Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture, HCMV Infection and Transient Transfection
2.2. Polyclonal Antisera and Monoclonal Antibodies
2.3. Coimmunoprecipitation (CoIP)
2.4. Quality Control of Purified Samples
2.5. Lentiviral Transduction and Selection of Stably Transduced HFFs
2.6. Proteomic Analyses
2.7. In Vitro Kinase Assay (IVKA)
2.8. Molecular Modeling
3. Results
3.1. Proteomic Detection of Protein Complexes Based on the Interaction between Cyclin B1 and Viral Kinase pUL97
3.2. In HCMV-Infected Cells, but Not in Ectopically pUL97-Expressing Cells, Cyclin H is Abundant in the pUL97 Interactome Compared to Other Types of Cyclins
3.3. Complex Patterns of Interaction Are Detectable for Human Cyclins and the Associated Viral Kinase pUL97 as Well as Additional Viral Proteins
3.4. Interaction of pUL97 with Cyclin B1, but Not with Other Cyclins, Occurs in a Kinase Activity-Dependent Manner, Suggesting a Cyclin Type-Specific Mode of Interaction
4. Discussion
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Gene | Entry | Length | Protein | WSC [bait]/WSC [Fc-control] | |||
---|---|---|---|---|---|---|---|
B1 | T1 | H | pUL97 | ||||
CDK5 | Q00535 | 292 | Cyclin-dependent like kinase 5 | 5 | - | - | - |
CCNB1 | P14635 | 433 | Cyclin B1 | 34 | - | - | 5 |
CDK1 | P06493 | 297 | Cyclin-dependent kinase 1 | 49 | - | - | 8 |
CDK2 | P24941 | 298 | Cyclin-dependent kinase 2 | 5 | - | - | b.c. |
CCNT1 | O60563 | 726 | Cyclin T1 | - | 49 | - | - |
CDK9 | P50750 | 372 | Cyclin-dependent kinase 9 | 4 | 41 | - | 4 |
CCNT2 | O60583 | 730 | Cyclin T2 | - | 5 | - | - |
CCNH | P51946 | 323 | Cyclin H | - | - | 47 | - |
CDK7 | P50613 | 346 | Cyclin-dependent kinase 7 | - | - | 27 | - |
MNAT1 | P51948 | 309 | Cyclin H assembly factor | - | - | 17 | - |
AFF4 | Q9UHB7 | 1163 | Major CDK9 EF-associated protein | 3 | 20 | b.c. | 12 |
UL97 | P16788 | 707 | HCMV protein kinase pUL97 | 27 | 4 | 5 | 34 |
CoIP Source Material | B1 | H | T1 | Fc |
---|---|---|---|---|
293T, pUL97 transient transfection | 100% | 4% | 9% | 1% |
HFF, HCMV AD169 infection ** | 100% | 160% | 77% | 8% |
HFF, pUL97 stable transduction | 100% | 6% | 42% | 4% |
Gene | Entry | Length | Protein | WSC [bait]/WSC [Flag-control] | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
A | B1 | B2 | D1 | E | H | T1 | pUL97 | Fc | ||||
HCMV proteins | ||||||||||||
IRS1 | P09715 | 846 | Early protein IRS1 | 5 | - | - | - | - | 11 | - | 4 | - |
TRS1 | P09695 | 788 | Tegument protein HHLF1 | - | - | - | - | - | 7 | - | 4 | - |
UL104 | P16735 | 697 | Portal protein pUL6 homolog | b.c. | - | - | - | b.c. | 3 | - | 7 | - |
UL122 | P19893 | 580 | IE2 | 3 | - | - | - | b.c. | 4 | - | 4 | - |
UL24 | P16760 | 358 | Tegument protein pUL24 | - | - | - | - | - | 5 | - | 6 | - |
UL25 | P16761 | 656 | Phosphoprotein pp85 | b.c. | - | - | b.c. | b.c. | 6 | b.c. | 4 | - |
UL26 | P16762 | 222 | Tegument protein pUL26 | - | - | - | - | b.c. | 8 | - | 6 | - |
UL29 | C0H677 | 701 | Early protein pUL29/pUL28 | - | - | - | - | - | 3 | - | 3 | - |
UL32 | P08318 | 1048 | Phosphoprotein pp150 | b.c. | - | - | - | - | 4 | - | 8 | - |
UL35 | P16766 | 640 | Tegument protein pUL35 | - | - | - | - | - | 5 | - | 7 | - |
UL43 | P16781 | 423 | Tegument protein pUL43 | - | - | - | - | - | 8 | - | 8 | - |
UL44 | P16790 | 433 | DNA polymerase processivity factor pUL44 | b.c. | b.c. | b.c. | b.c. | b.c. | 3 | b.c. | 3 | b.c. |
UL45 | P16782 | 906 | Late protein pUL45 | 4 | - | - | - | b.c. | 13 | b.c. | 9 | - |
UL47 | P16784 | 983 | Capsid assembly protein pUL37 homolog | b.c. | - | - | - | - | 4 | - | 7 | - |
UL48 | P16785 | 2241 | Deneddylase pUL48 | - | - | - | - | - | - | - | 18 | - |
UL50 | P16791 | 397 | Virion egress protein pUL34 homolog | 8 | - | - | - | b.c. | 3 | - | 10 | b.c. |
UL53 | P16794 | 376 | Virion egress protein pUL31 homolog | b.c. | - | - | - | - | b.c. | - | 3 | - |
UL69 | P16749 | 744 | mRNA export factor ICP27 homolog | b.c. | - | - | - | b.c. | 28 | b.c. | 19 | - |
UL83 | P06725 | 561 | Phosphoprotein pp65 | b.c. | b.c. | b.c. | b.c. | b.c. | 7 | b.c. | 7 | b.c. |
UL84 | P16727 | 586 | Early protein pUL84 (viral DNA replication) | - | - | - | - | - | 3 | - | b.c. | - |
UL86 | P16729 | 1370 | MCP | b.c. | - | - | - | 3 | b.c. | - | 6 | - |
UL97 | P16788 | 707 | Protein kinase pUL97 | 5 | b.c. | - | - | 3 | 13 | 3 | 32 | - |
US22 | P09722 | 576 | Early nuclear protein HWLF1 | 22 | - | - | - | b.c. | b.c. | b.c. | - | b.c. |
CDK/cyclin-specific proteins | ||||||||||||
CDK2 | P24941 | 298 | CDK2 | - | b.c. | - | - | 12 | - | - | - | - |
CDK1 | P06493 | 297 | CDK1 | - | 9 | 6 | - | - | - | - | - | - |
CCNB1 | P14635 | 433 | Cyclin B1 | - | 7 | - | - | - | - | - | - | - |
CCNB2 | O95067 | 398 | Cyclin B2 | - | - | 13 | - | - | - | - | - | - |
CCND1 | P24385 | 295 | Cyclin D1 | - | - | - | 4 | - | - | - | - | - |
CCNE1 | P24864 | 410 | Cyclin E1 | - | - | - | - | 8 | - | - | - | - |
CCNH | P51946 | 323 | Cyclin H | - | - | - | - | - | 36 | - | - | - |
CDK7 | P50613 | 346 | CDK7 | - | - | - | - | - | 23 | - | - | - |
AFF4 | Q9UHB7 | 1163 | Major CDK9 EF-associated protein | - | - | - | - | - | - | 9 | b.c. | - |
CCNT1 | O60563 | 726 | Cyclin T1 | - | - | - | - | - | - | 22 | - | - |
CDK9 | P50750 | 372 | CDK9 | - | - | - | - | - | - | 20 | - | - |
MNAT1 | P51948 | 309 | Cyclin H assembly factor | - | - | - | - | - | 22 | - | - | - |
Gene | Entry | Length | Protein | WSC [bait]/WSC [Flag-control] | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|
B1 | B1/MBV | H | H/MBV | T1 | T1/MBV | Fc | |||||
HCMV proteins | |||||||||||
IRS1 | P09715 | 846 | Early protein IRS1 | 3 | 5 | 21 | 15 | - | 6 | b.c. | |
IR11 | P16809 | 234 | Viral Fc-gamma receptor-like protein IR11 | b.c. | b.c. | b.c. | b.c. | 4 | 3 | 7 | |
IRL12 | P16810 | 416 | Uncharacterized protein IRL12 | b.c. | 3 | - | - | - | - | b.c. | |
TRS1 | P09695 | 788 | Tegument protein HHLF1 | 5 | 5 | 12 | 8 | 4 | 3 | b.c. | |
UL24 | P16760 | 358 | Tegument protein pUL24 | b.c. | b.c. | 17 | 9 | - | b.c. | - | |
UL25 | P16761 | 656 | Phosphoprotein pp85 | b.c. | b.c. | 10 | 9 | b.c. | b.c. | - | |
UL26 | P16762 | 222 | Tegument protein pUL26 | 6 | - | 16 | 16 | b.c. | - | - | |
UL29 | C0H677 | 701 | Early protein pUL29/pUL28 | b.c. | - | 9 | 9 | - | - | - | |
UL32 | P08318 | 1048 | Phosphoprotein pp150 | 21 | 14 | 25 | 27 | b.c. | 6 | - | |
UL35 | P16766 | 640 | Tegument protein pUL35 | b.c. | 3 | 10 | 8 | - | - | - | |
UL43 | P16781 | 423 | Tegument protein pUL43 | - | - | 11 | 15 | - | - | - | |
UL44 | P16790 | 433 | DNA polymerase processivity factor pUL44 | b.c. | b.c. | b.c. | 4 | b.c. | b.c. | b.c. | |
UL45 | P16782 | 906 | Late protein pUL45 | 11 | 4 | 54 | 51 | 6 | 6 | b.c. | |
UL46 | P16783 | 290 | Triplex capsid protein VP19C homolog | - | b.c. | - | 4 | - | b.c. | - | |
UL47 | P16784 | 983 | Capsid assembly protein pUL37 homolog | - | b.c. | b.c. | 3 | - | b.c. | b.c. | |
UL48 | P16785 | 2241 | Deneddylase pUL48 | b.c. | 3 | 11 | 16 | b.c. | 6 | - | |
UL50 | P16791 | 397 | Virion egress protein pUL34 homolog | 4 | 3 | 9 | 6 | b.c. | 3 | - | |
UL52 | P16793 | 668 | Packaging protein pUL32 homolog | - | 4 | b.c. | 5 | b.c. | 4 | - | |
UL53 | P16794 | 376 | Virion egress protein pUL31 homolog | 3 | 3 | 3 | b.c. | - | b.c. | - | |
UL54 | P08546 | 1242 | DNA polymerase catalytic subunit pUL54 | - | - | 3 | b.c. | - | - | - | |
UL69 | P16749 | 744 | mRNA export factor ICP27 homolog | b.c. | b.c. | 13 | 16 | b.c. | b.c. | - | |
UL70 | P17149 | 946 | DNA primase pUL70 | - | b.c. | 4 | - | - | b.c. | - | |
UL82 | P06726 | 559 | Tegument protein pp71 | - | b.c. | 3 | b.c. | - | - | - | |
UL83 | P06725 | 561 | Phosphoprotein pp65 | b.c. | b.c. | 16 | 15 | b.c. | b.c. | b.c. | |
UL85 | P16728 | 306 | Probable capsid protein VP23 | b.c. | 6 | 3 | 9 | b.c. | 5 | - | |
UL86 | P16729 | 1370 | Major capsid protein MCP | b.c. | b.c. | b.c. | 4 | b.c. | b.c. | b.c. | |
UL89 | P16732 | 674 | Tripartite terminase subunit pUL89 | - | b.c. | 4 | 7 | - | 5 | - | |
UL94 | P16800 | 345 | Capsid-binding protein pUL16 homolog | - | 3 | - | b.c. | - | b.c. | - | |
UL97 | P16788 | 707 | Protein kinase pUL97 | 6 | b.c. | 11 | 10 | 3 | 4 | - | |
UL98 | P16789 | 584 | Alkaline nuclease pUL98 | - | 7 | - | b.c. | b.c. | b.c. | - | |
UL112/UL113 | P17151 | 684 | Early phosphoprotein p84 | b.c. | b.c. | 4 | 5 | b.c. | b.c. | - | |
UL122 | P19893 | 580 | Immediate early protein 2 (IE2) | b.c. | b.c. | 5 | 6 | b.c. | 3 | - | |
UL123 | P13202 | 491 | Immediate early protein 1 (IE1) | - | 5 | - | b.c. | - | b.c. | - | |
US22 | P09722 | 576 | Early nuclear protein HWLF1 | b.c. | 4 | b.c. | b.c. | b.c. | b.c. | b.c. | |
US23 | P09701 | 592 | Tegument protein pUS23 | - | - | b.c. | 3 | - | - | - | |
CDK/cyclin-specific proteins | |||||||||||
CCNB1 | P14635 | 433 | Cyclin B1 | 36 | 29 | - | - | - | - | - | |
CDKN1B | P46527 | 198 | CDK inhibitor 1B | 7 | 6 | - | - | - | - | - | |
CDKN1C | B2D1N3 | 301 | CDK inhibitor 1C | 12 | 5 | - | - | - | - | - | |
CDK1 | P06493 | 297 | Cyclin-dependent kinase 1 | 42 | 51 | - | - | - | - | - | |
CDK2 | P24941 | 298 | Cyclin-dependent kinase 2 | 14 | 15 | - | - | - | - | - | |
CDK5 | Q00535 | 292 | Cyclin-dependent like kinase 5 | 5 | 5 | - | - | - | - | - | |
CDKN1A | P38936 | 164 | CDK inhibitor 1A (Cip1) | 3 | b.c. | - | - | - | - | - | |
CCNH | P51946 | 323 | Cyclin H | - | - | 33 | 39 | - | - | - | |
MNAT1 | P51948 | 309 | Cyclin H assembly factor | - | - | 15 | 17 | - | - | - | |
CDK7 | P50613 | 346 | Cyclin-dependent kinase 7 | - | - | 24 | 23 | - | - | - | |
AFF4 | Q9UHB7 | 1163 | Major CDK9 EF-associated protein | - | - | b.c. | 3 | 12 | 14 | - | |
CDK9 | P50750 | 372 | Cyclin-dependent kinase 9 | - | - | - | - | 36 | 39 | - | |
CCNT1 | O60563 | 726 | Cyclin T1 | - | - | - | - | 45 | 40 | - |
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Steingruber, M.; Kraut, A.; Socher, E.; Sticht, H.; Reichel, A.; Stamminger, T.; Amin, B.; Couté, Y.; Hutterer, C.; Marschall, M. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins. Viruses 2016, 8, 219. https://doi.org/10.3390/v8080219
Steingruber M, Kraut A, Socher E, Sticht H, Reichel A, Stamminger T, Amin B, Couté Y, Hutterer C, Marschall M. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins. Viruses. 2016; 8(8):219. https://doi.org/10.3390/v8080219
Chicago/Turabian StyleSteingruber, Mirjam, Alexandra Kraut, Eileen Socher, Heinrich Sticht, Anna Reichel, Thomas Stamminger, Bushra Amin, Yohann Couté, Corina Hutterer, and Manfred Marschall. 2016. "Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins" Viruses 8, no. 8: 219. https://doi.org/10.3390/v8080219