Cytomegalovirus replicon-based regulation of gene expression in vitro and in vivo.

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.

M94 is essential for the secondary envelopment of murine cytomegalovirus.

The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.

The m74 gene product of murine cytomegalovirus (MCMV) is a functional homolog of human CMV gO and determines the entry pathway of MCMV.

The glycoprotein gO (UL74) of human cytomegalovirus (HCMV) forms a complex with gH/gL. Virus mutants with a deletion of gO show a defect in secondary envelopment with the consequence that virus spread is restricted to a cell-associated pathway. Here we report that the positional homolog of HCMV gO, m74 of mouse CMV (MCMV), codes for a glycosylated protein which also forms a complex with gH (M75). m74 knockout mutants of MCMV show the same spread phenotype as gO knockout mutants of HCMV, namely, a shift from supernatant-driven to cell-associated spread. We could show that this phenotype is due to a reduction of infectious virus particles in cell culture supernatants. m74 knockout mutants enter fibroblasts via an energy-dependent and pH-sensitive pathway, whereas in the presence of an intact m74 gene product, entry is neither energy dependent nor pH sensitive. This entry phenotype is shared by HCMV expressing or lacking gO. Our data indicate that the m74 and UL74 gene products both codetermine CMV spread and CMV entry into cells. We postulate that MCMV, like HCMV, expresses alternative gH/gL complexes which govern cell-to-cell spread of the virus.

Engineering of cytomegalovirus genomes for recombinant live herpesvirus vaccines.

The advances of sequence knowledge and genetic engineering hold a great promise for a rational approach to vaccine development. Herpesviruses are important pathogens of all vertebrates. They cause acute and chronic infections and persist in their hosts for life. In man there are eight herpesviruses known and most of them can be linked to diseases. To date only one licensed vaccine against a human herpesvirus exists and there is no proven successful concept on rational design for herpesvirus vaccines available. Here, we use new reverse genetic systems, based on the 230-kb mouse cytomegalovirus genome to explore new methods of vaccine delivery and of virus attenuation. With regard to virus delivery, we show that the bacterial transfer of the infectious DNA in vivo is theoretically possible but not yet a practical option. With regard to a rational approach of virus attenuation, we consider a selective deletion of viral genes that modulate the immune response of the host.

The role of cell types in cytomegalovirus infection in vivo.

Human cytomegalovirus (HCMV) is the major viral cause of morbidity in immune compromised patients and of pre- and perinatal pathology in newborns. The clinical manifestations are highly variable and the principles which govern these differences cannot be understood without detailed knowledge on tissue specific aspects of HCMV infection. For decades the role of individual cell types during cytomegalovirus infection and disease has been discussed. The pathogenesis of mouse cytomegalovirus (MCMV) mirrors the human infection in many aspects. Although only MCMV infection is studied extensively at the level of organs, the relative contribution of specific cell types to viral pathogenesis in vivo has remained enigmatic. Here we discuss new approaches based on the cre/loxP-system to label nascent virus progeny or to lift a replication block. The salient aspect of this technique is the change of viral genome properties selectively in cells that express cre during infection in vivo. This allowed us to study the role of endothelial cells and hepatocytes for virus dissemination and will permit detailed studies on innate and adaptive immune responses to CMV.

Dominant-negative proteins in herpesviruses - from assigning gene function to intracellular immunization.

Investigating and assigning gene functions of herpesviruses is a process, which profits from consistent technical innovation. Cloning of bacterial artificial chromosomes encoding herpesvirus genomes permits nearly unlimited possibilities in the construction of genetically modified viruses. Targeted or randomized screening approaches allow rapid identification of essential viral proteins. Nevertheless, mapping of essential genes reveals only limited insight into function. The usage of dominant-negative (DN) proteins has been the tool of choice to dissect functions of proteins during the viral life cycle. DN proteins also facilitate the analysis of host-virus interactions. Finally, DNs serve as starting-point for design of new antiviral strategies.

Conditional gene expression systems to study herpesvirus biology in vivo.

Cytomegalovirus (CMV), a prototypic beta-herpesvirus, is an important human pathogen causing protean clinical manifestations in immature and immunocompromised patients. Mechanisms of infection can be studied in a mouse model. Mouse cytomegalovirus (MCMV) resembles in pathogenesis its human counterpart in many ways. Although MCMV infection is studied extensively on the level of organs, the contribution of specific cell types to viral replication in vivo is still elusive. Here we describe our approach based on the the Cre/loxP-system to investigate MCMV infection at the level of cell types in vivo. Using bacterial artificial chromosome (BAC)-technology, we created an MCMV virus containing an enhanced green fluorescent protein (egfp) reporter-gene which is not expressed due to a 'Stop' cassette flanked by two loxP-sites between promoter and coding sequence. Infection of cre-transgenic mice with this reporter virus results in the deletion of the 'Stop' cassette and expression of EGFP in a cell type-specific manner. Using this conditional gene expression system we are able to quantify viral productivity in specific cell types and to determine their contribution to viral dissemination in vivo. Furthermore, the deletion of viral genes can be used to screen for cell type-specificity of viral gene functions. Hence, conditional MCMV mutants allow the study of herpesvirus biology on the level of cell types in vivo.

The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host.

The course of systemic viral infections is determined by the virus productivity of infected cell types and the efficiency of virus dissemination throughout the host. Here, we used a cell-type-specific virus labeling system to quantitatively track virus progeny during murine cytomegalovirus infection. We infected mice that expressed Cre recombinase selectively in vascular endothelial cells or hepatocytes with a murine cytomegalovirus for which Cre-mediated recombination would generate a fluorescently labeled virus. We showed that endothelial cells and hepatocytes produced virus after direct infection. However, in the liver, the main contributor to viral load in the mouse, most viruses were produced by directly infected hepatocytes. Remarkably, although virus produced in hepatocytes spread to hepatic endothelial cells (and vice versa), there was no significant spread from the liver to other organs. Thus, the cell type producing the most viruses was not necessarily the one responsible for virus dissemination within the host.