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

Abstract

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 (pU(S)3) 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 pU(S)3 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 pU(S)3 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, 20U(S)3()K220A, were reduced when compared to parental virus similar to those of the U(S)3 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 pU(S)3. Enzymatically inactive pU(S)3()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 pU(S)3(). Furthermore, we demonstrated that pU(S)3 possesses anti-apoptotic activity, which is dependent on its kinase activity. Taken together, our results demonstrate that pU(S)3 and MDV-specific phosphoprotein pp38 represent a kinase-substrate pair and that growth impairment in the absence of pU(S)3 is caused by the absence of kinase activity. The unaltered disruption of F-actin by the K220A pU(S)3 mutant suggests that F-actin disassembly is unrelated to MDV growth restrictions in the absence of the unique-short protein kinase.

Rho’ing in and out of cells

“…Intact and kinase-inactive US3 of MDV display equal abilities to induce actin rearrangements, although these are less dramatic compared with those of other alphaherpesviruses, as MDV US3 induces actin stress fiber disassembly but has not been reported to induce cell projections. 130 Another indication that interference of MDV with Rho GTPase signaling is different compared with other alphaherpesviruses comes from the finding that, unlike for PRV, the Rac1/PAK signaling axis appears inhibitory to MDV spread, whereas the RhoA/ROCK signaling axis promotes spread. 131 Actin containing projections that contribute to viral spread have also been reported for the gammaherpesviruses Epstein Barr virus (EBV) and MHV-68.…”

Viral Serine/Threonine Protein Kinases

“…US3 possesses antiapoptotic properties in HSV, PRV, and MDV, but possibly not in bovine herpesvirus 1 (BoHV-1) (9,69,119,197,204). The kinase activity of HSV-1, PRV, and MDV US3 is necessary for this US3 function (33,52), and several phosphorylation targets implicated in the HSV-1 US3-mediated suppression of apoptosis have been identified, including Bad, Bid, and procaspase 3, pointing at the involvement of US3 in different antiapoptotic pathways (18,32).…”

“…The US3 kinase induces drastic rearrangements of the cytoskeleton, including disassembly of actin stress fibers and the formation of actin-and microtubulecontaining cellular projections, which are implicated in intercellular spread (63). The kinase activity of US3 is essential for these processes in HSV-2, PRV, and BoHV-1, but not in MDV, suggesting different or multiple cytoskeleton-affecting activities of US3 (27,66,156,197,215). Recent evidence with PRV US3 indicates that the kinase interferes with actin-controlling Rho-GTPase signaling to cause the cytoskeletal alterations.…”

Hyperphosphorylation of Histone Deacetylase 2 by Alphaherpesvirus US3 Kinases

“…Genetic disruption of US3 in VZV, HSV-1, HSV-2, PRV, and Marek's disease virus (MDV) (52) revealed that US3 protein is not essential for viral replication in tissue culture; however, US3 mutants are impaired for growth in certain cell types and have significantly impaired viral replication and virulence in animal models (1, 10, 14, 28, 38, 47-51, 55, 58, 61).…”