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. 2009 May 25;388(1):78-90.
doi: 10.1016/j.virol.2009.02.050. Epub 2009 Apr 5.

Rat cytomegalovirus infection depletes MHC II in bone marrow derived dendritic cells

Carmen C Baca Jones  1 , Craig N Kreklywich, Ilhem Messaoudi, Jennifer Vomaske, Erin McCartney, Susan L Orloff, Jay A Nelson, Daniel N Streblow
Affiliations

Rat cytomegalovirus infection depletes MHC II in bone marrow derived dendritic cells

Carmen C Baca Jones et al. Virology. .

Abstract

While cytomegalovirus (CMV) infects and replicates in a multitude of cell types, the ability of the virus to replicate in antigen presenting cells (APCs) is believed to play a critical role in the viral dissemination and latency. CMV infection of APCs and manipulation of their function are important areas of investigation. CMV down regulation of MHC II is reportedly mediated by the HCMV proteins US2, US3, UL83, UL111a (vIL10) or through the induction of cellular IL10. In this study, we demonstrate that rat CMV (RCMV) significantly reduces MHC II expression neither by mechanisms that do not involve orthologues of the known HCMV genes nor by an increase in cellular IL10. Rat bone marrow derived dendritic cells (BMDC) were highly susceptible to infection with RCMV and a recombinant RCMV expressing eGFP. RCMV infection of BMDCs depleted both surface and intracellular MHC II to nearly undetectable levels as well as reduced surface expression of MHC I. The effect on MHC II only occurred in the infected GFP positive cells and is mediated by an immediate early or early viral gene product. Furthermore, treatment of uninfected immature DCs with virus-free conditioned supernatants from infected cells failed to down regulate MHC II. RCMV depletion of MHC II was sensitive to treatment with lysosomal inhibitors but not proteasomal inhibitors suggesting that the mechanism of RCMV-mediated down regulation of MHC II occurs through endocytic degradation. Since RCMV does not encode homologues of US2, US3, UL83 or UL111a, these data indicate a novel mechanism for RCMV depletion of MHC II.

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Figures

Figure 1
Figure 1. Rat Bone Marrow Derived DC Cell Surface Marker Expression
Immature DC cultures were generated from total bone marrow cells isolated from the femurs of adult male Lewis rats and cultured for 9 days in complete media plus GM-CSF and IL-4. Nonadherent cells were removed by washing, and the remaining adherent DCs were incubated for 24 hours in fresh media without (A) and with (B) LPS. A) Cells were analyzed by flow cytometry for cell surface expression of CD8α, CD11b, CD11c, CD45R-B220, CD86 (B7.2), CD80 (B7.1), CD172a, OX42, OX62, and major histocompatibility marker complexes, MHC I (RT1A), and MHC II (RT1B). Isotype control (grey). B) Cells were analyzed at 24 hours post treatment by flow cytometry for cell surface expression of CD86, CD80, MHC I and II (RT1B). Isotype control (grey), LPS treated cells (solid black), Mock treated-no LPS (dashed).
Figure 2
Figure 2. Rat BMDCs Are Highly Susceptible To RCMV Infection
A) BMDCs were infected 24h post LPS treatment (mDC) or mock (iDC) at an MOI=1.0 (top panel) or 0.5 (middle and bottom panels) for 48h and then assessed by flow cytometry for GFP expression. Gate numbers represent the percent of the population positive for GFP. B) BMDC were infected with RCMV-GFP at an MOI=0.1. At 48 hours post-infection, the cells were fixed with 1% paraformaldehyde, permeabilized and stained with anti-RCMV IE antibody (red) and with Hoechst (blue), GFP (green). Mag=60x. C) Growth kinetics of RCMV-WT (squares) and RCMV-GFP (triangles) in BMDCs. At the indicated time points after infection (days post-infection) the presence of virus (combined intracellular and extracellular) in the cultures was determined by standard plaque assays on rat RFL6 fibroblasts. Viral titers are expressed as plaque forming units per ml and represent the average of three replicates.
Figure 3
Figure 3. RCMV Down Regulates MHC I And II Cell Surface Expression
A–C. iDCs were infected at an MOI equal to 1.0 with either RCMV Wt (thin black line), RCMV GFP (dashed black line) or mock (thick black line) for 48hrs and surface stained for MHC I (A), MHC II (RT1B) (B) or CD86 (C). Isotype controls were depicted with a thin grey line. In panels D and E, iDCs infected with RCMV-GFP at an MOI=0.1 were stained for surface expression of CD80 (D), or CD44H (E). Infected cultures were gated for GFP expression [GFP positive cells (thin dashed line), GFP negative cells (thin black line), mock treated cells (thick black line), and isotype controls (thin grey line)].
Figure 4
Figure 4. RCMV Infected BMDC Are Unable To Induce Allogeneic CD4 T Cell Proliferation In A Mixed Leukocyte Reaction
BMDC isolated from Lewis strain rats were infected with RCMV-Wt for 72h, and cocultured with CFSE labeled splenocytes taken from ACI strain rats for 5 days. Non-adherent cells were stained with anti-CD4 Ab and analyzed by FACS. CD4+ T cell proliferation was measured by CFSE dilution. T cell proliferation is represented as the percent of total CD4+ T cells that have divided. Isotype control (black fill), Uninfected (dark grey fill), RCMV infected (white fill). Shown in the right panels are histograms demonstrating CFSE fluorescence in CD4+ T cells mixed with infected or uninfected DCs.
Figure 5
Figure 5. Intracellular MHC II Is Undetectable In RCMV Infected BMDC
A. BMDCs were infected with RCMV-GFP. Cells were fixed, permeabilized and stained 48hpi with either an IgG isotype control antibody or antibodies directed against CD44H or MHC II (red). Deconvolution microscopy was used to visualize the stained cells. Mag=60x. B. BMDCs were infected with RCMV-Wt at an MOI=0.5. Cells were permeabilized and stained 48hpi with Hoechst (blue), MHC II (green) and anti-RCMV IE antibody (red). Deconvolution microscopy was used to visualize the stained cells. Mag=60x. C. BMDCs were infected with RCMV-WT at an MOI=1 and the cells were harvested in Laemmli’s sample buffer at 48 hours post-infection (lane 2). Uninfected rat RFL6 cells (lane 1) and uninfected BMDCs (lane 3) served as controls. Samples were analyzed by Western blot analysis for the presence of MHC II, RCMV-IE, or RhoA.
Figure 6
Figure 6. RCMV Induces A Stable Depletion Of Cell Surface MHC I And II
A. BMDC were infected with RCMV-GFP at an MOI=0.1 and harvested at the times indicated for analysis via FACS. Uninfected cells were harvested in parallel at the indicated times. Isotype control (grey line), Uninfected cells (thick black line), RCMV infected, GFP positive cells (thin black line), BMDC from the same infection culture but uninfected, GFP negative (dashed black line). B. Western blot analysis of MHC II depletion in RCMV-infected BMDC. BMDC were infected with RCMV-WT at an MOI=1 and the cells were harvested in Laemmli’s sample buffer at the indicated time points after infection. Uninfected rat RFL6 cells and uninfected BMDCs served as controls. Blots were stained for MHC II, RCMV-IE or GAPDH.
Figure 7
Figure 7. RCMV Infection Does Not Alter Gene Expression Of MHC I And II In Rat BMDCs
A. RT-PCR TaqMan was used to detect expression of MHC I and MHC II in uninfected and RCMV-infected rat BMDC at 12, 24, and 36 hpi (n=3). RT-PCR TaqMan detection mRNA from the gene for the ribosomal protein L32 was used to normalize the expression data (n=4). B. Pulse chase analysis was performed in mock- and RCMV-infected BMDC at 24hpi. Cells were starved and then labeled with 35S-methionine/cysteine at 0.2mCi/ml for 10 min. After the pulse, cells were diluted in chase media containing an excess of cold cysteine and methionine and then washed. Cells were then chased for 0.5, 1, 2 or 3 hrs at 37°C and then harvested by lysing the cell pellets in lysis buffer. MHC II was immunoprecipitated using the indicated antibody for 1hr at 4°C, followed by a 2hr incubation with Protein A/G beads. Beads were washed and resuspended in sample buffer and analyzed by SDS-PAGE and visualized by autoradiography. C. Western blot analysis of Ub in BMDC treated with epoxomicin (100nM) or lactacystin (50μM) for 36hrs. Cells were harvested in sample buffer at 36hrs post treatment. Untreated BMDCs (mock) served as controls. Blots were stained for Ubiquitinated proteins, MHC II RT1B and GAPDH. D. Pulse chase analysis was performed in mock- and RCMV-infected BMDC cultures treated with or without the proteosomal inhibitor epoxomicin (100nM). Cells were starved and then labeled with 35S-methionine/cysteine at 0.2mCi/ml for 10min. After the pulse, cells were diluted in chase media containing an excess of cold cysteine and methionine and then washed. Cells were then chased for 1hr at 37°C and then harvested by lysing the cell pellets in lysis buffer. MHC II was immunoprecipitated using the indicated antibody for 1hr at 4°C, followed by a 2hr incubation with Protein A/G beads. Beads were washed and resuspended in sample buffer and analyzed by SDS-PAGE and visualized by autoradiography. A fraction of the cellular lysate for each sample was also probed by western blotting for GAPDH to ensure equal loading.
Figure 8
Figure 8. RCMV Down Regulation Of MHC II Is Sensitive To Treatment With Bafilomycin
A. Western blot analysis of MHC II depletion in RCMV-infected BMDC treated with bafilomicin (100nM). BMDC were infected with RCMV-WT at an MOI=1 for 12 hours, treated with drug for an additional 36hrs and then harvested in sample buffer at 48hpi. Uninfected BMDCs served as controls. Blots were stained for MHC II RT1B and then the blot was stripped and probed for GAPDH. B. BMDCs were infected with RCMV-GFP for 12hrs and then treated with bafilomicin (100nM) for an additional 36 hrs. Cells were fixed at 48hpi, permeabilized and stained with antibodies directed against MHC II RT1B (red) and Hoechst DNA dye (blue). Infection was detected by GFP expression (green). Deconvolution microscopy was used to visualize the stained cells. Mag=60x.
Figure 9
Figure 9. RCMV Induced Reduction In MHC I and II Expression Is Not Mediated By Viral Binding and Entry, Late Gene Expression Or By A Secreted Factor
A. BMDC were infected at an MOI=1 with RCMV-Wt (thin black line) or Wt-UV inactivated (dashed black line) or mock infected (thick black line) for 48h and analyzed by FACS for cell surface expression of MHC I and II. Isotype control (grey). B. BMDC were pretreated with Foscarnet or mock, and then infected at an MOI equal to one with RCMV-Wt. Isotype control (grey), Uninfected + Foscarnet (thick black line), Infected + Foscavir (thin black line), Infected − Foscarnet (dashed black line). C. BMDC were infected with RCMV-Wt (thin black line) or mock (thick black line), or conditioned supernatants from RCMV infected BMDCs (dashed black line), for 48hrs and then assessed for cell surface expression of MHC II. Isotype control (grey line), RCMV-Wt infected (thin black line), uninfected (thick black line), conditioned supernatant treatment (dashed black line).
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