The protein encoded by the US3 orthologue of Marek's disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown

Abstract

Marek's disease virus (MDV) encodes a protein exhibiting high amino acid similarity to the US3 protein of herpes simplex virus type 1 and the gene 66 product of varicella-zoster virus. The MDV US3 orthologue was replaced with a kanamycin resistance gene in the infectious bacterial artificial chromosome clone BAC20. After transfection of US3-negative BAC20 DNA (20DeltaUS3), the resulting recombinant 20DeltaUS3 virus exhibited markedly reduced growth kinetics. Virus titers on chicken embryo cells were reduced by approximately 10-fold, and plaque sizes were significantly smaller (65% reduction) compared to parental BAC20 virus. The defect of the US3-negative MDV was completely restored in a revertant virus (20US3*) expressing a US3 protein with a carboxy-terminal FLAG tag. Electron microscopical studies revealed that the defect of the 20DeltaUS3 mutant to efficiently spread from cell to cell was concomitant with an accumulation in the perinuclear space of primarily enveloped virions in characteristic vesicles containing several virus particles, which resulted in reduced numbers of particles in the cytoplasm. The formation of these vesicles was not observed in cells infected with either parental BAC20 virus or the 20US3* revertant virus. The role of the MDV US3 protein in actin stress fiber breakdown was investigated by visualizing actin with phalloidin-Alexa 488 after infection or transfection of a US3 expression plasmid. Addition of the actin-depolymerizing drug cytochalasin D to cells transfected or infected with BAC20 resulted in complete inhibition of plaque formation with as little as 50 nM of the drug, while concentrations of nocodazole as high as 50 microM only had a relatively minor effect on MDV plaque formation. The results indicated that the MDV US3 serine-threonine protein kinase is transiently involved in MDV-mediated stress fiber breakdown and that polymerization of actin, but not microtubules, plays an important role in MDV cell-to-cell spread.

FIG. 1.

(A) Schematic illustration of the procedure to delete the U_S3 ORF from BAC20. Shown is the organization of the approximately 185-kbp BAC20 genome and the BamHI restriction map. The unique short region (U_S) with the introduced pHA1 sequence as well as the introduced kan^r gene is highlighted. (B) The construction of the rescuant viruses, harboring a FLAG tag at the carboxy terminus of the U_S3, is shown. Small letters indicate original amino acid sequences, while large letters denote the introduced FLAG tag. (C) Digitally scanned image of a Southern blot. DNA from BAC20, 20ΔU_S3, and 20U_S3* was cleaved with BamHI and transferred to a nylon membrane. The blot was incubated with a digoxigenin-labeled kan^r-specific probe. Specific hybridization was detected by chemiluminescence using CSPD (Roche Biochemicals).

FIG. 2.

(A) CEC were infected with BAC20, 20ΔU_S3, or 20U_S3* and fixed with 90% acetone at 5 days p.i. Plaques were analyzed by IIF using anti-gB MAb 2K11. Bound antibodies were detected anti-mouse IgG Alexa 488 (Molecular Probes). (B) For each virus, digital pictures of at least 100 plaques were taken and plaque sizes were measured. The mean plaque area of BAC20 virus was set to 100%, and average relative plaque areas of the 20ΔU_S3 and 20U_S3* viruses were calculated. Standard deviations are also given. Plaque areas and standard deviations were determined from three independent experiments. (C) Growth properties of BAC20, 20ΔU_S3, and 20U_S3* viruses recovered after transfection of BAC DNA. A total of 10⁶ CEC were infected with 150 PFU of the respective virus. At the given times p.i., cells were trypsinized, titrated, and coseeded with fresh CEC. Virus plaques were counted after IIF staining with MAb 2K11. Mean virus titers and standard deviations of the results of three independent experiments are shown.

FIG. 3.

Western blot analysis of CEC infected with BAC20, 20ΔU_S3, and 20U_S3*. Cells were harvested and lysed at 4 days p.i. Cell lysates were separated by SDS-10% PAGE and transferred to nitrocellulose. Identical blots were incubated with either anti-FLAG MAb M2 or anti-VP22 MAb L13. The molecular weights of a molecular weight marker (Fermentas) are given in thousands. Co, mock-infected CEC.

FIG. 4.

Electron microscopy of 20ΔU_S3- or 20U_S3*-infected CEC. Cells were infected with recombinant virus and fixed 3 days p.i. (A) Electron microscopic examination of the 20ΔU_S3 revealed an accumulation of enveloped virions in the perinuclear space in characteristic protrusions. (B) In cells infected with parental BAC20 (data not shown) or the 20U_S3* revertant virus, all stages of virion morphogenesis were detectable, without any accumulation of primarily enveloped virions in the perinuclear space. A newly synthesized A-type capsid in the nucleus is marked with a black arrow, while naked nucleocapsids and mature viruses in transport vesicles in the cytoplasm are marked with white arrows. Note that no extracellular virus is released from 20U_S3*- or 20ΔU_S3-infected cells.

FIG. 5.

(A) Disassembly of actin stress fibers in CEC infected with BAC20 at 48 h p.i. Cells were fixed with 90% acetone at 48 h after infection. MDV proteins were detected with polyclonal convalescent-phase serum that was visualized using an Alexa 568-conjugated anti-chicken IgG as the secondary antibody (red). Actin was visualized with phalloidin-Alexa 488 (green). Cells were finally inspected using a Zeiss Axiovert fluorescence microscope, and pictures were taken with a 40× objective before processing with Adobe Photoshop. (B) Percentage of infected cells with intact and depolymerized actin stress fibers. A total of 300 infected cells in three independent experiments were inspected.

FIG. 6.

(A) Subcellular localization of pU_S3* in CEC transfected with expression plasmid pcU_S3*. Cells were fixed at the given time points with 90% acetone, and FLAG-tagged U_S3 (red) was detected using the anti-FLAG M2 antibody and Alexa 568-conjugated anti-mouse IgG as the secondary antibody. Actin (green) was stained with phalloidin-Alexa 488. Red and green fluorescence signals were recorded separately by using appropriate filters with a confocal microscope (Olympus). Overlay of the pU_S3 and actin fluorescent signals is shown in the merge. (B) Percentages of pU_S3*- and pcMgB-transfected cells with intact actin stress fibers (100 cells were scored) at 24 and 48 h after transfection. Percentages represent means and standard deviations of three independent experiments.

FIG. 7.

(A) Effect of various concentrations of cytochalasin D (A to H) or nocodazole (I to P) on the actin cytoskeleton or microtubules in CEC, respectively. Cells were stained simultaneously with phalloidin-Alexa 488 to detect polymerized actin and with anti-α-tubulin-Alexa 546 to detect microtubules. Fluorescences were recorded individually by CLSM. A dose-dependent disassembly of actin stress fibers in the presence of cytochalasin D (A to C) and a gradual disassembly of microtubules with increasing concentrations of nocodazole (I to K) were observed. Note the presence of the microtubules in cytochalasin D-treated cells (E to G) and the intact actin stress fibers in cultures treated with nocodazole (I to K). Untreated control cells are in panels D, H, L, and P. (B) Number of plaques after transfection of BAC20 DNA in the presence or absence of cytochalasin D or nocodazole. CEC were transfected with 5 μg of BAC20 DNA, and the indicated concentrations of the drugs were added at 8 h after transfection. Media were changed twice daily to maintain drug concentrations. Five days after transfection, the number of plaques was determined after fixing the cells with 90% acetone and subsequent IIF using MAb 3F19, which recognizes the MDV major nucleocapsid protein VP5. Bars represent the number of plaques of four independent experiments performed in duplicate; standard deviations are given. (C) Plaque diameters after infection with BAC20 virus in the presence or absence of cytochalasin D or nocodazole. CEC were infected, and indicated concentrations of the drugs were added at 12 h after infection. Media were changed twice daily to maintain drug concentrations. Four days after infection, plaque sizes were determined after IIF staining with the MDV-specific chicken antiserum. Means and standard deviations of plaque areas of 100 plaques from two independent experiments are given.