Bodo Plachter

Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus

Human cytomegalovirus, a chief pathogen in immunocompromised people, can persist in a healthy immunocompetent host throughout life without being eliminated by the immune system. Here we show that pp65, the main tegument protein of human cytomegalovirus, inhibited natural killer cell cytotoxicity by an interaction with the activating receptor NKp30. This interaction was direct and specific, leading to dissociation of the linked CD3ζ from NKp30 and, consequently, to reduced killing. Thus, pp65 is a ligand for the NKp30 receptor and demonstrates a unique mechanism by which an intracellular viral protein causes general suppression of natural killer cell cytotoxicity by specific interaction with an activating receptor.

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.

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.

Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus

Marked interstrain differences in the endothelial cell (EC) tropism of human cytomegalovirus (HCMV) isolates have been described. This study aimed to define the step during the replicative cycle of HCMV that determines this phenotype. The infection efficiency of various HCMV strains in EC versus fibroblasts was quantified by immunodetection of immediate early (IE), early and late viral antigens. Adsorption and penetration were analysed by radiolabelled virus binding assays and competitive HCMV-DNA-PCR. The translocation of penetrated viral DNA to the nucleus of infected cells was quantified by competitive HCMV-DNA-PCR in pure nuclear fractions. The intracytoplasmic translocation of capsids that had penetrated was followed by immunostaining of virus particles on a single particle level; this was correlated with the initiation of viral gene expression by simultaneous immunostaining of viral IE antigens. The infectivity of nonendotheliotropic HCMV strains in EC was found to be 100-1000-fold lower when compared to endotheliotropic strains. The manifestation of this phenotype at the level of IE gene expression indicated the importance of initial replication events. Surprisingly, no interstrain differences were detected during virus entry. However, dramatic interstrain differences were found regarding the nuclear translocation of penetrated viral DNA. With nonendotheliotropic strains, the content of viral DNA in the cell nucleus was 100-1000-fold lower in EC when compared to endotheliotropic strains, thereby reflecting the strain differences in IE gene expression. Simultaneous staining of viral particles and viral IE antigen revealed that interstrain differences in the transport of penetrated capsids towards the nucleus of endothelial cells determine the EC tropism of HCMV.

Dense Bodies of Human Cytomegalovirus Induce both Humoral and Cellular Immune Responses in the Absence of Viral Gene Expression

ABSTRACT Infection of fibroblast cell cultures with human cytomegalovirus (HCMV) leads to the production of significant amounts of defective enveloped particles, termed dense bodies (DB). These noninfectious structures contain major antigenic determinants which are responsible for induction of both the humoral and the cellular immune response against HCMV. We tested the hypothesis that, by virtue of their unique antigenic and structural properties, DB could induce a significant immune response in the absence of infectious virus. Mice were immunized with gradient-purified DB, which were either left untreated or subjected to sequential rounds of sonication and freeze-thawing to prevent cellular entry. Titers of neutralizing antibodies induced by DB were in a range comparable to levels present in convalescent human sera. The virus-neutralizing antibody response was surprisingly durable, with neutralizing antibodies detected 12 months following primary immunization. The HCMV-specific major histocompatibility complex class I-restricted cytolytic T-cell (CTL) response was assayed using mice transgenic for the human HLA-A2 molecule. Immunization with DB led to high levels of HCMV-specific CTL in the absence of de novo viral protein synthesis. Maximal total cytolytic activity in mice immunized with DB was nearly as efficient as the cytolytic activity induced by a standard immunization with murine cytomegalovirus. Furthermore, DB induced a typical T-helper 1 (Th1)-dominated immune response in mice, as determined by cytokine and immunoglobulin G isotype analysis. Induction of humoral and cellular immune responses was achieved without the concomitant use of adjuvant. We thus propose that DB can serve as a basis for the future development of a recombinant nonreplicating vaccine against HCMV. Finally, such particles could be engineered for efficient delivery of antigens from other pathogens to the immune system.

Intracellular localization and expression of the human cytomegalovirus matrix phosphoprotein pp71 (ppUL82): evidence for its translocation into the nucleus

A polyclonal antiserum, raised against a pp71 fusion protein, was prepared in order to investigate the biosynthesis and localization of the matrix protein pp71 of human cytomegalovirus (HCMV), the UL82 gene product, during the HCMV infectious cycle in human fibroblasts. Transcription of the pp71-specific bicistronic 4.0 kb mRNA and pp71 biosynthesis exhibited a biphasic pattern during one round of the HCMV infectious cycle, with a first peak at 12 h and a second at 72 h post-infection (p.i.). Cycloheximide treatment of infected human fibroblasts revealed that the presence of pp71 in total cell extracts prior to 3 h p.i. was due to the input virus inoculum. Transcription of the two specific pp71 mRNAs commenced 5-7 h p.i. as shown by Northern blot analysis of total cellular RNA. Western blot analysis of isolated nuclei and indirect immunofluorescence experiments indicated that pp71, like the major tegument protein pp65, is present in the nucleus shortly after infection as well as during the late phase of viral morphogenesis. Also, after transient transfection of UL82 into U37 3MG cells, pp71 was found to be present in the nucleus of the transfected cells. By immunogold labelling, pp71 was detected in the nucleoplasm in association with nucleocapsids in electron-dense nuclear skein structures at late stages of the infection cycle. These findings suggest functions of pp71 in viral maturation in addition to that as an early transactivator of viral gene transcription described recently.

Quantification of replication of clinical cytomegalovirus isolates in cultured endothelial cells and fibroblasts by a focus expansion assay.

A method for quantitative analysis of the growth properties of human cytomegalovirus (HCMV) in various cell culture systems was developed. Recent HCMV isolates are, in most cases cell associated, causing only limited cytopathic effect. This renders comparative analysis of the biological properties of such isolates difficult. The focus expansion assay described in this study is based on cocultivation of infected fibroblasts with a cell type of choice, relying on cell mediated infectivity. The extent of replication of a given isolate in cell culture is quantified by determining the size of resulting infectious foci. Analysis of various clinical isolates and laboratory strains indicated that this assay is a reliable and valid method to define growth properties of HCMV in cell culture. Remarkable differences in the cytopathogenicity of these isolates in fibroblasts as well as in endothelial cells were found. The assay will be useful in studies regarding cell tropism and virulence of recent HCMV isolates and for the quick and easy phenotypic characterization of HCMV deletion mutants.

Inhibition of CD1 Antigen Presentation by Human Cytomegalovirus

ABSTRACT The betaherpesvirus human cytomegalovirus (HCMV) encodes several molecules that block antigen presentation by the major histocompatibility complex (MHC) proteins. Humans also possess one other family of antigen-presenting molecules, the CD1 family; however, the effect of HCMV on CD1 expression is unknown. The majority of CD1 molecules are classified on the basis of homology as group 1 CD1 and are present almost exclusively on professional antigen-presenting cells such as dendritic cells, which are a major target for HCMV infection and latency. We have determined that HCMV encodes multiple blocking strategies targeting group 1 CD1 molecules. CD1 transcription is strongly inhibited by the HCMV interleukin-10 homologue cmvIL-10. HCMV also blocks CD1 antigen presentation posttranscriptionally by the inhibition of CD1 localization to the cell surface. This function is not performed by a known HCMV MHC class I-blocking molecule and is substantially stronger than the blockage induced by herpes simplex virus type 1. Antigen presentation by CD1 is important for the development of the antiviral immune response and the generation of mature antigen-presenting cells. HCMV present in antigen-presenting cells thus blunts the immune response by the blockage of CD1 molecules.

The DNA-binding protein P52 of human cytomegalovirus reacts with monoclonal antibody CCH2 and associates with the nuclear membrane at late times after infection

Monoclonal antibody CCH2 is commonly used for the detection of human cytomegalovirus (HCMV) infected cells in tissue sections as well as in cultured cells. The specificity of CCH2 was determined by screening a recombinant lambda-gt11 cDNA gene bank from HCMV-infected fibroblasts. By sequencing a reactive clone, the antigen was identified to be the non-structural DNA binding protein p52 of HCMV (UL44 reading frame). The viral insert from the lambda clone was recloned in bacterial expression vectors. For this, a new vector, pRos-RS, was constructed. The resulting clones were tested in immunoblot analyses. They were reactive with CCH2 as well as with reconvalescent sera positive for antibodies against HCMV, by this proving the specificity of CCH2. Using this monoclonal antibody in confocal microscopy, the subcellular localization of p52 in infected cells was analyzed. In these analyses, p52 was found to be nuclear and to be associated with the nuclear membrane at late times after infection.

Immune evasion proteins gpUS2 and gpUS11 of human cytomegalovirus incompletely protect infected cells from CD8 T cell recognition.

Human cytomegalovirus (HCMV) encodes four glycoproteins, termed gpUS2, gpUS3, gpUS6 and gpUS11 that interfere with MHC class I biosynthesis and antigen presentation. Despite gpUS2-11 expression, however, HCMV infection is efficiently controlled by cytolytic CD8 T lymphocytes (CTL). To address the role of gpUS2 and gpUS11 in antigen presentation during viral infection, HCMV mutants were generated that expressed either gpUS2 or gpUS11 alone without coexpression of the three other proteins. Fibroblasts infected with these viruses showed reduced HLA-A2 and HLA-B7 surface expression. Surprisingly, however, CTL directed against the tegument protein pp65 and the regulatory IE1 protein still recognized and lysed mutant virus infected fibroblasts. Yet, suppression of IE1 derived peptide presentation by gpUS2 or gpUS11 was far more pronounced. The results show that gpUS2 and gpUS11 alone only incompletely protect HCMV infected fibroblasts from CTL recognition and underline the importance of studying infected cells to elucidate HCMV immune evasion.