Christian Sinzger

FIBROBLASTS, EPITHELIAL-CELLS, ENDOTHELIAL-CELLS AND SMOOTH-MUSCLE CELLS ARE MAJOR TARGETS OF HUMAN CYTOMEGALOVIRUS-INFECTION IN LUNG AND GASTROINTESTINAL TISSUES

High titre replication of human cytomegalovirus (HCMV) in cell culture is restricted to primary human fibroblasts. During acute infection in vivo, HCMV nucleic acids and antigens have been found in various organs. Using only morphological criteria, inconsistent data have been reported about the cell types that can be infected by HCMV. In particular, the role of fibroblasts in organ infections has remained unclear. To define accurately the target cells of HCMV in vivo, tissue sections from lung and gastrointestinal tract of patients suffering from acute HCMV infection were investigated using immunohistochemical double-labelling analyses. Monoclonal antibodies with defined specificity against immediate early (IE), early (E) and late (L) viral antigens and antibodies directed against cell marker proteins were employed to identify infected cells. The results demonstrated that a broad spectrum of cells was infected by HCMV in vivo. Consistent with their susceptibility in culture, fibroblasts formed a major population of HCMV-infected cells. In contrast, haemopoietic cells were only infrequently stained with virus-specific antibodies. Fibroblasts, epithelial cells, endothelial cells, smooth muscle cells and macrophages appeared to be permissive for HCMV replication. Contrary to this, polymorphonuclear cells showed only IE gene expression, indicating that these cells were abortively infected. The analysis of the distribution of infected cells in tissue supported the hypothesis that endothelial cells and monocytes/macrophages may play a crucial role in the haematogenous spread of HCMV; in contrast, fibroblasts, smooth muscle cells and epithelial cells may form the cell populations important for the multiplication and spread of the virus in infected tissues.

Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein

Human cytomegalovirus (HCMV) has evolved a multitude of molecular mechanisms to evade the antiviral immune defense of the host. Recently, using soluble recombinant molecules, the HCMV UL16 glycoprotein was shown to interact with some ligands of the activating immunoreceptor NKG2D and, therefore, may also function as a viral immunomodulator. However, the role of UL16 during the course of HCMV infection remained unclear. Here, we demonstrate that HCMV infection of fibroblasts induces expression of all known NKG2D ligands (NKG2DL). However, solely MICA and ULBP3 reach the cellular surface to engage NKG2D, whereas MICB, ULBP1 and ULBP2 are selectively retained in the endoplasmic reticulum by UL16. UL16‐mediated reduction of NKG2DL cell surface density diminished NK cytotoxicity. Thus, UL16 functions by capturing activating ligands for cytotoxic lymphocytes that are synthesized in response to HCMV infection.

Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E

Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

Cytomegalovirus Cell Tropism

The human cytomegalovirus (HCMV) can infect a remarkably broad cell range within its host, including parenchymal cells and connective tissue cells of virtually any organ and various hematopoietic cell types. Epithelial cells, endothelial cells, fibroblasts and smooth muscle cells are the predominant targets for virus replication. The pathogenesis of acute HCMV infections is greatly influenced by this broad target cell range. Infection of epithelial cells presumably contributes to inter-host transmission. Infection of endothelial cells and hematopoietic cells facilitates systemic spread within the host. Infection of ubiquitous cell types such as fibroblasts and smooth muscle cells provides the platform for efficient proliferation of the virus. The tropism for endothelial cells, macrophages and dendritic cells varies greatly among different HCMV strains, mostly dependent on alterations within the UL128-131 gene locus. In line with the classification of the respective proteins as structural components of the viral envelope, interstrain differences concerning the infectivity in endothelial cells and macrophages are regulated on the level of viral entry.

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-α-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.

Risk factors for treatment failures in patients receiving PCR-based preemptive therapy for CMV infection

PCR-based preemptive therapy with ganciclovir has been shown to reduce the incidence of CMV disease after BMT. Failures of this treatment strategy are CMV disease and secondary non-viral infections. Eighty-six consecutive patients at high risk for CMV disease who received PCR-based preemptive therapy with ganciclovir were assessed for treatment failures and possible risk factors. Ganciclovir was initiated in 57 of 86 patients (66%). Only 28 of 86 (32%) patients received 4 or more weeks of ganciclovir. Recurrence of CMV infection after successful treatment was more frequent among recipients of a BMT from an unrelated compared to a sibling donor (P = 0.004). three (3.5%) patients developed non-fatal early onset cmv disease and seven of 68 (10.3 %) late onset cmv disease (>100 days post transplant). Risk factors for late onset CMV disease were cGVHD (P = 0.0017) and duration of prior antiviral therapy >4 weeks (P = 0.0073). The incidence of secondary non-viral infections was 28% with the duration of antiviral treatment being a significant risk factor for secondary bacterial (P = 0.0045) and invasive fungal infections (P = 0.006). Thus, PCR-based preemptive treatment with ganciclovir reduces early onset CMV disease, but the duration of antiviral therapy prior to day +100 is a significant risk factor for late onset CMV disease as well as secondary non-viral infections. Bone Marrow Transplantation (2000) 25, 757–763.

Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome

Following extensive propagation in fibroblasts, human cytomegalovirus (HCMV) loses tropism for a number of otherwise natural host cells, in particular, endothelial cells. In this study, the hypothesis was tested that loss of endothelial tropism is associated with the appearance of genomic variants. Initial quantitative focus expansion assays on endothelial monolayers demonstrated that, while the laboratory strains AD169 and Towne failed to form detectable foci, 29 out of 30 recent clinical HCMV isolates had the potential to expand in endothelial cell culture. By long-term adaptation in fibroblast cultures, nonendotheliotropic strains could be selected from clinical HCMV isolates, while long-term endothelial-adapted strains of the same isolates retained both fibroblast tropism and endothelial tropism. Such differentially adapted isolate pairs always displayed genomic differences in restriction fragment length analyses. Coinfection of endothelial cells by two nonendotheliotropic HCMV strains yielded an endotheliotropic recombinant HCMV variant combining portions of the genomes of both parental viruses. When DNA purified from various isolates was transfected into fibroblasts, progeny virus retained the specific tropism of parental virus from which the DNA was isolated. These findings demonstrate that endothelial tropism is an inherent property of most clinical HCMV isolates and is determined by the viral genome. Although the specific determinants of HCMV cell tropism are still unknown, this study provides the first evidence for a genetic contribution.

Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus.

The susceptibility of monocyte-derived immature dendritic cells (DC) to infection by various strains of human cytomegalovirus (HCMV) was analysed. Immature DC were generated by incubation of peripheral blood monocytes with interleukin-4 and granulocyte-macrophage colony-stimulating factor for 7 days and were characterized by a CD1a+/CD40+/CD80+/CD86+/HLA-DR+/CD14- phenotype. Viral antigen expression and production of infectious progeny virus were analysed in infected immature DC cultures. Immature DC were 80-90 % susceptible to HCMV strains that had been propagated in endothelial cell culture, whereas the infection rate was negligible with fibroblast-adapted HCMV strains. Immature DC infection resulted in expression of viral immediate early, early and late genes. Productive infection was proven by the detection of infectious virus in single-step growth curves and in infectious centre assays. It is concluded that HCMV might interfere with the host immune reaction by permissive, lytic infection of immature DC.

Human Cytomegalovirus Cell Tropism and Pathogenesis

The human cytomegalovirus (HCMV) can cause lifelong infection with episodes of endogenous reactivation. Intrauterine fetal infection and infection of immunocompromised patients are well known to result in significant morbidity. Studies on HCMV cell tropism in vivo revealed three characteristics: (1) ubiquitously distributed cell types such as epithelial cells, endothelial cells and fibroblasts are the major targets of HCMV infection; (2) leukocytes circulating in the peripheral blood are susceptible to the virus, and (3) specialized parenchymal cells such as smooth muscle cells in the gastrointestinal tract and hepatocytes can also be infected. Questions to be resolved are, how the virus spreads throughout the organism, how it can impair the function of infected organs, and how it evades the hosts immune response to establish lifelong infection. This chapter is focused on the role of HCMV-infected target cells for the pathogenesis of HCMV-associated disease in the acutely infected immunocompromised host.

Cell Types Involved in Replication and Distribution of Human Cytomegalovirus

As the number of patients suffering from severe HCMV infections has steadily increased, there is a growing need to understand the molecular mechanisms by which the virus causes disease. The factors that control infection at one time and the events leading to virus multiplication at another time are only beginning to be understood. The interaction of HCMV with different host cells is one key for elucidating these processes. Through modern techniques, much has been learned about the biology of HCMV infections in culture systems. In addition to endothelial cells, epithelial cells, and smooth muscle cells, fibroblasts are one cell population preferentially infected in solid tissues in vivo. From these sites of multiplication, the virus may be carried by peripheral monocytes and circulating endothelial cells to reach distant sites of the body. This would explain the multiorgan involvement in acute HCMV infection and the modes of viral transmission. From what has been learned mainly from human fibroblast culture systems, future studies will focus on how HCMV regulates the expression of its putative 200 genes in different host cells at different stages of cell differentiation and activation to result in viral latency and pathogenesis.