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. 2012 Mar;86(5):2428-36.
doi: 10.1128/JVI.06719-11. Epub 2011 Dec 21.

Fluorescently tagged pUL47 of Marek's disease virus reveals differential tissue expression of the tegument protein in vivo

Keith W Jarosinski  1 , Sina Arndt, Benedikt B Kaufer, Nikolaus Osterrieder
Affiliations

Fluorescently tagged pUL47 of Marek's disease virus reveals differential tissue expression of the tegument protein in vivo

Keith W Jarosinski et al. J Virol. 2012 Mar.

Abstract

Marek's disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek's disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells.

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Figures

Fig 1
Fig 1
Generation of EGFP fusion proteins in rMDV. (A) Schematic representation of the MDV genome depicting the locations of the terminal repeat long (TRL) and short (TRS), internal repeat long (IRL) and short (IRS), and unique long (UL) and short (US) regions. (B) Positions and orientations of the UL47 and UL49 genes with respect to adjacent genes within the UL. (C) Schematic representation of EGFP fused to the C terminus of UL47 and UL49 with respect to their flanking genes. (D) RFLP analysis of BAC DNA obtained from the parental clone (rParental), one recombinant clone of rUL47-EGFP, and two recombinant clones of rUL49-EGFP digested with EcoRI or HindIII to confirm the expected band shift patterns. The addition of EGFP to the C termini of UL47 and UL49 generates genes of 3,144 and 1,467 bp, respectively. No extraneous alterations are evident in the three recombinant clones examined. The expected changes are indicated with arrows and are as follows: EcoRI, rParental, 10,748-bp fragment shifted to 11,465 bp in rUL47- and rUL49-EGFP; HindIII, rParental 24,155-bp fragment shifted to 24,872 bp in rUL47- and rUL49-EGFP. The molecular size marker used was the 1-kb Plus DNA Ladder from Invitrogen, Inc. (Carlsbad, CA) (not shown).
Fig 2
Fig 2
Plaque area assay of rMDV. Plaque areas were measured for parental virus (vParental) and viruses reconstituted from rUL47-EGFP (vUL47-EGFP) and rUL49-EGFP1 (vUL49-EGFP). They were significantly different (P = 1.7 × 10−6) using Student's t tests (indicated with an asterisk). The error bars indicate standard errors of the means.
Fig 3
Fig 3
Expression of pUL47- and pUL49-EGFP fusion proteins in vitro. (A) CKC cultures were infected with vParental, vUL47-EGFP, or vUL49-EGFP on glass coverslips and then fixed after 4 days p.i. Plaques were stained for multiple antigens, and four-color images were taken using the Axio Imager M1 system with AxioVision software. Shown is a representative plaque for each virus. Hoechst 33342 stain was used to identify nuclei (blue), anti-MDV chicken antibody was used to identify overall MDV antigen expression (red), and anti-Meq rabbit antibody was used to examine MDV antigen localization to the nucleus. For each plaque, the same parameters (lasers, excitation/emission wavelengths, time of exposure, magnification, etc.) were used to compare the fluorescence intensities. The merged images contain all four fluorescence channels. (B) High-magnification images of vUL47-EGFP-induced plaques. The arrows indicate cells that show abundant pUL47-EGFP expression. The images were recorded at the indicated magnifications using a Leica SP5 confocal microscope system.
Fig 4
Fig 4
Cellular expression of pUL47- and pUL49-EGFP. (A and C) vUL47-EGFP-induced (A) and vUL49-EGFP-induced (C) plaques were stained, and images of individual cells were taken at high magnification (×2,520 [A] and ×1,890 [C]) using a Leica SP5 confocal microscope system. Nuclear (N) and cytoplasmic (C) regions are indicated in the merged images, showing pUL47-EGFP expression in the nucleus (A), while pUL49-EGFP expression is seen throughout the cell (C). (B and D) Expression of pUL47-EGFP (B) and pUL49-EGFP (D) relative to nuclear staining (Hoechst). pUL47-EGFP is contained in large nuclear punctate domains (left, ×2,520; right, ×1,890), while pUL49-EGFP can be seen in the cytoplasm and nucleus (both, ×1,890).
Fig 5
Fig 5
Virulence of vUL47- and vUL49-EGFP in chickens. Groups of MD-susceptible P2a chickens were inoculated with vUL47-EGFP or vUL49-EGFP and housed in separate glove box isolators with contact (con) chickens for 10 weeks. MD incidence was determined by identification of gross lesions in dead or euthanized chickens, and the contact chickens were used to determine if rMDV was able to be transmitted to naïve chickens.
Fig 6
Fig 6
Tissue expression of pUL47-EGFP in chickens. (A) Spleen, tumor, and skin/feather tissues were collected from vUL47-EGFP-infected chickens at 21 days p.i. Slides with tissues were fixed and stained for multiple antigens, and four-color images were taken using the Axio Imager M1 system and AxioVision software. Hoechst 33342 stain was used to identify nuclei (blue), anti-pp38 monoclonal antibody was used to examine early lytic MDV protein expression (gray), and anti-Meq rabbit antibody was used to identify transformed or latently infected cells (red) in contrast to expression of pUL47-EGFP (green). Magnifications are indicated, and the white arrowheads indicate pUL47-EGFP-positive cells. The asterisks indicate Meq-positive cells in feather follicle sections that are most likely infected circulating T lymphocytes. (B) FFE cells infected with vUL47-EGFP were stained for pp38 (red) and nuclei (blue) with anti-pp38 antibody and Hoechst 33342, respectively, and z-stack images were collected at 1.5 μm per stack at ×2,520 using an SP5 LSCM system. pp38 expression is seen in both the nucleus and cytoplasm, while pUL47-EGFP expression is mostly in the cytoplasm. Some cells that express only pp38 or pUL47-EGFP can be seen, while others express both. The merging of colors had the following results: red plus green, yellow; red plus blue, magenta; blue plus green, cyan; and red plus blue plus green, white.
Fig 7
Fig 7
Dual-color staining of a whole feather follicle longitudinally cut down the feather follicle. On the left is shown a complete feather follicle in bright field and stained for pp38 (red) and pUL47-EGFP (green). The image was obtained by combining three images at ×25 with the Axio Imager M1 system. Boxes 1, 2, and 3 are shown on the right at ×200 to better visualize the differential staining. Also indicated are structural regions of the feather follicle, including the feather sheath, the follicle sheath, and FFE cells.
Fig 8
Fig 8
Visualization of pUL47-EGFP expression in skin and feathers of MDV-infected chickens. (A) Whole skin and feather tissues (∼1 cm2) were collected from vUL47-EGFP-infected P2a chickens or age-matched uninfected chickens and fixed in 10% buffered formalin. Shown are images taken with an Olympus SZX-12 stereoscope at either ×42 or ×96 under bright field or through an LP Green filter cube and merged by overlaying one image over the other and blending them to 50% in Adobe Photoshop CS2 version 9.0.2. The white arrowheads show pUL47-EGFP expression. The white circles show pUL47-EGFP expression on the axial side of the skin in the middle column. (B) Feathers were plucked from the wing and visualized as in panel A but without fixation, and only merged images are shown. (C) Feathers were plucked from the wing, or whole skin/feather tissues were collected, fixed in 10% buffered formalin, and stained for pp38 (red). The top row shows bright field, expression of pUL47-EGFP (green) and pp38 (red) individually, and then all three images merged (blended at 33% each with Adobe Photoshop CS2) at ×96. The boxed region in the merged image is shown below at ×376. The bottom row shows feathers protruding from the skin at ×360. The arrows indicate pp38-positive cells, while the arrowheads indicate pUL47-EGFP-positive cells.
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References

    1. Abramoff MD, Magalhaes PJ, Ram SJ. 2004. Image processing with ImageJ. Biophotonics Int. 11:36–42
    1. Ahmed M, Schidlovsky G. 1968. Electron microscopic localization of herpesvirus-type particles in Marek's disease. J. Virol. 2:1443–1457 - PMC - PubMed
    1. Anobile JM, et al. 2006. Nuclear localization and dynamic properties of the Marek's disease virus oncogene products Meq and Meq/vIL8. J. Virol. 80:1160–1166 - PMC - PubMed
    1. Brown AC, et al. 2009. Homodimerization of the Meq viral oncoprotein is necessary for induction of T-cell lymphoma by Marek's disease virus. J. Virol. 83:11142–11151 - PMC - PubMed
    1. Calnek BW. 2001. Pathogenesis of Marek's disease virus infection. Curr. Top. Microbiol. Immunol. 255:25–55 - PubMed

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