The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tailanchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction

Stable Marking and Transgene Expression Without Progression to Monoclonality in Canine Long-Term Hematopoietic Repopulating Cells Transduced with Lentiviral Vectors

Lentiviral gene transfer vectors have a number of potential advantages over gammaretroviral vectors including more efficient transduction of nondividing cells, a more favorable integration site profile, and the ability to accommodate large transgenes. Here, we present long-term follow-up data of animals that received lentivirus-transduced CD34-enriched cells. Six long-term surviving dogs were available for analysis. Transgene expression was analyzed from at least 12 months to more than 5 years after transplantation in peripheral blood cells and multiple cell lineages. All animals demonstrated long-term stable transgene expression in peripheral blood myeloid, lymphoid, and red blood cells as well as in platelets. Vector integration sites were analyzed by linear amplification-mediated polymerase chain reaction and showed a polyclonal repopulation pattern in all animals. There was no evidence of any development of monoclonality or leukemia in the animals. The stable long-term multilineage transgene expression, together with detection of the same integration site in myeloid and lymphoid cells, strongly suggests the transduction of long-term repopulating stem cells. Our data demonstrate safe and efficient transduction of multilineage long-term repopulating cells with lentiviral vectors and support the use of such vectors for gene therapy studies in patients

The M25 gene products are critical for the cytopathic effect of mouse cytomegalovirus

Abstract Cell rounding is a hallmark of the cytopathic effect induced by cytomegaloviruses. By screening a panel of deletion mutants of mouse cytomegalovirus (MCMV) a mutant was identified that did not elicit cell rounding and lacked the ability to form typical plaques. Altered cell morphology was assigned to the viral M25 gene. We detected an early 2.8 kb M25 mRNA directing the synthesis of a 105 kDa M25 protein, and confirmed that a late 3.1 kb mRNA encodes a 130 kDa M25 tegument protein. Virions lacking the M25 tegument protein were of smaller size because the tegument layer between capsid and viral envelope was reduced. The ΔM25 mutant did not provoke the rearrangement of the actin cytoskeleton observed after wild-type MCMV infection, and isolated expression of the M25 proteins led to cell size reduction, confirming that they contribute to the morphological changes. Yields of progeny virus and cell-to-cell spread of the ΔM25 mutant in vitro were diminished and replication in vivo was impaired. The identification of an MCMV gene involved in cell rounding provides the basis for investigating the role of this cytopathic effect in CMV pathogenesis

CD45 is internalized from the plasma membrane in m42-expressing cells.

<p>(A, B) An internalization assay for CD45 was set up by surface labelling RAW264.7 and RAW_m42 cells with primary Abs directed against CD45 (A) or CD71 (B). Cells were then incubated at 37°C for the indicated time periods, followed by labeling of the primary Ab that remained at the cell surface with corresponding PE-conjugated secondary Abs and flow cytometric analysis. The mean fluorescence intensity of the CD45 and CD71 signals was normalized to the values for time point 0. Results represent mean percentages ± SD of 4 independent experiments. (C) Representative images indicating localization of CD45 in RAW264.7 or RAW_m42 cells after Ab labeling of surface-resident CD45 and cultivation of the cells for the time periods are shown. After fixation and permeabilization of the cells, Ab-labelled CD45 molecules were stained with Alexa647-conjugated secondary Ab and visualized by confocal laser scanning microscopy. Cell nuclei were stained with Hoechst dye. Scale bars, 10 μm.</p

CD45 surface expression is reduced in MCMV-infected RAW264.7 macrophages.

<p>(A) RAW264.7 cells were either mock infected (open histogram) or infected with MCMVgfp (filled histograms) at an MOI of 3. 24 h p.i. CD45 surface expression was determined by flow cytometry for all cells of the cultures, except dead cells, which were excluded based on 7-AAD staining. Dotted line, isotype control. (B) Localization of CD45 was assessed 24 h p.i. by fluorescence microscopy in uninfected and infected (GFP+) RAW264.7 cells that were fixed, permeabilized and immunostained with a CD45-specific Ab. Cell nuclei were counterstained with Hoechst dye. Scale bars, 10 μm. (C) Schematic representation of the 230-kb MCMV genome (HindIII map), indicating the genes lacking in the respective deletion mutants. (D) RAW264.7 cells were mock-infected (open histograms) or infected (filled histograms) with the indicated deletion mutants, and 24 h p.i. immunostained to analyze CD45 surface levels. Dotted line, isotype control. For (D) gating was on living cells and for samples with infected cells additionally on GFP⁺ cells.</p

Detection of ORF m42-encoded proteins and kinetics of CD45 down-regulation.

<p>(A) Predicted amino acid sequence of the m42 protein with the LPTY (dotted line) and PPSY (underlined) motifs and the putative membrane anchor (gray) marked. (B) RAW264.7 cells were either mock-infected or infected with MCMVgfp, MCMVgfp-m42STOP or MCMVgfp-m42rev, and 24 h p.i. cells were analyzed by immunoblotting for CD45 and m42 expression. IE1 (immediate-early protein 1) served as infection marker and GAPDH as loading control. (C, D) RAW264.7 cells were infected with MCMVgfp, lysed at the indicated time points and expression of CD45 and the viral proteins m42, IE1, E1 and M57 was analyzed by immunoblotting with the respective antibodies. E1, early protein 1; M57, ssDNA-binding protein. (D) In parallel, CD45 surface expression in the MCMVgfp-infected RAW264.7 cells was assessed by flow cytometry. (E, left panel) CD45 surface levels of the parental RAW264.7 cells, the control cell line RAW_ctrl (obtained after transduction with the empty retroviral vector), and the m42-expressing cells were examined by flow cytometry. (right) Expression of CD45, m42 and GAPDH (loading control) in the respective cell lines was determined by immunoblotting.</p

Growth of the m42 mutant <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Murine embryonic fibroblasts (MEF) or bone marrow-derived macrophages (BMDM) were infected at an MOI of 0.1 with MCMVwt, MCMV-m42STOP or MCMV-m42rev. For the indicated time period cell culture supernatant was harvested daily and viral titers were determined by plaque assay on MEF. Each data point represents the average titer ± SD of triplicate cultures. (B) BALB/c mice were infected intraperitoneally with 2 × 10⁵ PFU of MCMVwt, MCMV-m42STOP or MCMV-m42rev. At the indicated time points viral titers of organ homogenates were determined by plaque assay on MEF. Data are representative of two independent experiments. (C) Viral DNA loads in organs of BALB/c mice infected with 2 × 10⁵ PFU via the footpad route with MCMV-m42STOP or MCMV-m42rev were measured on day 3 and 5 p.i. qPCR was performed for the viral gene M55 (encoding glycoprotein B) and data were normalized to 10⁵ cells by <i>pthrp</i>-specific qPCR. Each symbol corresponds to an individual mouse; horizontal bars indicate median values. <i>P</i> values < 0.05 were considered statistically significant.</p

Unaffected CD45 expression in RAW264.7 cell lines expressing m42 variants with a mutated PPSY motif.

<p>(A) Analysis of CD45 and m42 levels in RAW264.7 cells stably expressing m42 variants with disrupted PPxY motif. Lysates of RAW264.7, RAW_ctrl, RAW_m42, RAW_m42AASY and RAW_m42PPSA cells were examined by immunoblotting for expression of CD45 and m42. GAPDH served as loading control. (B) CD45 and CD71 surface expression of the indicated cell line was measured by flow cytometry. (C) Proposed model for m42-mediated down-regulation of CD45. By interaction of m42 with Nedd4-like ubiquitin ligases of the HECT family, a conformational change is induced. The thereby activated ubiquitin ligase marks CD45 for internalization and subsequent degradation.</p

MCMV infects the neonatal lung but not the mucosa of the gastrointestinal tract.

<p>Neonatal mice were (A–E) fed or (F–K) l.p. infected with either (A) latex microspheres, (C, E, F, H) mock inoculums, (H, I–K) 5×10⁴ PFU or (B, D, E, G, H) 10⁶ PFU of MCMV-3D and analyzed one day after application. (A) Histological analysis of distal colon after inoculation of latex microspheres. (B) Oral cavity with bony palate was analyzed by epifluorescence microscopy, green and red channels show autofluorescent tissue. (C–E) Intestines were explanted from the proximal esophagus to distal colon in one piece, (F–H) respiratory tract with trachea. (C) Mock inoculum and (D) MCMV-3D inoculum show only autofluorescent tissue but no mCherry⁺ cells in the intestine, inlay in (D) shows autofluorescence in the red channel in high magnification. Luciferase activity from homogenates of (E) flushed intestines or (H) lungs. (F) Mice after mock inoculation show autofluorescent tissue or (G) multiple mCherry⁺ cells, inlay displays single mCherry⁺ cells in high magnification. (I–K) Frozen sections of infected neonatal lungs. (I) Overview with solitary mCherry⁺ infected cells (arrows). (J, K) Solitary mCherry⁺ infected cell with (J) pro-surfactant protein C (proSPC) or (K) CD45 staining. (A–D, F, G, I–K) Representative from >3 experiments with n = 2–3 animals per experiment. (E) Pooled from two independent experiments, n = 4–5, median & range. (H) Pooled from two independent experiments, n = 3–4, median & range. Scale bars: (B–D, F, G) 1000 µm, (A, I) 100 µm and (J, K) 10 µm.</p

Visualization of cross-presentation within nodular inflammatory foci of neonatal mice.

<p>(A) Experimental setup: neonatal CD11c-YFP transgenic mice were infected with 5×10⁴ PFU SIINFEKL-encoding MCMV-3D, 4 days after infection 5×10⁶ naïve OTIxCFP T cells were adoptively i.p. transferred, 1 day after transfer lungs were analyzed by 2-photon microscopy. (B) Representative 2-photon microscopy image from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003828#ppat.1003828.s012" target="_blank">Movie S4</a> of nodular inflammatory foci in surpass mode and z-axis sequential images from one time-point of framed area are depicted, lines in z4 indicate distances from OTI to APC or infected cell, arrow indicates synapse between infected cell (red) and APC (yellow), arrow head indicates synapse between OTI T cell (blue) and APC (yellow). (C) Distances from OTIxCFP T cell center to surface of either mCherry⁺ or APCs at first time-point of Movies are depicted. (D) Contact-duration of OTIxCFP with APCs was estimated from 12–31 min Movies. (E) Percentage of OTIxCFP T cells with APC contacts where APCs are either <i>in</i> or <i>not in</i> contact with mCherry⁺ signal. (F) Neonatal mice were l.p. infected with 5×10⁴ PFU MCMV-3D, 5 days p.i. 5×10⁶ naïve OTIxGFP T cells were adoptively i.p. transferred and one day later mice were sacrificed and lungs explanted, frozen sections were stained with indicated antibodies and DAPI. (C–E) Means & SD. (B–E) Data from n = 4 animals from 2 independent experiments, (F) representative from n = 4 animals from 3 independent experiments. Scale bars (B) 15 µm, (F) 10 µm.</p