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. 2008 May 25;375(1):37-47.
doi: 10.1016/j.virol.2008.01.026. Epub 2008 Mar 4.

Enzymatically inactive U(S)3 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

Daniel Schumacher  1 , Caleb McKinney, Benedikt B Kaufer, Nikolaus Osterrieder
Affiliations

Enzymatically inactive U(S)3 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

Daniel Schumacher et al. Virology. .

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.

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Figures

Figure 1
Figure 1. The unique-short kinase of MDV and mutagensis of the active site
(A) Multiple amino acid sequence alignment of the VIB subdomain of MDV pUS3 and its orthologues in other alphaherpesviruses. The lysine that was altered in this study is shown in bold. Plus signs indicate similar amino acids and dots represent amino acids not shown in this alignment. (B) Nucleotide and deduced amino acid sequence before and after two-step Red en passant mutagenesis. The lysine residue at position 220 was substituted by an alanine by changing two adenines at position 657 and 658 into a guanine and a cytosine (see Materials and Methods).
Figure 2
Figure 2. Growth of US3 mutant viruses in cultured cells
(A) CEC were infected with v20, v20US3 or v20US3*K220A. At 5 days p.i., cells were fixed with 90% aceton and analyzed by IIF using a convalescent serum from a chicken infected with MDV-1. Antibodies were detected using a secondary chicken IgG Alexa488® (Molecular Probes). (B) For each virus digital pictures of 100 plaques were taken and plaque sizes were measured. The mean plaque area of the v20 virus was set to 100%, and average relative plaque areas of the 20ΔUS3 and the 20US3*K220A virus were calculated. Plaque areas and standard deviations were determined in three independent experiments. (C) Multi-step growth kinetics of v20, v20ΔUS3 and v20US3*K220A virus recovered after transfection of BAC DNA. 1 × 106 CEC were infected with 200 plaque-forming units of the respective virus. At the given times p.i., cells were trypsinized, titrated and co-seeded with fresh CEC. Virus plaques were counted after IIF staining with a convalescent MDV chicken serum. Mean virus titers and standard deviations of the results of three independent experiments are shown.
Figure 3
Figure 3. Electron microscopical analyses of CEC infected with v20US3*K220A
Cells were infected and fixed 3 days later. The v20US3*K220A kinase-negative mutant exhibited the same phenotype as the pUS3 null virus v20ΔUS3 (Schumacher et al., 2005). Primarily enveloped virions accumulated in the perinuclear space forming characteristic invaginations of the inner leaflet of the nuclear membrane. Bars represent 1 mm (left overview panel ) or 200 nm (right panel).
Figure 4
Figure 4. Immunoprecipitation analyses using FLAG-tagged pUS3 expressed by recombinant v20US3* virus
(A) Silver-stained SDS-polyacrylamide gel of immunoprecipitated proteins. Anti-FLAG® M2 affinity beads (Sigma-Aldrich) were used to precipitate pUS3* from CEC infected with v20US3*, v20 or mock infected cells. Cells were infected with the corresponding virus and harvested and lysed 4 days p.i. Lysates were separated by 12% SDS-PAGE and stained using SilverSNAP Stain Kit II (Pierce). (B) Western blot of proteins immunoprecipitated with anti-FLAG antibody beads and subsequently detected with antibodies H19 directed against pp38 or M2 to detect pUS3*. (C) Western blot detecting pp38 (left panel, H19 antibody) and pUS3* (right panel, M2 antibody) in immunoprecipitates obtained using antibody H19. Cell lysates infected with v20US3* or v20US3*K220A were used for the precipitations, which were then separated by SDS-12%-PAGE and transferred to nitrocellulose before western blot analyses were performed. The sizes of bands of a prestained molecular weight marker (Fermentas) are given in thousands.
Figure 5
Figure 5. Western blot analyses to examine the enzymatic relationship between pUS3 and pp38
(A) Western blot detecting pp38 in cell lysates from CEC cells infected with v20, v20ΔUS3 and v20US3*. (B) Detection of pp38 in cells infected with v20, v20ΔUS3 and multiple passages of v20US3*K220A. (C) Antibody H19 was used to detect pp38 in lysate of cells infected with v20US3*. Lysates were treated with λ phosphatase or mock treated for 30 or 60 minutes as detailed in Materials and Methods. The sizes of bands of a prestained molecular weight marker (Fermentas) are given in thousands.
Figure 6
Figure 6. Kinase activity of pUS3 is not required for depolymerization of actin stress fibers
(A) Percent-age of cells containing an intact actin cytoskeleton at 24h and 48h after the transfection of plasmids expressing FLAG-tagged pUS3*, FLAG-tagged US3*K220A or gB. CEC were transfected and fixed at 24 or 48 h after transfection. Expressing cells were detected using antibody M2 to detect FLAG-tagged versions of pUS3* or 2K11 to detect gB (red). Actin (green) was stained with phalloidin-Alexa488® (Molecular Probes). Data were obtained by analyzing 300 transfected cells for each plasmid and time point in three individual experiments in a blinded manner. (B) Disassembly of the actin cytoskeleton in pcUS3* and pUS3*K220A transfected cells after 24h. Cells were fixed at the given times after transfection. pUS3* and actin were stained as stated above. Red and green fluorescence signals were recorded separately by using appropriate filters with a confocal microscope (Olympus) before assembly using Adobe Photoshop. Overlay of the pUS3 (red) and actin (green) fluorescent signals are shown in the righost panels (merge).
Figure 7
Figure 7. pUS3 but not pUS3*K220A inhibits induction of apoptosis
CEC were transfected with pcUS3*, pcUS3*K220A or a control plasmid (pcORF9A) and apoptosis was induced with staurosporine. Apoptotic cells were detected with cleaved caspase-3 specific rabbit antibody, while transfected cells were identified using anti-FLAG or anti 6xHIS specific antibodies. The bar graph shows the ratio of apoptotic to non-apoptotic cells expressing the respective protein, normalized to the same ratio in non-transfected cells in the same experiment. Shown are means and standard deviations (error bars) from three independent experiments. The p value was computed using an unpaired Student’s t test.
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