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Results: 5

1.
Figure 2

Figure 2. Mbtps1wrt are susceptible to establishment of LCMV Cl13 infection in serum, spleen, liver and kidney, but have an early sustained defect in the bone marrow. From: Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell specific manner.

A) Mbtps1wrt are susceptible to LCMV Cl13 replication in serum, spleen, liver and kidney. Mean and SEM are shown from 4–6 mice/group at the indicated times post infection. B) In the bone marrow, monocytes and DC have defective establishment of LCMV Cl13 in Mbtps1wrt. Mean and SEM are shown from 5 mice/group, 5 dpi. *=P-value<0.05. **=P-value<0.005.

Daniel L. Popkin, et al. Cell Host Microbe. ;9(3):212-222.
2.
Figure 3

Figure 3. Mbtps1wrt splenic DC are susceptible to LCMV Cl13 infection. From: Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell specific manner.

A) Splenocytes were stained for the intracellular viral antigen NP conjugated to qDot655 and alexa488. Gated DCs shown were CD11c+, NK1.1−, CD3−. Flow cytometry dataset is summarized in B. B) Mean and SEM from 6 mice are shown. C) Splenocytes were sorted for B, T and dendritic cells. Cells bearing infectious virus were quantitated by infectious center assay. All mice were analyzed 9 dpi. Mean and SEM from 6 mice are shown. *=P-value<0.05. **=P-value<0.005.

Daniel L. Popkin, et al. Cell Host Microbe. ;9(3):212-222.
3.
Figure 1

Figure 1. Mbtps1wrt (wrt) are resistant to LCMV Cl13 infection despite immunodeficiency. From: Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell specific manner.

A) Mbtps1wrt are resistant to persistent LCMV Cl13 infection but the kinetics of viremia are not altered during infection with the acute LCMV Armstrong (ARM) parent strain. Mean and SEM are shown from 6–12 mice/group at the indicated times post infection. B) GP33 ex vivo peptide stimulation and C) viral peptide specific T cell enumeration with tetramers from splenocytes 5dpi. D) Mbtps1wrt mount a CTL immune response to LCMV Cl13, 9 dpi, when WT CTL exhibit exhaustion. GP33 and E) NP396 ex vivo peptide stimulation from splenocytes. Mean and SEM are shown from 6 mice/group. *=P-value<0.05. **=P-value<0.005.

Daniel L. Popkin, et al. Cell Host Microbe. ;9(3):212-222.
4.

Figure 5. Adoptive immunotherapy with Mbtps1wrt bmDC eliminates viral persistence. From: Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell specific manner.

A) bmDC were cultured from WT and wrt mice. Eight million bmDC were adoptively transferred i.v. into 8–10wk old recipient WT B6 mice. Either 1 hour after, or 2 days prior to infection with 2×106 pfu LCMV Cl13, bmDC were adoptively transfered to recipient B6 mice. Serum was harvested and titered at the indicated times. Mean and SEM from 8 mice/group are shown. B) wrt bmDC have higher levels of MHC molecules (class I and class II) and costimulatory molecules (CD80 and CD86). Flow cytometric analysis of bmDC from WT and wrt mice are shown with Mean and SEM for both % of cells in highly activated gate as well as gMFI for MHC and costimulatory molecules from one of 2 similar independent experiments (N=4). *=P-value<0.05.

Daniel L. Popkin, et al. Cell Host Microbe. ;9(3):212-222.
5.

Figure 4. Mbtps1wrt fibroblasts and macrophages support viral growth in contrast to dendritic cells in which viral growth is restricted by the S1P cleavage site, RRLA. From: Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell specific manner.

A) Ex vivo multistep LCMV Cl13 growth curves in wrt vs. WT primary fibroblasts. Mean and SEM are shown for one of 2 similar independent experiments (N=4). B) Western blot of LCMV GP from wrt and WT primary fibroblasts. GPC, GPC non glycosylated (NG) and processed GP2 are visualized with the monoclonal antibody 83.6. Concentrated LCMV stock was used as a positive control. N=3. C) Ex vivo LCMV Cl13 growth curves in wrt vs. WT primary bone-marrow derived macrophages and dendritic cells (bmMO and bmDC, respectively). Cells were inoculated at a multiplicity of infection (MOI) of 0.1 and 0.001 as shown. Mean and SEM of viral titers are shown for one of 2 similar independent experiments (N=4). D) Ex vivo LCMV growth curves in wrt vs. WT bmDC with recombinant virus containing the native LCMV Mbtps1 cleavage site (RRLA), designated S1P, vs. the Furin protease cleavage site (RRRR) in lieu of RRLA, designated FURIN. MOI=0.1. Similar results were seen using either the Armstrong or Cl13 viral backbones. Mean and SEM of viral titers are shown for one of 2 similar independent experiments (N=4). E) Western blot of LCMV GP from wrt and WT bmDC. Lysates were collected 24hpi and blotted for GPC and its cleavage products GP1 and GP2. One of 3 experiments is shown. F) Recombinant viruses containing either the native S1P cleavage site, RRLA, or the Furin recognition site, RRRR, were used to infect wrt and WT mice. Mice were bled on the indicated days post infection and titered for infectious virus. Mean and SEM of viral titers are shown for one of 2 similar independent experiments (N=4). Of note, Cl13FURIN serum levels were significantly higher at 5dpi in wrt mice as compared to the parent virus, Cl13S1P in wrt. *=P-value<0.05. **=P-value<0.005.

Daniel L. Popkin, et al. Cell Host Microbe. ;9(3):212-222.

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