Pero Lučin

A Mouse Cytomegalovirus Glycoprotein Retains MHC Class I Complexes in the ERGIC/cis-Golgi Compartments

The principle by which mouse cytomegalovirus blocks antigen presentation in the MHC class I pathway was investigated. The responsible gene m152, encoding a type I transmembrane glycoprotein of 40 kDa, is a member of a gene family located in the right-hand terminal region of the 230 kb virus genome. Expression of m152 in murine and human cells arrested the export of mouse class I complexes from the ER-Golgi intermediate compartment/cis-Golgi compartment and inhibited lysis by cytotoxic T cells. The plasma membrane transport of human MHC class I molecules was not affected. The deletion of the cytoplasmic tail of gp40 did not lift its effect on class I molecule export, indicating that this protein differs in its functions from known immunosubversive viral gene products and represents a novel principle by which a herpesvirus shuts off MHC class I function.

A cytomegalovirus glycoprotein re‐routes MHC class I complexes to lysosomes for degradation

Mouse cytomegalovirus (MCMV) early gene expression interferes with the major histocompatibility complex class I (MHC class I) pathway of antigen presentation. Here we identify a 48 kDa type I transmembrane glycoprotein encoded by the MCMV early gene m06, which tightly binds to properly folded β2‐microglobulin (β2m)‐associated MHC class I molecules in the endoplasmic reticulum (ER). This association is mediated by the lumenal/transmembrane part of the protein. gp48–MHC class I complexes are transported out of the ER, pass the Golgi, but instead of being expressed on the cell surface, they are redirected to the endocytic route and rapidly degraded in a Lamp‐1+ compartment. As a result, m06‐expressing cells are impaired in presenting antigenic peptides to CD8+ T cells. The cytoplasmic tail of gp48 contains two di‐leucine motifs. Mutation of the membrane‐proximal di‐leucine motif of gp48 restored surface expression of MHC class I, while mutation of the distal one had no effect. The results establish a novel viral mechanism for downregulation of MHC class I molecules by directly binding surface‐destined MHC complexes and exploiting the cellular di‐leucine sorting machinery for lysosomal degradation.

A mouse cytomegalovirus glycoprotein, gp34, forms a complex with folded class I MHC molecules in the ER which is not retained but is transported to the cell surface

Murine cytomegalovirus (MCMV) interferes with antigen presentation by means of retaining major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER). Here we identify and characterize an MCMV‐encoded glycoprotein, gp34, which tightly associates with properly conformed MHC class I molecules in the ER. Gp34 is synthesized in large quantities during MCMV infection and it leaves the ER only in association with MHC class I complexes. Many but not all class I molecules are retained in the ER during the early phase of MCMV infection, and we observe an inverse correlation between amounts of gp34 synthesized during the course of infection and class I retention. An MCMV deletion mutant lacking several genes, including the gene encoding gp34, shows increased class I retention. Thus, MCMV gp34 may counteract class I retention, perhaps to decrease susceptibility of infected cells to recognition by natural killer cells.

Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-gamma and tumour necrosis factor

We have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon-gamma (IFN-gamma). We now demonstrate that IFN-gamma and tumour necrosis factor alpha (TNF-alpha) display synergism in their antiviral activity. As little as 2 ng/ml of IFN-gamma and TNF-alpha reduced the virus yield by about three orders of magnitude. There was no effect on immediate early (IE) and early (E) gene expression as far as the candidate genes IE1, E1 and those encoding the major DNA-binding protein and the DNA polymerase were concerned. Late gene transcription, assayed by the candidate genes encoding glycoprotein B and the MCMV homologue of ICP 18.5, was blocked and MCMV DNA replication was found to be reduced but not halted. The most prominent finding of the cytokine effect, seen by electron microscopy, was an alteration of nucleocapsid formation. Altogether, the synergism is multifaceted and acts at more than one stage during viral morphogenesis. Because the cytokines clearly do not act at an early stage of infection we conclude that the mode of cytokine activity differs between alpha- and betaherpesviruses.

Cytomegaloviral control of MHC class I function in the mouse.

Summary: Cytomegaloviruses (CMVs) represent prototypic viruses of the (i‐subgroup of herpesviruses, Murine cytomegalovirus (MCMV) infects mice as its natural host. Among viruses, CMVs have evolved the most extensive genetic repertoire to subvert MHC class I functions. To date three MCMV proteins have been identified which affect MHC I complexes. They are encoded by members of large virus‐specific gene families located at either flanking region of the 235 kb MCMV genome. The MHC I subversive genes belong to the early class of genes and code for type I transmembrane glycoproteins. The ml52‐encoded 37/40 kDa glycoprotein interacts with MHC I transiently and retains class I complexes in the endoplasmic reticulum (ER) Golgi intermediate compartment on its journey to the endolysosome. In contrast, the m06‐encoded glycoprotein of 48 kDa complexes tightly with ternary MHC class I molecules in the ER, Due to sorting signals in its cytoplasmic tail, gp48 redirects MHC I to endolysosomal compartments for proteolytic destruction. Likewise, the 34 kDa glycoprotein encoded by mO4 binds tightly to MHC class I complexes in (he ER but the gp34/MHC I complex reaches the plasma membrane. The CD8+ T‐cell‐dependent attenuation of a m152 deletion mutant virus proves for the first time that inhibition of antigen presentation is indeed essential for the biological fitness of CMVs in vivo.

Participation of endogenous tumour necrosis factor α in host resistance to cytomegalovirus infection

Interferon gamma (IFN gamma) represents an essential cytokine involved in murine cytomegalovirus (MCMV) clearance from the salivary gland and the control of horizontal transmission. Because IFN gamma cannot be responsible for all cytokine effects during recovery from MCMV infection we have now tested the potential participation of tumour necrosis factor alpha (TNF alpha) in the antiviral defence. Neutralization of endogenous TNF alpha abolished the antiviral activity of CD4 T cells in immunocompetent as well as in CD8 subset-deficient mice. These data suggest that the antiviral effect of the CD4 subset requires the presence of at least two cytokines, namely IFN gamma and TNF alpha. Depletion of endogenous TNF alpha in adoptive cell transfer recipients diminished the antiviral function of CD8 T lymphocytes suggesting that TNF alpha also participates in CD8 T cell effector functions. Furthermore, endogenous cytokines were found to be required for survival after infection with lethal doses of MCMV, whereas immunotherapy with recombinant TNF alpha and IFN gamma could not limit virus replication in vivo. The results suggest that, similar to IFN gamma, TNF alpha is an integral part of the protective mechanisms involved in cytomegalovirus clearance.

Lack of MHC class I complex expression has no effect on spread and control of cytomegalovirus infection in vivo.

It has been claimed that MHC class I proteins serve as receptors for murine cytomegalovirus (MCMV) and that this interaction is the most important mechanism for virus entry in most cells. This claim is based on the observation that the MHC haplotype contributes to the susceptibility to cytomegalovirus (CMV) infection in vivo. Results from in vitro studies support the concept that stable expression of correctly folded MHC class I molecules contributes to infection, since the individual properties of MHC class I alleles, the availability of beta 2-microglobulin (beta 2m) and also the degree of peptide charging of the MHC class I heavy chain beta 2m heterodimers determined the infection phenotype of cell lines. To assess the biological relevance of proper MHC class I expression we investigated CMV infection in beta 2m-deficient mice which fail to express ternary MHC class I complexes and lack peripheral CD8+ T lymphocytes. We found that organ virus titres and virus clearance kinetics were not altered in beta 2m mutant mice. In addition, there was no indication of diminished virus propagation in beta 2m-/- embryonic fibroblasts. beta 2m-/- mice suffered from the lack of CD8+ T lymphocytes that was partially compensated for by the function of CD4+ T lymphocytes. An organ-specific anti-virus function of natural killer (NK) cells was observed, independent from the beta 2m deletion. The immune control unique for salivary gland infection was maintained. From the data presented here, we confirm the role of MHC class I molecules in the immune surveillance of CMV infection but question the biological impact of correct MHC class I complexes for productive infection.

Synthesis, spectroscopic characterization and antiproliferative evaluation in vitro of novel Schiff bases related to benzimidazoles.

A series of novel benzimidazole substituted Schiff bases were synthesized by reaction of aromatic aldehydes with corresponding 2-aminobenzimidazoles. Their structure has been studied by 1H and 13C NMR, IR and UV/Vis spectroscopy. Majority of prepared Schiff bases were tested on their antiproliferative activity in vitro and exerted non-specific antiproliferative activity on the tested cell lines at the highest tested concentration. Compounds 18 and 19 exerted the strongest non-specific antiproliferative effect on all cell lines and a concentration-dependent effect on HeLa and MCF-7 cell lines at micromolar concentrations but simultaneously being highly cytotoxic on human fibroblasts as well.

The establishment of cytomegalovirus latency in organs is not linked to local virus production during primary infection.

Recovery from primary cytomegalovirus (CMV) infection is associated with resolution of the productive infection without clearance of the virus genome from affected organs. The presence of latent CMV genome in multiple organs provides the molecular basis for recurrence of CMV within multiple organs, and explains the diversity in the organ manifestations of recrudescent CMV disease during states of immunodeficiency. As a part of a unifying concept of multifocal CMV latency and recurrence, previous work has demonstrated the importance of primary virus replication for the overall load of latent CMV in organs and the risk of recurrence. In the present report, the establishment of CMV latency was studied in a murine model in which the course of primary infection in the immunocompromised host after syngeneic bone marrow transplantation was modulated by a CD8+ T cell immunotherapy. The antiviral CD8+ effector cells limited virus replication in all organs and protected the recipients from lethal CMV disease, but after resolution of the productive infection virus DNA remained. Interestingly, the copy number of latent virus DNA in tissue did not quantitatively reflect the preceding virus production in the respective organ. Specifically, in contrast to the case in the lungs and the salivary glands, virus replication in the spleen was suppressed by CD8+ T cells to below the limit of detection; yet, virus DNA was also detected in the spleen during latency and accordingly, virus recurrence in the spleen could be induced. These findings demonstrate that the control of virus replication in a particular organ does not prevent the establishment of latency in that organ.

Early Changes in Frequency of Peripheral Blood Lymphocyte Subpopulations in Severe Traumatic Brain‐Injured Patients

Infections are leading causes of increased morbidity and mortality of severe traumatic brain‐injured (STBI) patients. The mechanism underlying the susceptibility to the infections is still unexplained. The purpose of the study was to investigate changes in frequency of leucocytes subpopulations in peripheral blood of patients with STBI during the course of intensive care treatment. Twenty patients with STBI were included in the study. Healthy age‐ and sex‐ volunteers served as control. Peripheral blood samples were taken from these patients at day 1, 4 and 7, and peripheral blood mononuclear cells (PBMC) were isolated. The percentage of T, B lymphocyte, NK and NKT cells as well as monocytes was analysed by simultaneous detection of surface antigens using fluorochrome‐conjugated monoclonal antibodies. The two major subsets of T lymphocytes (CD3+CD56−CD4+ and CD3+CD56−CD8+) and NK cells (CD3−CD56+dim and CD3−CD56+bright) were also analysed by flow cytometry. Extracranial infections were presented in 55% patients with STBI. At day 4, the percentage of T lymphocytes with cytotoxic phenotype significantly diminished and their numbers restored at day 7. The frequency of NKT cells showed the identical time‐dependent pattern, whereas the percentage of NK cells diminished on day 4 but did not restore after 7 days. The frequency of B lymphocytes did not change significantly during the time investigated, whereas the percentage of monocytes increased immediately after the injury and gradually diminished. The decrease in cells with cytotoxic phenotype might explain high incidence of susceptibility to infection of patients with STBI.