Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus

Abstract

The assembly of human cytomegalovirus (HCMV) virions is an orchestrated process that requires, as an essential prerequisite, the complex crosstalk between viral structural proteins. Currently, however, the mechanisms governing the successive steps in the constitution of virion protein complexes remain elusive. Protein phosphorylation is a key regulator determining the sequential changes in the conformation, binding, dynamics, and stability of proteins in the course of multiprotein assembly. In this review, we present a comprehensive map of the HCMV virion proteome, including a refined view on the virion phosphoproteome, based on previous publications supplemented by new results. Thus, a novel dataset of viral and cellular proteins contained in HCMV virions is generated, providing a basis for future analyses of individual phosphorylation steps and sites involved in the orchestrated assembly of HCMV virion-specific multiprotein complexes. Finally, we present the current knowledge on the activity of pUL97, the HCMV-encoded and virion-associated kinase, in phosphorylating viral and host proteins.

Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation

“…The study suggests a refined model of pUL50 functionality, in which the regulation of viral replication, specifically the nuclear egress, seems to allow some compensatory measures to balance alterations in the expression and phosphorylation levels of pUL50. It has not escaped our notice that pUL50 is not only present at low copy numbers in infectious virions [ 53 ], but also comprises additional, NEC-independent activities [ 54 , 55 ]. Thus, more detailed studies on the virion-associated state of pUL50 may help to understand this multifaceted regulatory aspect in the near future.…”

Protein Phosphatase 1 Regulates Human Cytomegalovirus Protein Translation by Restraining AMPK Signaling

“…The release of the tegument into the cell provides an instant supply of proteins upon lytic infection. As multiple studies have evaluated by now using proteomic techniques, the viral particle contains not only viral, but also many human proteins, many of which are robustly detected across multiple datasets (Varnum et al, 2004;Reyda et al, 2014;Rieder et al, 2017b;Couté et al, 2020). Also the human phosphatase PP1 has been previously described to be part of the tegument layer (Michelson et al, 1996).…”

“…Here, we show that PP1 is enriched in the virion compared to cellular lysates, implying an incorporation in a directed manner, although we did not discern the role of dense bodies in our experimental setup, and contamination with cellular microvesicles cannot be excluded. However, PP1 has also been detected in highly purified virions (Couté et al, 2020). PP1 specificity depends on the interaction with a regulatory subunit, thus we originally hypothesized that an HCMV tegument protein functions as a PP1 regulatory subunit for incorporation into the virion.…”

“…Asterisks indicate significant differences from Dunnett's ANOVA post hoc test compared to pooled controls. (F) Venn diagrams showing the gene name list of intersection between the indicated MS Datasets (Varnum et al, 2004;Reyda et al, 2014;Rieder et al, 2017b;Couté et al, 2020) and the PP1 interactor list from DEPOD (PP1-PPi) (Li et al, 2013).…”

“…Mass spectrometry (MS) analyses of purified HCMV virions from our group and others further indicate that in addition to PP1, HCMV integrates multiple other human proteins into its viral particle. From these data, we have identified 20 human proteins inside the virion that were reported as hits in 4 independent MS datasets (Varnum et al, 2004;Reyda et al, 2014;Rieder et al, 2017b;Couté et al, 2020) and that are also listed in the PP1 interactome according to the DEPOD database (Li et al, 2013) (Figure 1F). This list also contains members of the ribosomal translation elongation factor complex and 14-3-3 proteins, which were validated by western blot (Supplementary Figure 1D).…”