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

Abstract

Nuclear egress is a regulated process shared by α-, β- and γ-herpesviruses. The core nuclear egress complex (NEC) is composed of the membrane-anchored protein homologs of human cytomegalovirus (HCMV) pUL50, murine cytomegalovirus (MCMV) pM50, Epstein–Barr virus (EBV) BFRF1 or varicella zoster virus (VZV) Orf24, which interact with the autologous NEC partners pUL53, pM53, BFLF2 or Orf27, respectively. Their recruitment of additional proteins leads to the assembly of a multicomponent NEC, coordinately regulating viral nucleocytoplasmic capsid egress. Here, the functionality of VZV, HCMV, MCMV and EBV core NECs was investigated by coimmunoprecipitation and confocal imaging analyses. Furthermore, a recombinant MCMV, harboring a replacement of ORF M50 by UL50, was analyzed both in vitro and in vivo. In essence, core NEC interactions were strictly limited to autologous NEC pairs and only included one measurable nonautologous interaction between the homologs of HCMV and MCMV. A comparative analysis of MCMV-WT versus MCMV-UL50-infected murine fibroblasts revealed almost identical phenotypes on the levels of protein and genomic replication kinetics. In infected BALB/c mice, virus spread to lung and other organs was found comparable between these viruses, thus stating functional complementarity. In conclusion, our study underlines that herpesviral core NEC proteins are functionally conserved regarding complementarity of core NEC interactions, which were found either virus-specific or restricted within subfamilies.

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

“…Very central NEC functions are the virus-induced promotion of lamina disassembly, the capsid budding at the inner nuclear membrane (INM), and the recruitment of capsids to sites of nuclear egress (Figure 1). In the case of HCMV, a nuclear rim corecruitment of the NEC core proteins pUL50 and pUL53 has been described in detail and this property is shared by all other core NEC pairs investigated so far ( [8,25] with references therein). As an important feature, the NEC-associated HCMV protein kinase pUL97 interacts with p32/gC1qR, which bridges pUL97 to the pUL50-pUL53 core NEC [54,58].…”

“…As a result of this concerted activity of the multifunctional NEC, viral nuclear capsids are guided to lamina-depleted areas, where they undergo budding and primary envelopment at the inner nuclear membrane (INM). This scheme represents a refined version of a general herpesviral NEC model as analogously published before for the specific case of HCMV [25].…”

“…On the other hand, exchanging H71 and M84 for their D-stereoisomers had a much stronger effect than their replacement with alanine, indicating that changing the side chain orientation of these two amino acids results in peptide conformations that are incompatible of interaction with pUL50. Regarding the nuclear colocalization of core NEC pairs, specific imaging methods focusing on the hook-into-groove rim recruitment have been described by several researchers [9,10,12,21,25,43,48,53,55,[59][60][61][62][63][64][65][66][67][68][69]. Recently, we analyzed and compared the capacities of various NEC pairs of α-, βand γ-herpesviruses to interact in the form of heterodimeric complexes, both in the autologous manner (which was strongly detectable for each individual NEC pair of the virus species analyzed) and a nonautologous, cross-viral manner.…”

“…Members of the subfamilies α-, β- and γ- Herpesvirinae showed marked differences in sequence characteristics. While strains of HCMV showed highly conserved sequences for pUL50 and pUL53 (98.5%–99.5% and 98.4%–100%, respectively), the comparison between HCMVs and primate human cytomegaloviruses (CMVs) or rodent CMVs showed substantially decreasing conservation levels [ 25 ]. Even the comparison with human roseoloviruses (HHV-6A, HHV-6B and HHV-7) underlined the poor core NEC amino acid identities with human CMVs (≤ 25% for pUL50 and < 32% for pUL53).…”

“…Currently, a focus of many researchers is directed at the study of multiprotein complexes assembled between virus and host proteins, so that investigations of the HCMV-specific NEC appear highly relevant in this regard (for methods, see Supplementary Materials [25][26][27][28][29]). Novel information is accumulating for the biochemical and functional properties of NECs, raising the option to define mechanistic ways of a pharmacological interference with herpesviral nuclear egress, which may provide novel targeting strategies for NEC-directed small molecules.…”