Human cytomegalovirus (HCMV) expresses a variety of viral regulatory proteins that undergo close interaction with host factors including viral-cellular multiprotein complexes. The HCMV protein kinase pUL97 represents a viral CDK ortholog (vCDK) that determines the efficiency of HCMV replication via phosphorylation of viral and cellular substrates. A hierarchy of functional importance of individual pUL97-mediated phosphorylation events has been discussed, however, the most pronounced pUL97-dependent phenotype could be assigned to viral nuclear egress, as illustrated by genetic ORF-UL97 deletion or pharmacological pUL97 inhibition. Despite earlier data pointing to a cyclin-independent functionality, experimental evidence increasingly emphasized the role of pUL97-cyclin complexes. Consequently, the knowledge about pUL97 involvement in host interaction, viral nuclear egress and additional replicative steps led to the postulation of pUL97 as an antiviral target. Indeed, validation experiments in vitro and in vivo confirmed the sustainability of this approach. Consequently, current investigations of pUL97 in antiviral treatment go beyond the known pUL97-mediated ganciclovir prodrug activation and henceforward include pUL97-specific kinase inhibitors. Among a number of interesting small molecules analyzed on experimental and preclinical stages, maribavir is presently investigated in clinical studies and, in the near future, might represent a first kinase inhibitor applied in the field of antiviral therapy.
Property | General Description | Specific Feature | Own Findings (MM lab.) | Various References |
---|---|---|---|---|
Type of kinase | Ser/Thr | target site P + 5, target site LxSP |
[51,53,56,62,63,64] | [65,66,67,68,69] |
Molecular mass, basic features | 100/80/70 kDa | isoforms due to alternative translational start sites | [44,45,54] | [43,52,67] |
Expression pattern | three isoforms M1, M74, M15 (referring to other herpesviral protein isoforms) | differences in substrate binding, nuclear translocation and drug susceptibility | [44,70] | [71,72,73,74] |
Similarity and sequence conservation with other kinases | low | <35% identity with herpesviral kinases, <15% identity with cellular kinases | [45,63] | [48,49,50,75] |
Sequence conservation ORF-UL97 of HCMVs | high | no variation of translational start sites, NLS sequences or kinase domains | [44] | [76,77] |
Related to cell kinases | cyclin-dependent kinases (CDKs), viral CDK ortholog | functional overlap with CDKs, specific crosstalk with CDK9, CDK7 and CDK1, direct interaction with cyclins | [47,55,56,78,79,80,81,82,83] | [57,84,85] |
Coregulation of viral replication by pUL97 and cellular kinases | several novel cellular kinases, including CDKs, identified to be involved in HCMV replication | virus-supporting functions in signaling pathways and nuclear capsid egress | [55,56,86,87] | [88,89,90,91,92,93] |
Substrate proteins | viral, cellular | pUL44, pUL69, pp65, Rb, p32/gC1qR, nuclear lamins, EF-1δ, RNAP II, IFI16, SAMHD1 | [53,79,87,94,95,96,97,98] (references therein) | [57,75,84,99,100,101,102,103,104,105,106] (see also refs. in Figure 1) |
Involvement in intrinsic immunity evasion | stimulation of viral counterdefense of immunity | interaction with cellular restriction factors IFI16 and SAMHD1 | [96,107] | [108] |
Auto-phosphorylation | pronounced auto-phosphorylation activity, several N-terminal Ser and Thr residues | autophosphorylation most probably required for kinase activity/autoactivation | [44,54,56,94,109] | [65,66,110] |
Nucleoside phosphorylation | ganciclovir, valganciclovir, penciclovir, acyclovir, etc. | prodrug-activating monophosphorylation as an essential step in antiviral therapy | [51,111] | [59,112,113,114,115] |
Incorporation into virions | component of virion tegument | virion-derived pUL97 possesses highly detectable kinase activity | [45,95] | [43,116,117] |
Intracellular localization | mainly nuclear | two nuclear localization signals, NLS-1 (6–35), NLS-2 (164–213), classical importin-α pathway | [45,46,63,97,118] | [60] |
Inhibitors of pUL97 | small molecules (<500 Da, various chemical classes) | indolocarbazoles, benzimidazoles, quinazolines, others | [53] (references therein) [44,64,119] | [114,120,121] |
Phenotype of pUL97 inhibition or UL97 deletion | strongly reduced viral replication efficiency (100–1000-fold) | delayed replication kinetics; impaired genomic replication; impaired viral nuclear egress | [44,51,53,94,109,122,123] | [59,61,104,124] |
A HCMV-Infected Cells | B Recombinant Expression | ||||||||
---|---|---|---|---|---|---|---|---|---|
Cyclin Types | Cyclin IP MS|Wb | pUL97 IP MS|Wb | Colocalization in IF | Transfection | Yeast Two-Hybrid System | Phosphorylation by pUL97 in IVKA | |||
B-like | Cyclin A | + | ± | - | - | . | . | . | . |
Cyclin B1 | + | + | + | - | + | + | . | + | |
Cyclin B2 | - | + | - | - | - | . | . | . | |
Cyclin D1 | - | - | - | - | - | - | . | . | |
Cyclin E | ± | ± | - | - | . | . | . | . | |
Cyclin F | . | ± | - | - | . | . | . | . | |
C-like | Cyclin H | + | + | - | - | + | - | - | - |
Cyclin K | . | + | - | - | . | . | . | . | |
Cyclin L2a | . | - | - | - | . | . | . | . | |
Cyclin T1 | + | + | + | + | + | + | + | - | |
Y-like | Cyclin Y | . | ± | - | - | . | . | . | . |
Protein Origin | Designation | Function | Remarks | References |
---|---|---|---|---|
Viral | pUL50 | core nuclear egress protein (NEC) | forms the NEC groove, multiple PPIs, phosphorylated by viral and cellular kinases | [62,99,126,127,134,135] |
Viral | pUL53 | core nuclear egress protein (NEC) | forms NEC hook, possibly docking to capsids, phosphorylated by viral kinase | [99,136,137] |
Viral | pUL44 | DNA polymerase pUL54 processivity factor | phosphorylation might regulate activity | [104,122] |
Viral | pp65 | major tegument protein | massively phosphorylated and virion-associated with pUL97 | [44,60,95] |
Viral | pUL69 | RNA transport regulator | phosphorylation regulates activity | [78,79,138,139] |
Viral | pUL97 | CDK-like serine/threonine protein kinase, multifunctional | dimers/oligomers, autophosphorylation | [50,53,54,65,110,114,132] |
Cellular | p32/gC1qR | multiligand binding protein, multifunctional | NEC bridging factor | [94,98,140] |
Cellular | lamins A/C | structural and regulatory components of the nuclear envelope | lamin phosphorylation is a rate-limiting step of viral nuclear egress | [57,84,94,97,129,141] |
Cellular | Rb | retinoblastoma protein, cell cycle check-point regulator | multiply phosphorylated by CDKs and pUL97 | [48,57,85,103,106] |
Cellular | IFI16 and SAMHD1 | intrinsic immune restriction factors of virus infections | interferon-induced, phosphorylation-controlled | [96,105,107,108,142] |
Cellular | RNAP II | main cellular mRNA transcriptase | activity-regulated by C-terminal phosphorylation (CTD) | [59,61,90,100] |
Cellular | EF-1 | translation elongation factor 1 delta | activity-regulated by phosphorylation | [53,101] |
Cellular | cyclins | regulatory subunits of CDKs | types B1, H, T1 were found pUL97-associated (possibly also B2, K, others) | [55,56,58,84,92] |
Kinase Characteristics | pUL97 | CDK1 | CDK7 | CDK9 |
---|---|---|---|---|
Amino acids (aa) | 707 | 297 | 345 | 372 |
Aa sequence identity to pUL97 | 100% | 4.5% | 4.2% | 8.6% |
Cyclin binding partner [56,146,147] | cyclin B1 cyclin H cyclin T1 |
cyclin A1/A2 cyclin B1/B2/B3 cyclin D1/D3 cyclin F cyclin K (activating) |
cyclin H cyclin A2 cyclin B1/B2 cyclin E (activating) |
cyclin T1/T2 cyclin H cyclin K (activating) |
Region in the kinase required for cyclin binding [55,148,149] | cyclin T1: 231ESQDSAVASGPGRIPQPLSGSSGEESATAVEADSTSHDDVHCTCSNDQII280 and in silico-predicted binding interfaces for cyclins B1, H and T1 spanning aa 328–647 | cyclin B1: a positively charged region in the N-lobe (containing K6, K9, K34, R36, R75, excluding the PSTAIRE helix) cyclin A2: 45PSTAIRE51 |
cyclin H: 56NRTALRE62 | cyclin T1/T2, K: 60PITALRE66 |
Cyclin phosphorylation [56,82,146,150,151,152,153] | cyclin B1 | cyclin B1 S126 by CDK1 S128 by CDK1 | cyclin H by CDK7/CDK8-cyclin C (inhibitory) | n.d.* |
T-loop phosphorylation [56,154,155,156,157,158,159,160,161] | no, (possibly S483) | T161 by CAK (activating) | S164 and T170 by CDK1/CDK2 (activating) | T186 by CaMK1D or CDK9 (S175 by CAK, not essential for activity) |
Autophosphorylation [110,155,156] | yes | no | (yes) outside the T-loop | yes within the T-loop |
Rb phosphorylation [66,75,82,145,162,163] | S780, S807, S811, T821, T823, T826 | S249, T252, T373, S807, S811 | no | C-terminus (793–834) |
p53 phosphorylation [164,165,166] | n.d. | S315 | S33 (MAT1-dependent) | S33, S315, S392 |
Lamin A/C phosphorylation [84,141,167,168] | S22 (inhibitory) | S22, S392 (inhibitory) | no | no |
CTD RNAP II phosphorylation [100,169,170] | S2, S5 (activating) | no | S2, S5, S7 (activating) | S2, S5, S7 (activating) |
SAMHD1 phosphorylation [171,172,173] | yes | T592 | n.d. | n.d. |
HCMV pUL69 phosphorylation [78,79] | yes | yes | yes | yes |
HCMV pUL50 phosphorylation [127] | yes | yes | n.d. | n.d. |
This entry is adapted from the peer-reviewed paper 10.3390/microorganisms8040515