Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong
latent infection, which places the infected host at risk for numerous
cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68
infection regulated by 2-fold or greater ca. 86% of identified
phosphopeptides - a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the
infection-associated induction or repression of specific cellular
proteins globally altered the flow of information through the host
phosphoprotein network, yielding major changes to functional
protein clusters and ontologically associated
proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the
infection-associated phosphoproteome and identified 155 host
proteins, such as the
transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as
kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68
cyclin D ortholog as a
viral protein that contributes to the prominent MAPK/CDK signature of the
infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate
infection and replication.