Modulation of Clr Ligand Expression and NKR-P1 Receptor Function during Murine Cytomegalovirus Infection

Abstract

Viruses are known to induce pathological cellular states that render infected cells susceptible or resistant to immune recognition. Here, we characterize an MHC-I-independent natural killer (NK) cell recognition mechanism that involves modulation of inhibitory NKR-P1B:Clr-b receptor-ligand interactions in response to mouse cytomegalovirus (MCMV) infection. We demonstrate that mouse Clr-b expression on healthy cells is rapidly lost at the cell surface and transcript levels in a time- and dose-dependent manner upon MCMV infection. In addition, cross-species infections using rat cytomegalovirus (RCMV) infection of mouse fibroblasts and MCMV infection of rat fibroblasts suggest that this response is conserved during host-pathogen interactions. Active viral infection appears to be necessary for Clr-b loss, as cellular stimulation using UV-inactivated whole virus or agonists of many innate pattern recognition receptors failed to elicit efficient Clr-b downregulation. Notably, Clr-b loss could be partially blocked by titrated cycloheximide treatment, suggesting that early viral or nascent host proteins are required for Clr-b downregulation. Interestingly, reporter cell assays suggest that MCMV may encode a novel Clr-b-independent immunoevasin that functionally engages the NKR-P1B receptor. Together, these data suggest that Clr-b modulation is a conserved innate host cell response to virus infection that is subverted by multiple CMV immune evasion strategies.

Interferon-Dependent Induction of Clr-b during Mouse Cytomegalovirus Infection Protects Bystander Cells from Natural Killer Cells via NKR-P1B-Mediated Inhibition

“…Previous studies have shown that infection of various mouse and rat cells with a number of viruses (MCMV, RC-MV-E, vaccinia, ectromelia) promotes a rapid loss of mouse Clr-b ( Clec2d ) and the rat Clr-b homolog rClr-11 ( Clec2d11 ) at both the steady-state transcript and cell surface levels [6,16,18,19] . To distinguish between infected and uninfected (bystander) cells at the single cell level, we infected mouse NIH3T3 fibroblasts over an early time course using a modified MCMV-GFP reporter virus in which an enhanced GFP transgene is driven by an immediate early gene (ie1/3) promoter in the MCMV-Smith (VR-194) strain [19][20][21] .…”

“…To distinguish between infected and uninfected (bystander) cells at the single cell level, we infected mouse NIH3T3 fibroblasts over an early time course using a modified MCMV-GFP reporter virus in which an enhanced GFP transgene is driven by an immediate early gene (ie1/3) promoter in the MCMV-Smith (VR-194) strain [19][20][21] . While we consistently observed a loss of Clr-b surface expression on the infected population at later time points (GFP + , 12-24 h postinfection), at early time points the uninfected bystander population expressed elevated Clr-b surface levels relative to mock-infected parental cells (GFP -, 3-12 h postinfection; Fig.…”

“…MCMV is a β-herpesvirus with a large double-stranded DNA genome capable of accommodating numerous immunoevasin genes that subvert host immune responses. Previous studies have shown that MCMV, RCMV-E, and vaccinia virus infections promote a rapid loss of mouse Clr-b/ Clec2d and rat Clr-11/ Clec2d11 on fibroblasts [16,18,19] . Interestingly, at early time points during MCMV infection in vitro, uninfected fibroblasts actually upregulate Clr-b expression, as do cells exposed to passaged viral supernatants.…”

“…Clr-b ( Clec2d ) is a type-II transmembrane C-type lectin-like glycoprotein expressed on most hematopoietic and some nonhematopoietic cells in a pattern similar to that of MHC-I molecules [8] . However, under pathological conditions such as oncogenesis, virus infection, and genotoxic stress, Clr-b is rapidly downregulated at both the cell surface and steady-state transcript levels, rendering these cells more sensitive to NK cell-mediated "missing-self" recognition [8,[16][17][18][19] . The regulatory mechanisms governing the modulation of Clr-b expression remain to be elucidated, although previous work has suggested roles for transcriptional and posttranscriptional, as well as ubiquitin-proteasomal and endolysosomal, control [16][17][18][19] .…”

Expansion and Protection by a Virus-Specific NK Cell Subset Lacking Expression of the Inhibitory NKR-P1B Receptor during Murine Cytomegalovirus Infection

“…Its interaction with NKR-P1B inhibits NK cells and increases rat CMV virulence in rats (42). The MCMV genome also appears to encode for Clr-bindependent ligands, which can functionally engage NKR-P1B receptor in reporter cell assays (43). However, their identity, and whether they bear homology to Clr-b, remains to be determined.…”