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Interaction of Human Cytomegalovirus pUL52 with major components of the viral DNA encapsidation network underlines its essential role in genome cleavage-packaging

ABSTRACT: Cleavage of human cytomegalovirus (HCMV) genomes and their packaging into capsids requires at least seven essential viral proteins, yet it is not completely understood how these proteins cooperate to accomplish this task. Besides the portal protein pUL104 and the terminase subunits pUL51, pUL56, and pUL89, the UL52 protein is also necessary for HCMV genome encapsidation, however, knowledge about pUL52 is scant. In the absence of pUL52, viral concatemers are not cleaved into unit-length genomes and no DNA-filled capsids are observed, yet no viral or cellular proteins interacting with pUL52 have been identified that would explain how pUL52 exerts its essential role in the HCMV infection cycle. In this study, we aimed at a comprehensive definition of pUL52-interacting proteins in infected cells. Using suitable HCMV mutants, we employed three complementary state-of-the-art proteomic approaches, namely biotin ligase-dependent proximity labeling, affinity purification and cross-linking mass spectrometry. These experiments, combined with thorough validation by immunoblotting, pointed to several viral DNA-associated proteins and key players pivotal for genome encapsidation as interactors of pUL52. The most noticeable direct pUL52 interaction partners were the terminase subunits pUL56 and pUL89 as well as the portal protein pUL104. Hence, we suggest a model of pUL52 function in which pUL52 mediates association of HCMV genomes with the terminase subunits and the capsid portal. Taken together, our data contribute to the understanding of an essential viral process previously recognized as a prominent antiviral target. Disturbing the identified pUL52 interactions may provide a starting point to develop novel antiviral medication.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Human Cytomegalovirus (hhv-5) (human Herpesvirus 5)

TISSUE(S): Foreskin Fibroblast Cell Line, Fibroblast

SUBMITTER: Boris Bogdanow

LAB HEAD: Fan Liu

PROVIDER: PXD060825 | Pride | 2025-02-17

REPOSITORIES: pride

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			Action	DRS
	F1_20210121_BB_EB_HA_52B2_1.raw	Raw
	F1_20210121_BB_EB_HA_52B2_2.raw	Raw
	F1_20210121_BB_EB_HA_52B2_3.raw	Raw
	F1_20210121_BB_EB_HA_HG_1.raw	Raw
	F1_20210121_BB_EB_HA_HG_2.raw	Raw

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Project description:We have established that human cytomegalovirus (HCMV) infection modulates the biology of target primary blood monocytes, allowing HCMV to use monocytes as 'vehicles' for its systemic spread. HCMV infection of monocytes results in rapid induction of PI(3)K and NF-kB activity. Integrins, which are upstream of the PI(3)K and NF-kB pathways, were shown to be involved in HCMV binding to and entry into fibroblasts, suggesting that receptor-ligand-mediated signaling following viral binding to integrins on monocytes could trigger the functional changes seen in infected monocytes. We now show that integrin engagement and the activation of the integrin/Src-signaling pathway is essential for the induction of HCMV-infected monocyte motility. To investigate how integrin engagement by HCMV triggers monocyte motility, we examined the infected monocyte transcriptome and found that the integrin/Src-signaling pathway regulates the expression of paxillin, which is an important signal transducer in the regulation of actin rearrangement during cell adhesion and movement. Functionally, we observed that paxillin is activated via the integrin/Src-signaling pathway and is required for monocyte motility. Because motility is intimately connected to cellular cytoskeletal organization, a process that is also important in viral entry, we investigated the role paxillin regulation plays in the process of viral entry of monocytes. New results confirmed that HCMV`s ability to enter target monocytes is significantly inhibited in cells deficient in paxillin expression or that had their integrin/Src/paxillin signaling pathway blocked. From our data, HCMV-cell interactions emerge as an essential trigger for the cellular changes that allow for HCMV entry and hematogenous dissemination. Monocytes were mock-infected, HCMV-infected, or pretreated with PP2 inhibitor prior to HCMV infection. There were three samples analyzed per individual replicate. Three replicates are included. comparative studies with a use of the specific Src kinase activity inhibitor

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Interaction of Human Cytomegalovirus pUL52 with major components of the viral DNA encapsidation network underlines its essential role in genome cleavage-packaging

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