Species-Specific Inhibition of Necroptosis by HCMV UL36.

Viral infection activates cellular antiviral defenses including programmed cell death (PCD). Many viruses, particularly those of the Herpesviridae family, encode cell death inhibitors that antagonize different forms of PCD. While some viral inhibitors are broadly active in cells of different species, others have species-specific functions, probably reflecting the co-evolution of the herpesviruses with their respective hosts. Human cytomegalovirus (HCMV) protein UL36 is a dual cell death pathway inhibitor. It blocks death receptor-dependent apoptosis by inhibiting caspase-8 activation, and necroptosis by binding to the mixed lineage kinase domain-like (MLKL) protein and inducing its degradation. While UL36 has been shown to inhibit apoptosis in human and murine cells, the specificity of its necroptosis-inhibiting function has not been investigated. Here we show that UL36 interacts with both human and murine MLKL, but has a higher affinity for human MLKL. When expressed by a recombinant mouse cytomegalovirus (MCMV), UL36 caused a modest reduction of murine MLKL levels but did not inhibit necroptosis in murine cells. These data suggest that UL36 inhibits necroptosis, but not apoptosis, in a species-specific manner, similar to ICP6 of herpes simplex virus type 1 and MC159 of molluscum contagiosum virus. Species-specific necroptosis inhibition might contribute to the narrow host range of these viruses.

Copy-Paste Mutagenesis: A Method for Large-Scale Alteration of Viral Genomes.

The cloning of the large DNA genomes of herpesviruses, poxviruses, and baculoviruses as bacterial artificial chromosomes (BAC) in Escherichia coli has opened a new era in viral genetics. Several methods of lambda Red-mediated genome engineering (recombineering) in E. coli have been described, which are now commonly used to generate recombinant viral genomes. These methods are very efficient at introducing deletions, small insertions, and point mutations. Here we present Copy-Paste mutagenesis, an efficient and versatile strategy for scarless large-scale alteration of viral genomes. It combines gap repair and en passant mutagenesis procedures and relies on positive selection in all crucial steps. We demonstrate that this method can be used to generate chimeric strains of human cytomegalovirus (HCMV), the largest human DNA virus. Large (~15 kbp) genome fragments of HCMV strain TB40/E were tagged with an excisable marker and cloned (copied) in a low-copy plasmid vector by gap repair recombination. The cloned fragment was then excised and inserted (pasted) into the HCMV AD169 genome with subsequent scarless removal of the marker by en passant mutagenesis. We have done four consecutive rounds of this procedure, thereby generating an AD169-TB40/E chimera containing 60 kbp of the donor strain TB40/E. This procedure is highly useful for identifying gene variants responsible for phenotypic differences between viral strains. It can also be used for repair of incomplete viral genomes, and for modification of any BAC-cloned sequence. The method should also be applicable for large-scale alterations of bacterial genomes.

Laboratory diagnostics of murine blood for detection of mouse cytomegalovirus (MCMV)-induced hepatitis.

Mouse models are important and versatile tools to study mechanisms and novel therapies of human disease in vivo. Both, the number and the complexity of murine models are constantly increasing and modification of genes of interest as well as any exogenous challenge may lead to unanticipated biological effects. Laboratory diagnostics of blood samples provide a comprehensive and rapid screening for multiple organ function and are fundamental to detect human disease. Here, we adapt an array of laboratory medicine-based tests commonly used in humans to establish a platform for standardized, multi-parametric, and quality-controlled diagnostics of murine blood samples. We determined sex-dependent reference intervals of 51 commonly used laboratory medicine tests for samples obtained from the C57BL/6J mouse strain. As a proof of principle, we applied these diagnostic tests in a mouse cytomegalovirus (MCMV) infection model to screen for organ damage. Consistent with histopathological findings, plasma concentrations of liver-specific enzymes were elevated, supporting the diagnosis of a virus-induced hepatitis. Plasma activities of aminotransferases correlated with viral loads in livers at various days after MCMV infection and discriminated infected from non-infected animals. This study provides murine blood reference intervals of common laboratory medicine parameters and illustrates the use of these tests for diagnosis of infectious disease in experimental animals.