Human cytomegalovirus UL97 kinase alters the accumulation of CDK1

Abstract

The UL97 protein kinase is a serine/threonine kinase expressed by human cytomegalovirus (CMV) that phosphorylates ganciclovir. An investigation of the subcellular localization of pUL97 in infected cells indicated that, early in infection, pUL97 localized to focal sites in the nucleus that transitioned to subnuclear compartments and eventually throughout the entire nucleus. When UL97 kinase activity was eliminated with a K355M mutation or pharmacologically inhibited with maribavir, the expansion and redistribution of pUL97 foci within the nucleus was delayed, nuclear reorganization did not occur and assembly complexes in the cytoplasm failed to form normally. As UL97 kinase and its homologues appear to be functionally related to CDK1, a known regulator of nuclear structural organization, the effects of the UL97 kinase on CDK1 were investigated. Expression of CDK1 in infected cells appeared to be induced by UL97 kinase activity at the level of transcription and was not tied to other virus life-cycle events, such as viral DNA replication or virion assembly. These results suggest that, in addition to phosphorylating CDK1 targets, the UL97 kinase modifies G 2 /M cell-cycle checkpoint regulators, specifically CDK1, to promote virus replication.

Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

“…The important finding that pUL97 is able to phosphorylate CDK substrates in vitro and in cell-based assays confirms this idea at the functional level [13,26,27,28,35,62,63,64]. The first experimental evidence that pUL97 interacts with human cyclins was provided by Graf et al, (2013); [58], showing that pUL97-cyclin T1 can be coimmunoprecipitated from cotransfected as well as HCMV-infected cells, that interaction can be detected in a yeast two-hybrid assay and that pUL97 colocalizes with cyclin T1 in nuclear compartments.…”

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

“…Primary mouse monoclonal Abs used included anti-lamin A/C (636 [IgG2B]; Santa Cruz Biotechnology), anti-lamin A (133A2 [IgG3]; Abcam), anti-UL97 (IgG2A; a kind gift from Dr. Mark Prichard, University of Alabama School of Medicine, Birmingham)(Gill et al, 2012; Milbradt et al, 2014), anti-UL44 (1202S [IgG1]; Goodwin Institute), anti-UL53 (IgG1; a kind gift from Dr. Stipan Jonjic, University of Rijeka, Croatia)(Milbradt et al, 2014), anti-MCP (a kind gift from Dr. Bill Britt, University of Alabama, Birmingham) (Lai and Britt, 2003; Sanchez et al, 2000), anti-Pan Actin (Ab-5; NeoMarkers) and anti-Flag (9A3 [IgG1]; Cell Signaling). Rabbit Abs used included anti-UL50 (a kind gift from Dr. James Alwine, University of Pennsylvania, Philadelphia) (Buchkovich et al, 2010; Milbradt et al., 2014; Schmeiser et al, 2013), anti-Phospho-Rb (Ser780; 9307; Cell Signaling), anti-HA (C29F4; Cell Signaling), anti-phospho-lamin A/C (Ser22; Cell Signaling) and anti-lamin B1 (Proteintech).…”

Inhibition of cellular STAT3 synergizes with the cytomegalovirus kinase inhibitor maribavir to disrupt infection

“…The antibodies against hCMV proteins were anti-pUL123 1B12, anti-pUL37 2A1D, and anti-pUL38 3D12 (generously provided by Dr. Tom Shenk), anti-pUL97 4-1 (Gill et al, 2012) (generously provided by Dr. Mark Prichard) and anti-pUL44 (Virusys). Secondary antibodies include anti-mouse and anti-rabbit HRP (Jackson ImmunoResearch).…”