High-resolution crystal structures of two prototypical β- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity

Abstract

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1–BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these β- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.

Patterns of Autologous and Nonautologous Interactions between Core Nuclear Egress Complex (NEC) Proteins of α-, β- and γ-Herpesviruses

“…Our initial data derived from coexpression experiments with domain swap constructs, in which distinct parts of the hook element were artificially exchanged between HCMV and EBV (pUL53::BFLF2 fusions), indicated that very little variability in the hook element is acceptable for retaining a detectable level of high-affinity interaction with the respective groove proteins. In most cases of domain swap, the autologous hook element lost reactivity in NEC interaction [16]. A third aspect considered as potentially facilitating nonautologous NEC interaction was seen in the recruitment of a number of NEC-associated host proteins, a property known for all herpesviral NECs analyzed so far.…”

“…Furthermore, a colocalization of HSV-1 proteins pUL34 and pUL31 with VZV Orf24 and Orf27, respectively, was detectable ( Figure 4D, d and h). In contrast, nonautologous combinations between homologs of the different herpesviral subfamilies did not develop colocalization ( Figure 4D, u and y; Figure 4E) [16]. The nonautologous colocalization between HCMV and MCMV nuclear egress proteins was additionally quantitated.…”

“…The X-ray-based structures of four different αand β-herpesviral core NECs have been resolved by independent groups [8,12,14,15]. Very recently, we reported the first structure of γ-herpesviral core NEC, namely the 1.75 Å structure of Epstein-Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53 [16]. A biochemical comparative analysis of the βand γ-herpesviral NECs characterized the unique hook-into-groove NEC interaction by applying several different experimental approaches [16].…”

“…Very recently, we reported the first structure of γ-herpesviral core NEC, namely the 1.75 Å structure of Epstein-Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53 [16]. A biochemical comparative analysis of the βand γ-herpesviral NECs characterized the unique hook-into-groove NEC interaction by applying several different experimental approaches [16]. However, there is still little information, to which extent the functionality of the core NEC, in terms of binding activities, crossviral complementarity and intranuclear transport, is conserved.…”

Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties

“…Regarding the currently available data on core NEC structures, four 3D crystal structures have been published, i.e., those of HCMV, HSV-1, PRV and EBV [16,18,20,[22][23][24]. Many structural properties of core NEC proteins were found conserved and qualitatively mostly consistent.…”