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U H Koszinowski

Publications and source records attributed to U H Koszinowski.

At least 19 recordsLinked to original sources
PubMed2002

K3-mediated evasion of CD8(+) T cells aids amplification of a latent gamma-herpesvirus.

The murine gamma-herpesvirus-68 (MHV-68) K3 protein, like that of the Kaposi's sarcoma associated herpesvirus, down-regulates major histocompatibility complex (MHC) class I expression. However, how this contributes to viral replication in vivo is unclear. After intranasal MHV-68 infection, K3 was transcribed both during acute lytic infection in the lung and during latency establishment in lymphoid tissue. K3-deficient viruses were not cleared more rapidly from the lung, but the number of latently infected spleen cells was reduced and the frequency of virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) was increased. CTL depletion reversed the viral latency deficit. Thus, a major function of K3 appears to be CTL evasion during viral latency expansion.

3T3 Cells↗
PubMed2002

Strategies for the identification and analysis of viral immune-evasive genes--cytomegalovirus as an example.

Co-evolution of herpesviruses with their hosts has resulted in multiple interactions between viral genes and cellular functions. Some interactions control genomic maintenance and replication in specific tissues, other affect the immune control at various stages. Few immunomodulatory functions of genes can be predicted by sequence homology. The majority of genes with immunomodulatory properties only become apparent in functional assays. This chapter reviews procedures which have been used for successful identification of immunomodulatory genes in the past and deals with recent methods which may be applicable for the identification of additional immunomodulatory functions unknown so far.

Animals↗
PubMed2001

Secreted virus-encoded proteins reflect murine cytomegalovirus productivity in organs.

Mouse infection with murine cytomegalovirus (MCMV) is an established model for studying human cytomegalovirus infection. In this study, the relationship was analyzed between MCMV activity in organs of infected mice and the presence of infectious virus (viremia), viral genomes (DNAemia), or secreted virus-encoded proteins in the blood. For the latter, 2 recombinant viruses were constructed that encode for the hepatitis B virus surface antigen and the secreted alkaline phosphatase, respectively, as secreted marker proteins. The secreted markers correlated better with the infection in organs than DNAemia and viremia. The marker protein assays can serve as practical and sensitive tools for longitudinal monitoring of MCMV infection in individual mice.

Alkaline Phosphatase↗
PubMed2001

HIV envelope protein inhibits MHC class I presentation of a cytomegalovirus protective epitope.

CTL recognize peptides that derive from viral protein Ags by proteolytic processing and are presented by MHC class I molecules. In this study we tested whether coexpression of viral Ags in the same cell leads to competition between them. To this end, two L(d)-restricted epitopes derived from HIV-1 envelope gp160 (ENV) and from CMV pp89 phosphoprotein were coexpressed. HIV ENV strain IIIB, but not MN variant, impaired recognition by specific CTL of CMV pp89 epitope 9pp89. Susceptibility to inhibition after ENV coexpression was inversely related to the amount of antigenic 9pp89 peptide processed from different antigenic constructs. In line with it, competition decreased the yield of naturally processed antigenic 9pp89 peptide bound to MHC class I molecules in coinfected cells. Also, point mutants of the presenting MHC class I molecule differed in their competition pattern. Collectively, the data imply that competition operates at the step of MHC-peptide complex assembly or stabilization. We conclude that, although not the rule, in certain combinations there is interference between different Ags expressed in the same cell and presented by the same MHC class I allele. These studies have implications for vaccine development and for understanding immunodominance.

Animals↗
PubMed2001

The multiple immune-evasion genes of murine cytomegalovirus are not redundant: m4 and m152 inhibit antigen presentation in a complementary and cooperative fashion.

Both human cytomegaloviruses (HCMVs) and murine cytomegaloviruses (MCMVs) encode multiple genes that interfere with antigen presentation by major histocompatibility complex (MHC) class I, and thus protect infected targets from lysis by virus-specific cytotoxic T lymphocytes (CTLs). HCMV has been shown to encode four such genes and MCMV to encode two. MCMV m152 blocks the export of class I from a pre-Golgi compartment, and MCMV m6 directs class I to the lysosome for degradation. A third MCMV gene, m4, encodes a glycoprotein which is expressed at the cell surface in association with class I. Here we here show that m4 is a CTL-evasion gene which, unlike previously described immune-evasion genes, inhibited CTLs without blocking class I surface expression. m152 was necessary to block antigen presentation to both K(b)- and D(b)-restricted CTL clones, while m4 was necessary to block presentation only to K(b)-restricted clones. m152 caused complete retention of D(b), but only partial retention of K(b), in a pre-Golgi compartment. Thus, while m152 effectively inhibited D(b)-restricted CTLs, m4 was required to completely inhibit K(b)-restricted CTLs. We propose that cytomegaloviruses encode multiple immune-evasion genes in order to cope with the diversity of class I molecules in outbred host populations.

Animals↗
PubMed2001

The murine cytomegalovirus immune evasion protein m4/gp34 forms biochemically distinct complexes with class I MHC at the cell surface and in a pre-Golgi compartment.

We have recently demonstrated that the murine CMV (MCMV) gene m4 is an immune evasion gene that protects MCMV-infected targets from some virus-specific CTL clones. m4 encodes m4/gp34, a 34-kDa glycoprotein that binds to major histocompatibility complex class I in the endoplasmic reticulum and forms a detergent-stable complex that is exported to the surface of the cell. To investigate how m4/gp34 promotes CTL evasion, we analyzed the assembly and export of m4/gp34-K(b) complexes. We found that 50-70% of K(b) exported over the course of MCMV infection was m4/gp34 associated. Because these complexes are present at the cell surface, it is possible that m4 mediates CTL evasion by interfering with contact between class I and receptors on the T cell. In addition, we found that K(b) retained by the MCMV immune evasion gene m152 formed a novel type of complex with Endo H-sensitive m4/gp34; these complexes are distinguished from the exported complexes by being stable in 1% digitonin and unstable in 1% Nonidet P-40. Because this association occurs in a pre-Golgi compartment, m4/gp34 might also interfere with Ag presentation by affecting some aspect of class I assembly, such as peptide loading. Although m4/gp34 requires beta(2)-microglobulin to bind class I, there was no significant binding of m4/gp34 to beta(2)-microglobulin in the absence of class I H chain, demonstrating that m4/gp34 forms Nonidet P-40-stable complexes specifically with folded conformations of class I. We conclude that m4/gp34 promotes immune evasion by a novel mechanism involving altered assembly and/or T cell recognition of class I molecules.

Animals↗
PubMed2001

A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism.

Human cytomegalovirus infects vascular tissues and has been associated with atherogenesis and coronary restenosis. Although established laboratory strains of human cytomegalovirus have lost the ability to grow on vascular endothelial cells, laboratory strains of murine cytomegalovirus retain this ability. With the use of a forward-genetic procedure involving random transposon mutagenesis and rapid phenotypic screening, we identified a murine cytomegalovirus gene governing endothelial cell tropism. This gene, M45, shares sequence homology to ribonucleotide reductase genes. Endothelial cells infected with M45-mutant viruses rapidly undergo apoptosis, suggesting that a viral strategy to evade destruction by cellular apoptosis is indispensable for viral growth in endothelial cells.

3T3 Cells↗
PubMed2001

Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences.

We studied the in vivo biological properties of viruses reconstituted from the genome of murine gammaherpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus RgammaHV68A98.01, containing BAC vector sequences, is attenuated in vivo as determined by (i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen cells reactivating virus in an ex vivo reactivation assay. Since the BAC vector sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus RgammaHV68A98.02, with biological properties comparable to those of wild-type MHV-68. On the basis of these data we conclude (i) that excision of BAC vector sequences from cloned MHV-68 genomes is critical for reconstitution of the wild-type phenotypic properties of this virus and (ii) that the BAC-cloned MHV-68 genome is suitable for the construction of mutants and mutant libraries whose phenotypes can be reliably assessed in vivo.

Animals↗
PubMed2000

The luminal part of the murine cytomegalovirus glycoprotein gp40 catalyzes the retention of MHC class I molecules.

Murine cytomegalovirus (MCMV) interferes with the MHC class I pathway of antigen presentation. The type I transmembrane glycoprotein gp40, encoded by the gene m152, retains major histocompatibility complex (MHC) class I complexes in the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC)/cis-Golgi. These MHC class I complexes are stable, show an extended half-life and do not exchange beta(2)-microglobulin, whereas gp40 reaches an endosomal/lysosomal compartment where it subsequently is degraded. The analysis of regions within the viral protein that are essential for MHC class I retention revealed that a secreted form of gp40, lacking the cytoplasmic tail and the transmembrane region, still has the capacity to retain MHC class I complexes. Continuous expression of gp40 is not required for MHC class I retention. Our data indicate that the retention of MHC class I complexes in the ERGIC/cis-Golgi is triggered by gp40 and does not require the further presence of the viral protein.

3T3 Cells↗
PubMed2000

The proteasome regulator PA28alpha/beta can enhance antigen presentation without affecting 20S proteasome subunit composition.

PA28alpha/beta is a regulatory complex of the 20S proteasome which consists of two IFN-gamma inducible subunits. Both subunits, alpha and beta, contribute equally to the formation of hexa- or heptameric rings which can associate with the 20S proteasome. Previously, we have shown that overexpression of the PA28alpha subunit enhanced the MHC class I-restricted presentation of two viral epitopes and that purified PA28alpha/beta accelerated T cell epitope generation by the 20S proteasome in vitro, indicating a role for PA28alpha/beta in antigen presentation. This conclusion was recently confirmed in PA28beta gene targeted mice which were severely deficient in MHC class I-restricted antigen presentation. These mice displayed a defect in the assembly of immunoproteasomes, suggesting that a lack of the proteasome subunits LMP2, LMP7, and MECL-1 may account for the deficiency in antigen presentation. In this study we investigated whether the effect of PA28alpha/beta on antigen presentation is dependent on a change of proteasome subunit composition. We have analyzed the assembly and subunit composition of proteasomes in fibroblast transfectants overexpressing both, alpha and beta subunits of PA28. In these transfectants we found a marked enhancement in the presentation of the immunodominant H-2Ld-restricted pp89 epitope of murine cytomegalovirus, although the 20S proteasome composition was the same as in recipient cells. We, therefore, conclude that PA28alpha/beta can enhance antigen processing independently of changes in 20S proteasome subunit composition or assembly.

Animals↗
PubMed2000

Viral mechanisms of immune evasion.

During the millions of years they have coexisted with their hosts, viruses have learned how to manipulate host immune control mechanisms. Viral gene functions provide an overview of many relevant principles in cell biology and immunology. Our knowledge of viral gene functions must be integrated into virus-host interaction networks to understand viral pathogenesis, and could lead to new anti-viral strategies and the ability to exploit viral functions as tools in medicine.

Animals↗
PubMed2000

Forward with BACs: new tools for herpesvirus genomics.

The large, complex genomes of herpesviruses document the high degree of adaptation of these viruses to their hosts. Not surprisingly, the methods developed over the past 30 years to analyse herpesvirus genomes have paralleled those used to investigate the genetics of eukaryotic cells. The recent use of bacterial artificial chromosome (BAC) technology in herpesvirus genetics has made their genomes accessible to the tools of bacterial genetics. This has opened up new avenues for reverse and forward genetics of this virus family in basic research, and also for vector and vaccine development.

Alleles↗
PubMed2000

Viral mechanisms of immune evasion.

During the millions of years they have coexisted with their hosts, viruses have learned how to manipulate host immune control mechanisms. Viral gene functions provide an overview of many relevant principles in cell biology and immunology. Our knowledge of viral gene functions must be integrated into virus-host interaction networks to understand viral pathogenesis, and could lead to new anti-viral strategies and the ability to exploit viral functions as tools in medicine.

Antibody Formation↗
PubMed2000

Viral mechanisms of immune evasion.

During the millions of years they have coexisted with their hosts, viruses have learned how to manipulate host immune control mechanisms. Viral gene functions provide an overview of many relevant principles in cell biology and immunology. Our knowledge of viral gene functions must be integrated into virus-host interaction networks to understand viral pathogenesis, and could lead to new anti-viral strategies and the ability to exploit viral functions as tools in medicine.

Animals↗
PubMed2000

Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome.

Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) in Escherichia coli. During propagation in E. coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked by loxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAC-cloned MHV-68. Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.

Animals↗
PubMed2000

Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes.

We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone. We applied this method to retrieve mutants of HCMV envelope glycoprotein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fibroblasts. In contrast to BACs with mutations in the genes coding for gB, gH, gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frame UL4 (gp48) or UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit. Thus, gO (UL74), a component of the glycoprotein complex III, is dispensable for viral growth. We conclude that our approach of PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes.

Cytomegalovirus↗
PubMed2000

Macrophages escape inhibition of major histocompatibility complex class I-dependent antigen presentation by cytomegalovirus.

The mouse cytomegalovirus (MCMV) m152- and m06-encoded glycoproteins gp40 and gp48, respectively, independently downregulate major histocompatibility complex (MHC) class I surface expression during the course of productive MCMV infection in fibroblasts. As a result, presentation of an immediate-early protein pp89-derived nonapeptide to H-2L(d)-restricted CD8(+) cytotoxic T cells is completely prevented in fibroblasts. Here we demonstrate that MCMV-infected primary bone marrow macrophages and the macrophage cell line J774 constitutively present pp89 peptides during permissive MCMV infection to cytotoxic T lymphocytes (CTL). In contrast to fibroblasts, expression of the m152 and m06 genes in macrophages does not affect surface expression of MHC class I. Assessment of pp89 synthesis and quantification of extracted peptide revealed a significantly higher efficiency of macrophages than of fibroblasts to process pp89 into finally trimmed peptide. The yield of pp89 peptide determined in MCMV-infected tissues of bone marrow chimeras confirmed that bone marrow-derived cells represent a prime source of pp89 processing in parenchymal organs. The finding that macrophages resist the viral control of MHC I-dependent antigen presentation reconciles the paradox of efficient induction of CMV-specific CD8(+) CTL in vivo despite extensive potential of CMVs to subvert MHC class I.

Animals↗