Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus

Abstract

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting

“…This region overlaps with a minimal binding region for cyclin T1. Recent modeling approaches based on the in silico prediction of binding interfaces suggested extended binding interfaces for cyclins T1, B1 and H. Moreover, pUL97 is involved in the multiple regulatory steps during HCMV replication through the phosphorylation of viral and cellular substrates (see horizontal bars), as reported by several independent groups [44][45][46]54,55,57,75,[79][80][81][82][83][84][94][95][96][97][98][99][100][101]122,[126][127][128][129][130][131][132]. Substrates include the viral DNA polymerase cofactor pUL44, viral RNA transport factor pUL69, major tegument protein pp65, nuclear egress core protein heterodimer pUL50-pUL53, cellular multi-ligand binding protein p32/gC1qR, tumor suppressor/checkpoint protein Rb, nuclear lamins A/C, RNA polymerase II, translation factor EF-1δ, interferon-inducible proteins IFI16 and SAMHD1, as well as the therapeutically applied nucleoside analog ganciclovir (GCV; [47,56,82] and references therein).…”

“…Notably, pUL97 phosphorylates several viral and cellular proteins (see horizontal bars in Figure 1 for those binding regions within pUL97 that have been mapped thus far), including the viral DNA polymerase cofactor pUL44 [122], viral RNA transport factor pUL69 [79], major tegument protein pp65 [95], nuclear egress core proteins pUL50-pUL53 [99,127], cellular multiligand binding protein p32/gC1qR [98,122], tumor suppressor protein Rb [75], nuclear lamins A/C [57,84,94,98,129], RNAP II [100], translation factor EF-1δ [63,101,130], interferon-inducible, intrinsic immune restriction factors IFI16 [96] and SAMHD1 [105] (Figure 2; Table 3; compare with Tables S1, S2 and S3). It should be emphasized that the pUL97 substrate proteins belong to several functionally different groups (Table 3), thus underlining the multifunctional nature of this singly expressed viral protein kinase.…”

Patterns of Autologous and Nonautologous Interactions between Core Nuclear Egress Complex (NEC) Proteins of α-, β- and γ-Herpesviruses

“…Initially, pUL50 (HCMV) or its homologs in MCMV, EBV and VZV (pM50, BFRF1 or Orf24, respectively), anchored within the inner side of the NE, interacts with the nucleoplasmic cofactor pUL53 (pM53, BFLF2 or Orf27, respectively). The recruitment of several cellular and viral proteins, like protein kinases (pUL97, PKC, CDK1 and possibly others in case of HCMV; [1,2]), by the core NEC leads to the formation of the multicomponent NEC. Site-specific phosphorylation of the nuclear lamins by NEC-associated kinases results in massive rearrangement of the NE and particularly the formation of lamina-depleted areas (LDAs), the sites where viral nuclear capsids gain access to the NE [2][3][4][5].…”

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

“…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].…”