Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confers in vivo genetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.

Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.

We report the primary sequence analyses of two loci, hel and ccl, whose gene products are required specifically for the biogenesis of c-type cytochromes in the Gram-negative photosynthetic bacterium Rhodobacter capsulatus. Genetic and molecular analyses show that the hel locus contains at least four genes, helA, helB, helC, and orf52, and the ccl locus contains two genes, cell and ccl2, that are essential for cytochromes c biogenesis. HelA is homologous to a class of proteins called ABC transporters and helA, belB, and helC are proposed to encode an export complex. Cytochrome C2-alkaline phosphatase gene fusions were used to show that apocytochrome C2 synthesis and secretion are not affected by the hel and ccl defects. Cell and Ccl2 possess typical signal sequences to direct them to the periplasm. The periplasmic orientation of Cell was confirmed using a CcU-alkaline phosphatase gene fusion. The CcU-alkaline phosphatase gene fusion analysis also demonstrated that Cell does not require hel genes for its synthesis and secretion. Cell is homologous to proteins encoded by chloroplast and mitochondrial genes, suggesting analogous functions in these organelles. Taken together, these results support the hypothesis that the hel-encoded proteins are required for the export of heme to the periplasm where it is subsequently ligated to the c-type apocytochromes.

No abstract

Rhodobacter capsulatus is a

Cytokines and chemokines activate and direct effector cells during infection. We previously identified a functional group of five cytokines and chemokines, namely, IFN-γ, activation-induced T cell-derived and chemokine-related cytokine/lymphotactin, macrophage-inflammatory protein 1α, macrophage-inflammatory protein 1β, and RANTES, coexpressed in individual activated NK cells, CD8+ T cells, and CD4+ Th1 cells in vitro and during in vivo infections. However, the stimuli during infection were not known. In murine CMV (MCMV) infection, the DAP12/KARAP-associated Ly49H NK cell activation receptor is crucial for resistance through recognition of MCMV-encoded m157 but NK cells also undergo in vivo nonspecific responses to uncharacterized stimuli. In this study, we show that Ly49H ligation by m157 resulted in a coordinated release of all five cytokines/chemokines from Ly49H+ NK cells. Whereas other cytokines also triggered the release of these cytokines/chemokines, stimulation was not confined to the Ly49H+ population. At the single-cell level, the production of the five mediators showed strong positive correlation with each other. Interestingly, NK cells were a major source of these five cytokines/chemokines in vitro and in vivo, whereas infected macrophages produced only limited amounts of macrophage-inflammatory protein 1α, macrophage-inflammatory protein1β, and RANTES. These findings suggest that both virus-specific and nonspecific NK cells play crucial roles in activating and directing other inflammatory cells during MCMV infection.

No abstract

The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.