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Study of Anaphase Promoting Complex Inactivation by Human Cytomegalovirus Reveals Cellular Mechanism for Downregulating Anaphase Promoting Complex Subunits

  • Author(s): Clark, Elizabeth
  • et al.
Abstract

Human Cytomegalovirus (HCMV) is a ubiquitous pathogen causing illness in immunosuppressed individuals and is the top viral cause of birth defects. HCMV deregulates the cell cycle by several means, including inactivation of the Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase. Viral proteins UL97and UL21a, respectively, affect the APC/C by phosphorylation of APC/C co-activator Cdh1 and by inducing degradation of subunits APC4 and APC5, which with APC1 form the APC/C platform sub-complex. We have further characterized this mechanism, showing the relative contributions of UL21a and UL97 to APC/C substrate accumulation and to viral growth. Further, we demonstrate that UL21a causes the proteasome-dependent degradation of APC1 in addition to APC4 and 5. We see that in uninfected Human Foreskin Fibroblast (HF) cells in G0, UL21a expression, but not UL97, leads to accumulation of APC/C substrates. We also demonstrate that there is a previously unreported cellular mechanism for a specific decrease in the levels of all three platform subunits, APC1, APC4, and APC5, upon depletion of any one of these subunits or subunit APC8. A UL21a/UL97 double mutant virus and cells that express UL21a, UL97, or both in the presence of Cre recombinase were made in order to propagate the double mutant while avoiding compensatory mutations. The double mutant is severely growth-restricted at the stage of late protein expression, and depleting subunits 5 or 8 from the APC/C can increase titers of the double mutant. When the double mutant infects complementing cells expressing UL21a and/or UL97, UL97 provides a more complete rescue of viral growth than UL21a, and that the presence of UL21a is more beneficial at low multiplicity of infection (MOI) than at high multiplicity. A method for using the Fucci cell cycle indicator system to screen for inhibitors of the APC/C is presented, along with the method for selecting highly-expressing transduced cells, which proves superior to use of a selectable marker only. Finally, the interaction of the APC/C with UL21a and with Cdh1 appears to be mutually exclusive

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