C/D and HCMV lattices also have overlapping dimeric and trimeric interfaces, whereas in the HSV-1 A/B lattice, the trimeric and dimeric interactions are almost completely reversed. Interfaces in the PRV lattice differ from those in the other three lattices.

Fig. 7

Subtomogram averaging of PRV NEC coat. Slices through the membrane-proximal (MP) and membrane-distal (MD) regions are shown to highlight two layers of the NEC hexagonal lattice. The MD slice corresponds to the lattices seen in crystal structures of HSV-1 and HCMV NEC as well the hexagonal lattice seen previously by cryoET in HSV-1 NEC coats formed in vitro. The MP slice shows a distinct lattice likely formed by MP regions, absent from all crystallized NEC constructs. It links the NEC core structure to the UL34 transmembrane anchor and to the membrane. A 90 degree-related side view below shows the MP region (pink) forming an archway in the vicinity of the membrane whereas the MD region (purple) is mainly involved in forming the hexagonal NEC lattice. Reprinted from Hagen, C., Dent, K.C., Zeev-Ben-Mordehai, T., Grange, M., Bosse, J.B., Whittle, C., Klupp, B.G., Siebert, C.A., Vasishtan, D., Bauerlein, F.J., Cheleski, J., Werner, S., Guttmann, P., Rehbein, S., Henzler, K., Demmerle, J., Adler, B., Koszinowski, U., Schermelleh, L., Schneider, G., Enquist, L.W., Plitzko, J.M., Mettenleiter, T.C., Grunewald, K., 2015. Structural basis of vesicle formation at the inner nuclear membrane. Cell, 163, 1692–1701. http://dx.doi.org/10.1016/j.cell.2015.11.029, under Creative Commons Attribution License, https://creativecommons.org/licenses/by/4.0/.

Fig. 8

Position of mutant residues that disturb HSV-1 NEC activity within the hexagonal lattice. Two protomers within the hexameric ring are shown, with the interface colored in hotpink. The hexameric interface is critical for NEC function because mutations of residues at this interface result in a nonbudding phenotype. Mutations at the interhexameric interfaces (lower panel: dimeric interface) in dark blue, trimeric interface in teal also reduce budding efficiency but to a lower extent. The suppressor mutation that showed an enhanced budding activity (R229L) is also shown at the dimeric interface. Adapted from Bigalke, J.M., Heldwein, E.E., 2015b. Structural basis of membrane budding by the nuclear egress complex of herpesviruses. EMBO J., 34, 2921–2936.

Fig. 9

Model of NEC-mediated budding during nuclear egress. UL34 localizes to the INM by means of its C-terminal transmembrane helix. Soluble UL31 is present in the nucleus and binds to UL34 at the INM. Hexameric rings assemble and eventually connect to form a lattice. Upon capsid binding, possibly through UL17/UL25 or the major capsid protein, the NEC forms a coat that deforms the membrane around the capsid. To form a spherical object, disruptions in the hexagonal lattice are likely required. NEC coats lack obvious icosahedral symmetry, and these disruptions may be of irregular nature. Reprinted from Bigalke, J.M., Heldwein, E.E., 2016. Nuclear exodus: herpesviruses lead the way. Annu. Rev. Virol., 3.