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9.A.82. The Viral Nuclear Egress Complex (V-NEC) Family 

This system plays an essential role in virion nuclear egress, the first step of virion release from infected cell. Within the host nucleus, NEC1 interacts with the newly formed capsid through the vertexes and directs it to the inner nuclear membrane by associating with NEC2. The former induces the budding of the capsid at the inner nuclear membrane as well as its envelopment into the perinuclear space. There, the NEC1/NEC2 complex promotes the fusion of the enveloped capsid with the outer nuclear membrane and the subsequent release of the viral capsid into the cytoplasm where it will reach the secondary budding sites in the host Golgi or trans-Golgi network.

Herpesviral nuclear egress is a regulated process of viral capsid nucleocytoplasmic release. Due to the large capsid size, regular transport via the nuclear pores is not feasible, so that a multistage-regulated export pathway through the nuclear lamina and both leaflets of the nuclear membrane has evolved. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. The transmembrane NEC protein pUL50 serves as a multi-interacting determinant that recruits regulatory proteins by direct and indirect contacts. The nucleoplasmic core NEC component pUL53 is strictly associated with pUL50 in a structurally defined hook-into-groove complex and is considered as the potential capsid-binding factor. Tillmanns et al. 2023 validated the concept of blocking the pUL50-pUL53 interaction by small molecules as well as cell-penetrating peptides or an overexpression of hook-like constructs, which can lead to a pronounced degree of antiviral activity. More recently, they extended this strategy by utilizing covalently binding warhead compounds, originally designed as binders of distinct cysteine residues in target proteins, such as warheads that target viral NEC proteins. The findings were as follows: (i) warhead compounds exhibited a pronounced anti-HCMV potential in cell-culture-based infection models; (ii) computational analysis of NEC primary sequences and 3D structures revealed cysteine residues exposed to the hook-into-groove interaction surface; (iii) several of the active hit compounds exhibited NEC-blocking activity, as shown at the single-cell level by confocal imaging; (iv) the clinically approved warhead drug ibrutinib exerted a strong inhibitory impact on the pUL50-pUL53 core NEC interaction, as demonstrated by the NanoBiT assay system; and (v) the generation of recombinant HCMV ∆UL50-SigmaUL53, allowing the assessment of viral replication under conditional expression of the viral core NEC proteins, was used for characterizing viral replication and a mechanistic evaluation of ibrutinib antiviral efficacy. Combined, the results point to a rate-limiting importance of the HCMV core NEC for viral replication and to the option of exploiting this determinant by the targeting of covalently NEC-binding warhead compounds (Tillmanns et al. 2023).

The nuclear egress complex (NEC) is required for efficient translocation of newly synthesized herpesvirus nucleocapsids from the nucleus to the cytosol. It consists of the type II membrane protein pUL34 which interacts with pUL31 at the inner nuclear membrane (INM). To map regions within pUL34 required for nuclear membrane targeting and pUL31 interaction, Passvogel et al. (2013) constructed deletion/substitution mutations.  50 C-terminal amino acids (aas) of pseudorabies virus (PrV) pUL34, including the transmembrane domain, could be functionally replaced by cellular lamina-associated polypeptide 2beta (Lap2beta) sequences. In contrast, replacement of the C-terminal 100 aa abrogated complementation but not the pUL31 interaction. To further delineate essential sequences within this region, C-terminal pUL34 truncations of 60, 70, 80, 85, and 90 aa fused to Lap2beta sequences were generated. While truncations up to 85 aa were functional, deletion of the C-terminal 90 aa abrogated function, which indicates that the important region is located between aas 171 and 176. Amino acids 173 to 175 represent RQR, a motif suggested to mediate INM targeting. Mutagenesis to RQG revealed that the mutant protein exhibited pronounced Golgi localization, but a fraction still reached the INM. Deletion mutations in the N-terminal domain of pUL34 demonstrated that absence of the first 4 aa was tolerated, while removal of 9 or more residues resulted in a nonfunctional protein. In addition, mutation of three conserved cysteines did not abrogate pUL34 function, whereas alteration of a conserved glutamine/tyrosine sequence yielded a nonfunctional protein (Paßvogel et al. 2013).

References associated with 9.A.82 family:

Paßvogel, L., P. Trübe, F. Schuster, B.G. Klupp, and T.C. Mettenleiter. (2013). Mapping of sequences in Pseudorabies virus pUL34 that are required for formation and function of the nuclear egress complex. J. Virol. 87: 4475-4485. 23388710
Tillmanns, J., S. Häge, E.M. Borst, J. Wardin, J. Eickhoff, B. Klebl, S. Wagner, C. Wangen, F. Hahn, E. Socher, and M. Marschall. (2023). Assessment of Covalently Binding Warhead Compounds in the Validation of the Cytomegalovirus Nuclear Egress Complex as an Antiviral Target. Cells 12:. 37190072