Antagonistic Relationship between Human Cytomegalovirus pUL27 and pUL97 Activities during Infection

Abstract

Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclindependent kinase (CDK) inhibitor p21Cip1 . In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97 and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21Cip1 . Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibition of the kinase resulted in elevated levels of p21 Cip1 in wild-type virus but not a pUL27-deficient virus. We manipulated the p21 Cip1 levels to evaluate the functional consequence to MBV. Overexpression of p21 Cip1 restored MBV activity against a pUL27-deficient virus, while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21Cip1 in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C, resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered the nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21Cip1 and support the idea that CDKs can complement some activities of pUL97. IMPORTANCEHCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist, yet they exhibit high levels of toxicity, are not approved for use during pregnancy, and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21Cip1 . In contrast, we provide evidence that p21 Cip1 functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21Cip1 activators might be useful in combination with MBV to effectively inhibit HCMV infections. H uman cytomegalovirus (HCMV) infects the majority of the world's population (1). Infection of immunocompetent children and adults is usually asymptomatic or associated with minor disease. In contrast, HCMV infection in immunocompromised patients results in serious disease, especially in organ transplant recipients receiving immunosuppressants (2). HCMV is also the leading congenital infection in the developed world (3). Currently, the...

Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components

“…R25 blocks activity of CDK1, CDK2 and CDK5) (Sandal et al, 2002;Soni & Jacobberger, 2004). Consistent with our data, R25 treatment has been shown to reduce viral titres by approximately 1.2 log units, even at a much lower dose of 150 nM (Bigley et al, 2015). The identification of the CDK1-pUL50 interaction by CoIP analysis in the present study further suggests that at least CDK1 is directly involved in core NEC formation.…”

Network mechanisms and dysfunction within an integrated computational model of progression through mitosis in the human cell cycle

“…Studies by Hamirally and Kamil et al [52] have demonstrated that phosphorylation of LMNA contributes to nuclear egress of HCMV particles. The antiviral compound, maribavir inhibits this process and its antiviral activity is further supported by increased steady-state levels of CDKN1A (p21 CIP1 ) ( Fig 6C) [51,54,70]. It is important to note that the kinetics of mitotic entry are regulated by the HCMV protein pUL21a which suppresses Cyclin A levels ( Fig 9A) [55].…”

“…This state includes disruption of the nuclear lamina by and k 0 f 11 are high, respectively. https://doi.org/10.1371/journal.pcbi.1007733.g008 phosphorylation of LMNA by both HCMV pUL97 kinase and MPF kinase (CCNB1:CDK1) which is required for virion particle nuclear egress [51][52][53][54]. Disrupting these kinases is the defined mechanism of action for the antiviral, Maribavir [52,54].…”

“…https://doi.org/10.1371/journal.pcbi.1007733.g008 phosphorylation of LMNA by both HCMV pUL97 kinase and MPF kinase (CCNB1:CDK1) which is required for virion particle nuclear egress [51][52][53][54]. Disrupting these kinases is the defined mechanism of action for the antiviral, Maribavir [52,54]. Entry of the infected cell into mitosis is regulated HCMV pUL21a which limits Cyclin A levels and subsequently CCNB1 levels ( Fig 9A) [55].…”

Human Cytomegalovirus Can Procure Deoxyribonucleotides for Viral DNA Replication in the Absence of Retinoblastoma Protein Phosphorylation

“…Thus, in UL27-positive cells, p21 levels are high, cellular Cdks are inhibited, and Rb is phosphorylated during infection only if UL97 is present and active. In UL27-negative cells, p21 levels are low (54), and as a result, cellular Cdks are active and able to phosphorylate Rb. Rb phosphorylation by cellular Cdks or UL97 can be easily distinguished by monitoring the residues serine 249 and threonine 252, which are phosphorylated by cellular Cdks but not UL97 (2).…”