Enzymatically inactive US3 protein kinase of Marek's disease virus (MDV) is capable of depolymerizing F-actin but results in accumulation of virions in perinuclear invaginations and reduced virus growth

Marek's disease (MD) is a highly contagious, lymphoproliferative disease of chickens caused by the cell-associated MD virus (MDV), a member of the alphaherpesvirus subfamily. In a previous study we showed that the absence of the serine/threonine protein kinase (pUS3) encoded in the MDV unique-short region resulted in accumulation of primarily enveloped virions in the perinuclear space and significant impairment of virus growth in vitro. It was also shown that pUS3 is involved in actin stress fiber breakdown [Schumacher, D., Tischer, B. K., Trapp, S., and Osterrieder, N. (2005). Here, we constructed a recombinant virus to test the importance of pUS3 kinase activity for MDV replication and its functions in actin rearrangement. Disruption of the kinase active site was achieved by substituting a lysine at position 220 with an alanine (K220A). Titers of a kinase-negative MDV mutant, 20US3⁎K220A, were reduced when compared to parental virus similar to those of the US3 deletion mutant. We were also able to demonstrate complete absence of phosphorylation of MDV-specific phosphoprotein pp38 in cells infected with the kinase-deficient virus, indicating that pp38 phosphorylation depends entirely on the kinase activity of pUS3. Enzymatically inactive pUS3⁎K220A was, however, still capable of mediating breakdown of the actin cytoskeleton in transfection studies, and this activity was indistinguishable from that of wild-type pUS3⁎. Furthermore, we demonstrated that pUS3 possesses anti-apoptotic activity, which is dependent on its kinase activity. Taken together, our results demonstrate that pUS3 and MDV-specific phosphoprotein pp38 represent a kinase–substrate pair and that growth impairment in the absence of pUS3 is caused by the absence of kinase activity. The unaltered disruption of F-actin by the K220A pUS3 mutant suggests that F-actin disassembly is unrelated to MDV growth restrictions in the absence of the unique-short protein kinase.