Abstract
The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.
Highlights
The human cytomegalovirus (HCMV) is a pathogen with worldwide distribution [1].While primary infection in immunocompetent individuals mostly remains asymptomatic, HCMV can Viruses 2016, 8, 219; doi:10.3390/v8080219 www.mdpi.com/journal/virusesViruses 2016, 8, 219 cause a common opportunistic infection in immunocompromised individuals, including transplant recipients, tumor and AIDS patients
We described the interaction between the HCMV regulatory protein pUL69 [13,14,36] as well as HCMV kinase pUL97 and human cyclins [58]
The first experimental evidence that pUL97 interacts with human cyclins was provided by Graf et al, (2013); [58], showing that pUL97-cyclin T1 can be coimmunoprecipitated from cotransfected as well as HCMV-infected cells, that interaction can be detected in a yeast two-hybrid assay and that pUL97 colocalizes with cyclin T1 in nuclear compartments
Summary
While primary infection in immunocompetent individuals mostly remains asymptomatic, HCMV can Viruses 2016, 8, 219; doi:10.3390/v8080219 www.mdpi.com/journal/viruses. Viruses 2016, 8, 219 cause a common opportunistic infection in immunocompromised individuals, including transplant recipients, tumor and AIDS patients. These complex clinical situations are frequently associated with end-organ disease, graft rejection or cardiovascular symptoms [2,3]. HCMV is the leading cause of congenital infection and fetal malformation in developed countries and has been associated with excess mortality in the general population [3,4,5]. The currently licensed anti-HCMV drugs comprise the nucleoside analogue ganciclovir (GCV), its prodrug valganciclovir (VGCV), foscarnet (FOS) and cidofovir (CDV), all of which inhibit viral DNA synthesis [6]. Antiviral therapy with these compounds often results in drug-resistant virus variants during long-term treatment and causes severe side effects such as myelo- and nephrotoxicity [7], so that there is a continued need for improved antivirals [8]
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