Investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection

AbstractMurine norovirus (MNV) is a recently discovered pathogen that has become a common contaminant of specific pathogen-free mouse colonies. MNV-1 induces a robust interferon-β response and causes histopathology in some mouse strains, suggesting that it may impact other mouse models of infection. Despite many concerns about MNV-1 contamination, there is little information about its impact on immune responses to other infections. This study addresses whether MNV-1 infection has an effect on a model of murine cytomegalovirus (MCMV) infection. Exposure to MNV-1 resulted in a decreased CD8 T cell response to immunodominant MCMV epitopes in both BALB/c and C57BL/6 mice. However, MNV-1 did not impact MCMV titers in either mouse strain, nor did it stimulate reactivation of latent MCMV. These data suggest that while MNV-1 has a mild impact on the immune response to MCMV, it is not likely to affect most experimental outcomes in immunocompetent mice in the MCMV model

Cross-Presentation of a Spread-Defective MCMV Is Sufficient to Prime the Majority of Virus-Specific CD8+ T Cells

CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection

Correction: Cross-presentation of a spread-defective MCMV is sufficient to prime the majority of virus-specific CD8+ T cells.

[This corrects the article DOI: 10.1371/journal.pone.0009681.]

Cross-priming of antigen from fibroblasts infected with the ΔgL virus results in a similar immundominance hierarchy as wild-type infected fibroblasts.

<p>A) Balb-3T3s, which do not express H-2^b MHC and thus cannot directly stimulate CD8+ T cells from B6 mice, were left uninfected or infected with ΔgL virus for 3 hours. After infection, cells were washed with citrate buffer as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009681#s3" target="_blank">materials and methods</a> and frozen immediately. The indicated peptides, representing a range of CD8+ T cell responses, or thawed lysate of infected cells (equivalent to the number of cells injected into each animal in B and C below) were used to stimulate splenic CD8+ T cells from a chronically infected C57BL/6 mouse and IFN-γ production was measured. B) K^bD^b-/- murine embryonic fibroblasts were infected with K181 or ΔgL viruses and washed as described. Infected cells were injected i.p. into C57BL/6 mice and IFN-γ production by T cells in the spleen was measured 7 days later after stimulation with the indicated peptides (n = 3 mice per group injected with wild-type infected cells and n = 4 mice per group injected with ΔgL infected cells in this experiment). ND  =  not done. The shaded area reflects the background from unstimulated cells in this experiment. Similar results were obtained from peripheral blood CD8+ T cells in an independent experiment. C) Balb-3T3s were infected with ΔgL and washed as above with a citrate buffer. After washing, cells were either left untreated or were frozen and thawed to produce a lysate. C57BL/6 mice were injected with live, ΔgL infected cells or the equivalent amount of frozen and thawed lysate from the same infection. 7 days later, splenic CD8+ T cells were stimulated with the indicated peptides and IFN-γ production was measured.</p

Deletion of glycoprotein L from MCMV can be complemented by gL-expressing cell line.

<p>A) Schematic of the strategy to functionally delete gL from the MCMV genome. B) ΔgL virus produced plaques on complementing cells. ΔgL virus was used to infect gL-expressing NIH-3T3 at the indicated dilutions and plaques were revealed at 10⁻² and 10⁻³ dilutions by crystal violet staining 6 days later. C) The gL gene was detected by PCR in DNA extracted from wild-type or ΔgL viral preparations. D) The ΔgL virus grows with similar kinetics as the wild-type K181 virus in complementing cells. gL-3T3 were infected with the indicated virus at an moi = 2 or moi = 0.1. Cells were harvested at the indicated times and the presence of infectious virus was measured by plaque assay. Filled circles: K181 virus infected gL-3T3. Open circles: ΔgL infected gL-3T3. Filled triangles: K181 infected Balb-3T3. Data represents an individual experiment performed in duplicate.</p

MCMV can not spread from cell to cell without the gL.

<p>A) Virions are produced in non-complementing cells. Non-complementing murine embryonic fibroblasts were infected with the ΔgL virus for 25 hours, fixed and prepared for electron microscopy. Images represent a magnification of 7100x. The insets are enlargements of the areas indicated by black boxes. B) The ΔgL virus does not spread in a culture of murine embryonic fibroblasts. Non-complementing murine embryonic fibroblasts (MEFs) were infected with wild-type or ΔgL virus at an moi = 0.3 and images of viral cytopathic effect were captured 3 days later. Images represent a magnification of 10x. Arrowheads indicate examples of cells displaying the typical cytopathic/rounding effect induced by MCMV infection. The inset in the left panel shows an enlarged image of the cytopathic/rounding effect in MEFs infected for 2 days with the ΔgL virus in an independent experiment. C) Non-complementing cells that survive infection can grow for at least 3 weeks. Non-complementing Balb-3T3 cells were infected with 1×10⁵ pfu (moi = 0.67) of the ΔgL virus. After 3 days rounded cells displaying the typical MCMV cytopathic effect are evident (upper left panel). After 6 days, the cells had grown to confluency (day 6 – upper right panel), were split into a larger volume containing all of the original supernatent and re-plated. Subsequent images were taken 2 days (lower left panel) and 13 days (lower right panel) after re-plating the ΔgL infected fibroblasts. Data are representative of 7 independent experiments. D) The ΔgL virus can not spread in immuno-compromised mice. Balb/c-SCID mice were infected i.p. with 1×10⁵ pfu of ΔgL or wild-type virus. 14 days after infection, DNA was extracted from the spleen and salivary gland (S.G.) and tested for the presence of MCMV by qPCR. Each symbol represents an individual mouse. Copies within the shaded area not distinguishable from background. Similar data was obtained in 2 other independent experiments.</p

Correction: Cross-presentation of a spread-defective MCMV is sufficient to prime the majority of virus-specific CD8+ T cells

[This corrects the article DOI: 10.1371/journal.pone.0009681.]

The ΔgL and K181 wild-type viruses induce a similar immunodominance hierarchy after direct i.p. infection.

<p>A) There are fewer splenocytes in ΔgL infected mice. C57BL/6 mice were infected i.p. with 1×10⁵ pfu K181 or ΔgL virus. 7 days after infection the total number of splenocytes and CD8+ T cells were quantified. Statisitical significance was determined by a Student's t-test. Data represents an average of 3 separate mice in this experiment. B) Immunodominant CD8+ T cell responses are readily detected in the blood of ΔgL infected mice. C57BL/6 mice were infected with the indicated viruses and the CD8+ T cell responses in the peripheral blood were examined by tetramer staining 7 days later (n = 7 mice per group combined from 2 independent experiments). Data is representative of at least 5 independent experiments. C) The immunodominance hierarchies induced by wild-type and ΔgL infections are remarkably similar. C57BL/6 mice were infected i.p. with 1×10⁵ pfu of the indicated viruses and spleens were harvested 7 days later (n = 3 mice per group). Cells were stimulated with the indicated peptides and the production of IFN-γ was measured as described. The shaded area reflects the background from unstimulated cells in this experiment. Similar data was obtained in an independent experiment that included a more limited subset of MCMV peptides (not shown).</p