Biochemical, Biophysical, and Mutational Analyses of Subunit Interactions of the Human Cytomegalovirus Nuclear Egress Complex

Abstract

Nuclear egress, the trafficking of herpesvirus nucleocapsids from the nucleus to the cytoplasm, involves two conserved viral proteins that form a complex at the nuclear envelope, referred to as the nuclear egress complex. In human cytomegalovirus, these two proteins are called UL50 and UL53. To study UL50 and UL53 in molecular detail, these proteins were expressed in bacteria and purified. To obtain highly expressed, pure proteins, it was necessary to truncate both constructs based on sequence conservation and predicted secondary structural elements. Size exclusion chromatography and analytical ultracentrifugation studies indicated that the truncated form of UL50 is a monomer in solution, that the truncated form of UL53 is a homodimer, and that, when mixed, the two proteins form a heterodimer. To identify residues of UL53 crucial for homodimerization and for heterodimerization with UL50, we constructed and expressed mutant forms of UL53 containing alanine substitutions in a predicted helix. Isothermal titration calorimetry was used to measure the binding affinities of the UL53 mutants to UL50. UL53 residues, the replacement of which reduced binding to UL50, form a surface on one face of the predicted helix. Moreover, most of the substitutions that reduce UL53-UL50 interactions also reduced homodimerization. Substitutions that reduced the interaction between UL50 and UL53 in vitro also reduced colocalization of full-length UL50 and UL53 at the nuclear rim in transfected cells. These results demonstrate direct protein-protein interactions between these proteins that are likely to be mediated by a helix, and they have implications for understanding nuclear egress and for drug discovery.The delivery of newly assembled herpes capsids from the nucleus to the cytoplasm is a process known as nuclear egress (12). This process is still rather poorly understood. Although it is generally recognized that early steps of nuclear egress entail nucleocapsids' gaining access to the inner nuclear membrane, followed by primary envelopment of the nucleocapsids and budding through the inner nuclear membrane, there are currently three models for the remaining steps (13,14). Even the early steps in nuclear egress are not understood mechanistically although the formation of a specific nuclear egress complex (NEC) (10,11,19,21) has been shown to be crucial. In alphaherpesviruses such as herpes simplex virus (HSV) and pseudorabies virus (PRV), the NEC consists of two proteins called UL34 and UL31 (11). Throughout the alpha, beta, and gamma subfamilies of herpesviruses, highly conserved homologs of these two proteins are found to form an NEC at the nuclear rim in all studied cases (1,4,5,8,10,(17)(18)(19)25). Remarkably, in transfected cells, the PRV NEC, without any other viral proteins, is capable of remodeling the nuclear membrane to form vesicles that resemble the primary enveloped particles observed during productive infection (7).The human cytomegalovirus (HCMV) NEC is composed of UL50 and UL53. UL50 of HCMV was predicted to have a p...

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

“…Specific Structural Determinants Accomplish a Tight Interlocking of the pUL50-pUL53 Heterodimer-pUL50 and pUL53 readily form a complex when mixed in solution (38). The crystal structure reveals the hallmarks of the interaction and at the same time corroborates previous findings from mutagenesis experiments (14,38).…”

“…The crystal structure reveals the hallmarks of the interaction and at the same time corroborates previous findings from mutagenesis experiments (14,38). In the pUL50-pUL53 complex, each protein contributes as much as 1880 Å 2 of solvent-accessible surface area to the protein interface.…”

“…Tertiary and Quaternary Arrangements of the pUL50-pUL53 Heterodimers Are Important for Providing a Scaffold Structure for Multimeric NEC Assembly-The fragments of pUL50 and pUL53 used in this study readily form heterodimers in vitro (data not shown) (38). In the crystals, however, we observe even larger symmetric assemblies, namely hexameric ring-like structures that result from the application of the P6 crystal symmetry to the pUL50-pUL53 heterodimer present in the crystallographic asymmetric unit (Fig.…”

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

“…Previous studies indicated that the interaction interface on M53 and UL53 includes a predicted amphipathic helical segment interrupted by a proline comprising residues 60-82 (Fig. S4A) and requires both hydrophobic residues and charged residues within this predicted helix (17,30). We speculate that at least part of this helical segment fits within the groove in such a way as to allow burying of bulky hydrophobic sidechains present in the peptide (Fig.…”

“…To gain insight into this interaction, we hypothesized that the strong sequence conservation between both subunits of the HCMV and MCMV NECs would allow M50 to bind a peptide derived from a region of HCMV UL53 that was previously shown to be important for heterodimer formation (Figs. S1B and S4A) (30). We titrated this synthetic UL53 peptide into 15 N-labeled M50 (1-168) and monitored the binding using a series of 1 H-15 N heteronuclear single-quantum coherence (HSQC) experiments.…”