Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

Thorsen, Michael; Lai, Alex L.; Lee, Michelle W.; Hoogerheide, David P.; Wong, Gerard C. L.; Freed, Jack H.; Heldwein, Ekaterina E.

doi:10.1128/mbio.01548-21

Cited by 18 publications

(45 citation statements)

References 141 publications

(223 reference statements)

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“…Consequently, site-specific Ser22-phosphorylated lamins are recognized by the cellular peptidyl-prolyl cis/trans isomerase Pin1, which isomerizes the lamins towards conformational reorganization and, thus, disassembles the lamina network to provide the capsids access to the INM [ 26 , 41 , 93 ]. Concerning the membrane-specific effects of the nuclear egress pathway, several highly valuable model systems have been established, in particular representing α-herpesvirus infections [ 6 , 7 , 94 , 95 , 96 ]. While the step toward the distinct recruitment of viral capsids to the NE is still insufficiently understood, several studies investigated the budding process in which core NEC proteins are involved by inducing membrane fission.…”

Section: Main Functionality and Regulatory Roles Shared By Herpesvira...mentioning

confidence: 99%

“…For HSV-1 and HCMV, it has been demonstrated that their heterodimeric core NECs can oligomerize to form hexameric rings, which may associate to honeycomb-like lattices and, thus, enable a membrane curvature and, subsequently, the budding of capsids [ 33 , 72 ]. In addition, specific in vitro experiments demonstrated that HSV-1 core NEC proteins are sufficient to induce a negative membrane curvature, which is an essential prerequisite for viral scission and budding [ 64 , 96 ].…”

Section: Main Functionality and Regulatory Roles Shared By Herpesvira...mentioning

confidence: 99%

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‘Come together’—The Regulatory Interaction of Herpesviral Nuclear Egress Proteins Comprises Both Essential and Accessory Functions

Häge

Marschall

2022

Cells

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Herpesviral nuclear egress is a fine-tuned regulatory process that defines the nucleocytoplasmic release of viral capsids. Nuclear capsids are unable to traverse via nuclear pores due to the fact of their large size; therefore, herpesviruses evolved to develop a vesicular transport pathway mediating the transition across the two leaflets of the nuclear membrane. The entire process involves a number of regulatory proteins, which support the local distortion of the nuclear envelope. In the case of the prototype species of β-Herpesvirinae, the human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the core proteins pUL50 and pUL53 that oligomerize, form capsid docking lattices and mediate multicomponent assembly with NEC-associated viral and cellular proteins. The NEC-binding principle is based on the hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. Thus far, the function and characteristics of herpesviral core NECs have been well studied and point to the groove proteins, such as pUL50, as the multi-interacting, major determinants of NEC formation and egress. This review provides closer insight into (i) sequence and structure conservation of herpesviral core NEC proteins, (ii) experimentation on cross-viral core NEC interactions, (iii) the essential functional roles of hook and groove proteins for viral replication, (iv) an establishment of assay systems for NEC-directed antiviral research and (v) the validation of NEC as putative antiviral drug targets. Finally, this article provides new insights into the conservation, function and antiviral targeting of herpesviral core NEC proteins and, into the complex regulatory role of hook and groove proteins during the assembly, egress and maturation of infectious virus.

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Section: Main Functionality and Regulatory Roles Shared By Herpesvira...mentioning

confidence: 99%

Section: Main Functionality and Regulatory Roles Shared By Herpesvira...mentioning

confidence: 99%

‘Come together’—The Regulatory Interaction of Herpesviral Nuclear Egress Proteins Comprises Both Essential and Accessory Functions

Häge

Marschall

2022

Cells

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“…Data of the present study indicate that formation of dimers, hexamers and higher oligomers may represent a strong intrinsic assembly property of the viral core NEC that may be significant for later steps of binding activities and regulation. Specifically, the NEC association of protein kinases, such as vCDK/pUL97, CDK1, CDK2, protein kinase C (PKC) and possibly even more, is another important characteristic shared between herpesviral NECs that leads to the phosphorylation-driven regulatory activities of the multicomponent extensions of these complexes [ 8 , 9 , 10 , 12 , 13 , 19 , 36 , 49 ]. Some of these events of site-specific phosphorylation of NEC-associated proteins have been functionally described, such as lamin A/C pSer22 phosphorylation [ 7 , 20 , 38 ], while others remain to be further investigated, such as the phosphorylation of core NEC proteins themselves, p32/gC1qR phosphorylation and the association of a variety of further phosphoproteins [ 4 ].…”

Section: Discussionmentioning

confidence: 99%

“…This process of site-specific lamin phosphorylation is a very typical and common property of herpesviral NEC activities [ 4 , 7 , 8 ]. Beyond this, the phosphorylation of further NEC-relevant proteins is also an important hallmark of nuclear egress [ 9 , 10 , 11 , 12 ]. In our recent study, we demonstrated the oligomeric interaction of the HCMV core NEC proteins, a finding that directly referred to the hexameric NEC assemblies identified by our crystallization-based structural analyses [ 13 , 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors

Kicuntod

Häge

Hahn

et al. 2022

Viruses

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The nucleo-cytoplasmic capsid egress of herpesviruses is a unique regulated process that ensures the efficiency of viral replication and release. For human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50–pUL53 heterodimer that is able to oligomerize and thus to build hexameric lattices. These structures determine capsid binding and multicomponent protein interaction including NEC-associated host factors. The underlying characteristic of the core NEC formation is based on the N-terminal hook structure of pUL53 that binds into an alpha-helical groove of pUL50, and is thus described as a hook-into-groove interaction. This central regulatory element has recently been validated as a target of antiviral strategies, and first NEC-targeted prototypes of inhibitory small molecules were reported by our previous study. Here, we further analyzed the oligomerization properties of the viral NEC through an approach of chemical protein cross-linking. Findings were as follows: (i) a cross-link approach demonstrated the oligomeric state of the HCMV core NEC using material from HCMV-infected or plasmid-transfected cells, (ii) a Western blot-based identification of NEC-associated kinases using the cross-linked multicomponent NECs was successful, and (iii) we demonstrated the NEC-inhibitory and antiviral activity of specific inhibitors directed to these target kinases. Combined, the results strongly underline the functional importance of the oligomerization of the HCMV-specific NEC that is both phosphorylation-dependent and sensitive to antiviral kinase inhibitors.

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“…Being too large to egress via nuclear pores, capsids leave by budding into and then out of the perinuclear space, the former step being catalysed by the herpesvirus nuclear egress complex (NEC) [65]. A recent study identified that the NEC induces lipid ordering to generate the negative curvature required for capsid budding [66]. It is tempting to speculate that the accelerated replication kinetics of pUL21 V382E HSV-1 arises, at least in part, from an increased rate of nuclear capsid egress owing to increased Cer abundance.…”

Section: Discussionmentioning

confidence: 99%

Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT

Benedyk

Connor

Caroe

et al. 2022

Preprint

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Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide transport protein CERT. CERT mediates non-vesicular transport of ceramide from the ER to the trans-Golgi network, whereupon ceramide is converted to sphingomyelin and other sphingolipids that play important roles in cell proliferation, cell signalling and membrane trafficking. Using click chemistry to profile the kinetics of sphingolipid metabolism in cultured cells, we show that pUL21-mediated dephosphorylation activates CERT and increases the rate of ceramide to sphingomyelin conversion. Purified pUL21 and full-length CERT interact with sub-micromolar affinity and we map the domains responsible for the interaction. Solving the solution structure of the pUL21 C-terminal domain in complex with the CERT PH and START domains using small-angle X-ray scattering allows us to identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. Sphingolipid profiling demonstrates that ceramide to sphingomyelin conversion is severely diminished in the context of HSV-1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. The accumulation of ceramide in the pUL21 mutant virus infection is correlated with accelerated virus replication, illuminating how HSV-1 manipulates lipid metabolism via altering protein phosphorylation to fine-tune its replication.

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Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

Cited by 18 publications

References 141 publications

‘Come together’—The Regulatory Interaction of Herpesviral Nuclear Egress Proteins Comprises Both Essential and Accessory Functions

‘Come together’—The Regulatory Interaction of Herpesviral Nuclear Egress Proteins Comprises Both Essential and Accessory Functions

The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors

Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT

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