Properties of Oligomeric Interaction of the Cytomegalovirus Core Nuclear Egress Complex (NEC) and Its Sensitivity to an NEC Inhibitory Small Molecule

Abstract

Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural–functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50–pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

“…4B, panels 5-8), and likewise HSV-1 pUL31 was recruited by VZV Orf24 to this rim colocalization (panels 9-12). The converse setting, using VZV Orf27 combined with HSV-1 pUL34 (panels [29][30][31][32], provided an almost identical rim staining pattern. However, all other combinations between VZV Orf24 or Orf27 with βor -herpesviral NEC protein counterparts lacked a comparable rim staining pattern.…”

Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors

“…In a related approach, the antiviral activity of merbromin was investigated by testing its effect on the formation of cytoplasmic viral assembly complexes (cVACs), which correlates with the efficiency of viral nuclear egress. Three different human cell types, i.e., MRC-5, ARPE-19, and HFF, were infected with three different strains of HCMV, i.e., TB40 UL32-GFP, TB40E, and AD169 (Table 1) [11]. In this assay, merbromin was found to dose-dependently reduce the cVAC count for all three viral strains, further substantiating the inhibitory effect of merbromin on HCMV nuclear egress.…”

“…b Three different human cell types and three different viral strains were used as follows: MRC-5, human lung fibroblasts reaching senescence after 45-60 passages; ARPE-19, human retinal epithelial cells; HFF, primary human foreskin fibroblasts; HCMV, recombinant TB40 UL32-GFP fibroblast-adapted; TB40E, epithelial cell-adapted strain; AD169, fibroblast-adapted laboratory reference strain. For primary data, see [11].…”

“…The formation of the core nuclear egress complex (core NEC), composed of viral proteins pUL50 and pUL53, which is anchored to the inner nuclear membrane, is a central component of nuclear egress, as it serves as a scaffold for the assembly of a multimeric NEC composed of viral and cellular proteins [8,9]. Extensive structural analysis of the pUL50-pUL53 interaction, through x-ray crystallography of the protein complex, has provided detailed information on the structural features of the proteins, as well as the protein interface [10][11][12][13][14]. While both proteins adopt a globular fold with mixed secondary structure elements, the hallmark element of the pUL50-pUL53 interaction is an N-terminal α-helical, hook-like extension in pUL53, which is independent of the overall globular protein fold, and contributes approximately 80% to the total interface area with pUL50 [10].…”

“…While both proteins adopt a globular fold with mixed secondary structure elements, the hallmark element of the pUL50-pUL53 interaction is an N-terminal α-helical, hook-like extension in pUL53, which is independent of the overall globular protein fold, and contributes approximately 80% to the total interface area with pUL50 [10]. Interestingly, the pUL50-pUL53 heterodimeric complex partially assembles into hexameric ring-like structures [10], and is also able to oligomerize in vitro [11]. We have recently shown that a 29-mer synthetic peptide presenting the pUL53 N-terminal hook specifically interacts with soluble pUL50, i.e., it does not bind to BFRF1, which is the Epstein-Barr virus homolog of pUL50 [12,13].…”