ß-Diazocarbonyl Compounds: Synthesis and their Rh(II)-Catalyzed 1,3 C-H Insertions.

Herein, we describe the first electrophilic diazomethylation of ketone silyl enol ethers with diazomethyl-substituted hypervalent iodine reagents that gives access to unusual ß-diazocarbonyl compounds. The potential of this unexplored class of diazo compounds for the development of new reactions was demonstrated by the discovery of a rare Rh-catalyzed intramolecular 1,3 C-H carbene insertion that led to complex cyclopropanes with excellent stereocontrol.

Modulation of natural killer cells by human cytomegalovirus.

Human cytomegalovirus (HCMV) causes lifelong, persistent infections and its survival is under intense, continuous selective pressure from the immune system. A key aspect of HCMV's capacity for survival lies in immune avoidance. In this context, cells undergoing productive infection exhibit remarkable resistance to natural killer (NK) cell-mediated cytolysis in vitro. To date, six genes encoding proteins (UL16, UL18, UL40, UL83, UL141 and UL142) and one encoding a microRNA (miR-UL112) have been identified as capable of suppressing NK cell recognition. Even though HCMV infection efficiently activates expression of ligands for the NK cell activating receptor NKG2D, at least three functions (UL16, UL142 and miR-UL112) act in concert to suppress presentation of these ligands on the cell surface. Although HCMV downregulates expression of endogenous MHC-I, it encodes an MHC-I homologue (UL18) and also upregulates the expression of cellular HLA-E through the action of UL40. The disruption of normal intercellular connections exposes ligands for NK cell activating receptors on the cell surface, notably CD155. HCMV overcomes this vulnerability by encoding a function (UL141) that acts post-translationally to suppress cell surface expression of CD155. The mechanisms by which HCMV systematically evades (or, more properly, modulates) NK cell recognition constitutes an area of growing understanding that is enhancing our appreciation of the basic mechanisms of NK cell function in humans.

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141.

Natural killer (NK) cells are crucial in the control of cytomegalovirus infections in mice and humans. Here we show that the viral UL141 gene product has an immunomodulatory function that is associated with low-passage strains of human cytomegalovirus. UL141 mediated efficient protection of cells against killing by a wide range of human NK cell populations, including interferon-alpha-stimulated bulk cultures, polyclonal NK cell lines and most NK cell clones tested. Evasion of NK cell killing was mediated by UL141 blocking surface expression of CD155, which was previously identified as a ligand for NK cell-activating receptors CD226 (DNAM-1) and CD96 (TACTILE). The breadth of the UL141-mediated effect indicates that CD155 has a key role in regulating NK cell function.