Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome

Abstract

Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC-labeled human fibroblasts during infection with either wild-type HCMV or a pUL97 kinase-dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high-resolution MS. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication.

Keywords: Cell biology; Herpesvirus; Human cytomegalovirus; Nuclear pore complex; TREX complex; Viral kinase.

Figure 1

Experimental design and validation. (A) SILAC workflow for enriching phosphopeptides. (B) Representative MS1 spectrum of a pUL53 phosphopeptide showing relative abundances of heavy and light SILAC precursor ions. (C) Corresponding MS2 spectrum showing b- and y- fragment ions used for precursor ion sequence identification and phosphosite mapping.

Figure 2

Phosphopeptide data and ratio modeling. (A) Scatterplots comparing corrected and uncorrected phosphopeptide ratios. (B) Phosphopeptide ratio distributions and fitting. Solid red line; density plot of Johnson distribution fitted by maximum likelihood estimation. Dashed red line; 2-fold significance threshold. Dashed green line; p0.95 significance threshold.

Figure 3

Putative UL97 kinase motifs and pathway analysis. (A) Enriched phosphorylation motifs and statistics. All significantly regulated peptides (≥2-fold [UL97wt/UL97mut]) across all time points were analyzed non-redundantly using Motif-X [41, 90] and MMFPH [43]. (B) Euler diagram displaying overlap of enriched phosphorylation motifs in all phosphopeptides showing 2-fold or greater occupancy decrease upon mutation of pUL97. Note that the numbers of peptides in each class differ from those identified by Motif-X and MMFPH as these tools discard all peptides absent from their background databases. (C) Selected ontology terms enriched in putative pUL97 substrates. This list is a subset of the full list of ontology terms used to construct the network in panel D. All enriched ontology terms, their p-values, and constituent proteins are listed in Supporting Information Table S4. (D) Cluego/Clupedia ontology and STRING network. Hexagonal nodes represent significantly enriched gene ontology terms (p>0.005). Circular nodes represent proteins. Connecting lines (edges) indicate interrelatedness. Multicolored lines connecting proteins highlight different STRING protein-protein interaction evidence codes. Stars indicate that a protein contains an LxSP phosphorylation motif.

Figure 4

Protein level ontology network analysis. (A) SILAC protein ratios modeled as in Fig. 2B. (B) Selected ontology terms enriched in pUL97 substrates at the protein level. This list is a subset of the full list of ontology terms used to construct the network in panel C. All enriched ontology terms, their p-values, and constituent proteins are listed in Supporting Information Table S5. (C) Cluego network analysis of all proteins with a 2-fold or greater SILAC ratio.