Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Abstract

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.

Keywords: NMR; cytomegalovirus; drug target; herpesvirus; protein–protein interactions.

Fig. 1.

Solution-state structure of M50 (1–168) as determined by NMR. (A) The 15 lowest energy structures generated by the AMBER water refinement are shown overlaid using backbone atoms for residues 10–158 in side-by-side stereoview (Protein Data Bank ID code 5A3G). (B) Three viewpoints of a ribbon diagram of M50 (1–168) showing nine β-strands (red) and six α-helices (blue). Faces A and B of the β-taco are delineated in Left by the dotted outlines and labeled. *Helix-α4 is recognized as helical by PROCHECK (50) but not by default PyMOL (51) secondary structure identification. (C) Schematic of the topology of M50 (1–168).

Fig. S1.

Protein schematics and effect of RDC refinement. (A) Schematic of M50. The region demarcated in blue represents the residues expressed and used in the described NMR and ITC experiments. The M50 C-terminal transmembrane domain (TM) from residues 285–307 as predicted by TMHMM Server, version 2.0 (www.cbs.dtu.dk/services/TMHMM/) is demarcated in black. (B) Schematic of UL53. The region demarcated in orange represents the residues represented in the synthetic peptide used for the described NMR-based titration experiments. (C) The 15 lowest energy structures from the CYANA structure calculation of M50 (residues 1–168) in red aligned with the 15 lowest energy structures after the RDC and AMBER water refinements in blue. (D) T₁, T₂, and hetero-NOE values as derived from the associated NMR experiments (acquired at 291 K and plotted vs. residue number). These data show that M50 shows flexibility primarily in the loop regions of the protein but not in regions with defined secondary structure. Secondary structure elements (α-helices in blue and β-sheets in red) as determined by PROCHECK from the structure are depicted above the plots.

Fig. S2.

Clustal Omega (61) alignment of human herpesvirus homologs of M50. Colons indicate conservation between groups of strongly similar properties (scoring >0.5 in the Gonnet PAM 250 matrix). Periods indicate conservation between groups of weakly similar properties (scoring ≤0.5 in the Gonnet PAM 250 matrix). Residues with nonzero normalized ratios of peak intensities lower than 0.4 in titration experiments with 100% UL53 peptide and isotopically enriched M50 (1–168) are highlighted in red. Secondary structural elements (based on PROCHECK topology) from the M50 (1–168) lowest energy fold are blue in areas of α-helical character and red in areas of β-stranded character. TM, transmembrane domain. *Positions that have an identical residue.

Fig. S3.

Stereo views of the PyMol (51) alignment of the Phyre2 (www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (62) HCMV UL50 (purple) and HSV-1 UL34 threading results (red) with the M50 (1–168) lowest energy model (blue). Left and Center form a wall-eyed stereoview, and Center and Right form a cross-eyed stereoview. The free end of the structure depicted at the bottom is the N terminus. The orientation is a 90° clockwise rotation from Fig. 1B, Center.

Fig. 2.

UL53 peptide binding site. (A) Heteronuclear single-quantum coherence spectrum of ¹⁵N M50 (1–168) with equimolar unlabeled UL53 peptide titrated into the sample. The expanded view shows Y57 as an example of a peak that shows chemical shift perturbation on the addition of the peptide and S82 (marked with an asterisk) as an example of a peak that shows no chemical shift perturbation. (B) Bar graph of the normalized ratio of peak intensities with equimolar unlabeled UL53 peptide present to peak intensities with no unlabeled UL53 peptide present for each residue. Areas highlighted by red boxes are residues that show marked reduction in peak intensity on addition of the peptide, and these areas correspond to the residues shown in red in C and D. (C) Ribbon representation of M50 (1–168), with residues showing a nonzero normalized ratio of peak intensity lower than 0.4 in titration experiments highlighted in red. (D) Surface representation of M50 (1–168), with residues showing a nonzero normalized ratio of peak intensity lower than 0.4 in titration experiments highlighted in red. Only a subset of residues has been labeled in C and D for clarity of presentation.

Fig. S4.

UL53 peptide and example ITC traces. (A) Clustal Omega alignment of the UL53 peptide (residues 58–85) and M53 (GenBank accession no. ACE95567.1) sequences shows a high degree of conservation between the HCMV and MCMV in the minimal conserved region previously identified (30, 61). Colons indicate conservation between groups of strongly similar properties (scoring >0.5 in the Gonnet PAM 250 matrix). Periods indicate conservation between groups of weakly similar properties (scoring ≤0.5 in the Gonnet PAM 250 matrix). Residues highlighted in cyan are those with aliphatic hydrophobic sidechains. Residues highlighted in green are those with aromatic hydrophobic sidechains. *Positions that have an identical residue. (B) ITC trace for M50 (residues 1–168) titrated into M53 (residues 50–292). (C) ITC trace for M50 (residues 1–168) V52A titrated into M53 (residues 50–292).

Fig. S5.

Substitution mutants, viral replication, UL53 distribution, and nuclear lamina disruption. (A) UL50 mutant viruses. V52A (□) and S125A (△) were compared with WT BADGFP virus (◆) after inoculation at a multiplicity of infection of 0.1 pfu per cell of 10⁵ HFF cells per well in 24-well plates. For each data point in A and B, the supernatants from two wells were pooled on the indicated day postinfection, and titers were calculated by averaging plaque counts from duplicate titrations. Counts from duplicate titrations differed by less than a factor of two for all data points. Error bars are not shown. (B, Upper) Rescued derivatives of the UL50 null mutant 50NR (◆), the E56A mutant E56AR (△), and the L130 mutant L130AR (×). (B, Lower) The point mutants V52A (■) and S125A (▲) and their rescued derivatives V52AR (□) and S125AR (△), respectively, were compared with WT virus (BADGFP; □ in Upper and ○ in Lower) for replication after inoculation at a multiplicity of infection of 0.1 pfu per cell of 10⁵ HFF cells per well in 24-well plates. (C) HFFs were electroporated with WT (i–iii) UL53-FLAG pBADGFP or (iv–vi) UL50 null UL53-FLAG pBADGFP or the E56A (vii–ix) UL53-FLAG pBADGFP or (x–xii) its rescued derivative E56AR UL53-FLAG pBADGFP. Cells were fixed on day 7 and stained with anti-FLAG antibody (red) or DAPI (blue), and virus-infected cells were visualized by confocal microscopy. (D) HFFs were (i–iii) mock-electroporated or electroporated with (iv–vi) WT, (vii–ix) UL50 null, (x–xii) E56A, or (xiii–xv) E56AR pBADGFP. The cells were fixed on day 7 and stained with antibody against lamin A/C (red), and the nucleus was stained with DAPI (blue). Virus-infected cells were observed for the appearance of the nuclear lamina using confocal microscopy. White arrows point to disruptions of nuclear lamina.

Fig. 3.

Effects of substitutions on distribution of UL53 and disruption of nuclear lamina. HFFs were electroporated with (i–iv) the mutant L130A UL53-FLAG pBADGFP or (v–viii) the rescued derivative L130AR UL53-FLAG pBADGFP. Cells were fixed on day 7, stained with DAPI, and stained with either (A) anti-FLAG antibody (red) or (B) antibody against lamin A/C (green). Cells positive for GFP were visualized by confocal microscopy. In B, the red arrow points to an uninfected cell, and the white arrows point to gaps in nuclear lamina in cells infected with the rescued derivative.