Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

Abstract

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase

“…In particular, UL97-mediated phosphorylation of lamin A/C has been posited to play a role in altering the nuclear lamina to promote nuclear egress (15,(32)(33)(34). A defect in nuclear egress largely explains the replication defect of UL97-null mutants in cycling cells (15), which would in turn explain why HPV16 E7 expression does not complement the loss of UL97 in these cells.…”

“…To detect E7, monoclonal antibodies ED19 (57) or ED17 (Santa Cruz Biotechnology), in combination with 8C9 (Invitrogen), were used. pRb, tubulin, UL97, IE1, UL44, and actin were detected as previously described (7,18,33). UL57 and pp28 were detected using mouse monoclonal antibodies (Virusys Inc).…”

Therapeutics to prevent congenital cytomegalovirus during pregnancy: what is available now and in the future?

“…During the nuclear phase of HCMV replication, viral capsids are packaged and exported to the cytoplasm by transition through the nuclear envelope (nuclear egress) for further virion maturation. Nuclear egress is a multi-step regulatory process that involves a phosphorylationtriggered distortion of the nuclear lamina [42][43][44][45] . Pivotal for nuclear egress is the role of the two viral nuclear egress proteins pUL50 and pUL53 that heterodimerise and form a core for the multimeric viralcellular NEC 46 …”

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

“…UL44 is the putative processivity subunit of the HCMV DNA polymerase (Ertl & Powell, 1992), and several different phosphorylated forms of UL44 are present in the infected cell (Silva et al, 2011). At least one form of UL44 accumulates at the periphery of replication compartments throughout HCMV replication, and UL44 is also found at considerably lower levels within certain domains inside replication compartments, plus in the nucleus outside replication compartments and also in the cytoplasm (Hamirally et al, 2009;Penfold & Mocarski, 1997; Strang et al, 2012b). Viral DNA synthesis occurs at foci within the layer of UL44 at the periphery of replication compartments (Fig.…”

“…The viral nuclear egress complex (UL50 and UL53) recruits the viral kinase UL97 to the nuclear lamina in HCMV-infected cells (Sharma et al, 2014) (Table 1). Phosphorylation of the lamina component lamin A/C by UL97 promotes lamina disruption, akin to that observed during cell division, resulting in thinning and the appearance of gaps in the lamina (Hamirally et al, 2009). This should facilitate capsid movement through the lamina to the nuclear membrane.…”