Podcasts about ie1

Die bisherige Analyse der mini-LCL-stimulierten T-Zelllinien konzentrierte sich hauptsächlich auf die zytotoxischen CD8+ T-Zellen. Klinische Studien haben aber gezeigt, dass für eine lang anhaltende Immunität nach Stammzelltransplantation (SCT) der adoptive Transfer von CMV-spezifischen CD8+ und CD4+ T-Zellen wichtig ist. Neuere Studien unterstreichen die Rolle der CD4+ T-Zellen bei der Etablierung eines funktionierenden CD8+ T-Zellgedächtnis. Darüber hinaus wirken CD4+ T-Zellen antiviral durch die Sekretion von Zytokinen, wie IFN-g und TNF-a, und können ebenfalls zytotoxisch aktiv gegen Virus-befallene Zellen werden. Die vorliegende Arbeit sollte untersuchen, ob das mini-EBV-System geeignet ist T-Zellen zu generieren, die viele verschiedene CMV-Epitope - durch MHC-Klasse-I- und -II-Moleküle präsentiert - erkennen. T-Zellkulturen von Spendern mit unterschiedlichen HLA-Typen sollten etabliert und analysiert werden. Dabei stand eine eingehende Analyse der Epitop-Spezifitäten der generierten T-Zellen im Vordergrund. Des Weiteren sollte geklärt werden, ob sich mLCLs zur Primärstimulation von naiven T-Zellen eignen. Da neben pp65 auch IE1 ein weiteres wichtiges CMV-Antigen darstellt, sollten zwei neue mEBV-Plasmide konstruiert werden, damit auch Spender erreicht werden können, die keine ausgeprägte Immunantwort gegen pp65 besitzen. Es gibt nur wenige gesunde Seropositive, die keine Immunität gegen zumindest eines dieser beiden Antigene besitzen. Ein Vektor sollte eine Expressionskassette für IE1 enthalten, der zweite eine Kassette für ein Fusionsprotein bestehend auch pp65 und IE1 (pp65_IE1). Mit Hilfe der generierten IE1-mLCLs und pp65_IE1-mLCLs sollten autologe T-Zellen stimuliert und ihre Eigenschaften eingehend charakterisiert werden.

We have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon- (IFN-). We now demonstrate that IFN- and tumour necrosis factor alpha (TNF-) display synergism in their antiviral activity. As little as 2 ng/ml of IFN- and TNF- 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.

The product of the ie 1 gene, the regulatory immediate early protein pp89 of murine cytomegalovirus (MCMV), interacts with core histones, which can mediate the association of pp89 with DNA. We report the capacity of pp89 to interact directly with DNA in the absence of cellular proteins. After separation of proteins by SDS–PAGe, pp89 bound ds- and ssDNA, with a preference for ssDNA. Binding to specific DNA sequences in the MCMV genome was not detected. The DNA-binding region of pp89 was located to amino acids 438 to 534 by analysis of deletion mutants expressed as -galactosidase or TrpE fusion proteins. This region is identical to the highly acidic C-terminal region spanning amino acids 424 to 532. The human cytomegalovirus IE1 protein, which contains a similar extended C-terminal acidic region, does not react with DNA under the same experimental conditions.

The regulatory immediate-early (IE) protein pp89 of murine cytomegalovirus induces CD8+ T lymphocytes that protect against lethal murine cytomegalovirus infection. The IE1 epitope is the only epitope of pp89 that is recognized by BALB/c cytolytic T lymphocytes (CTL). Using synthetic peptides, the optimal and minimal antigenic sequences of the IE1 epitope have been defined. To evaluate the predictive value of data obtained with synthetic peptides, recombinant vaccines encoding this single T-cell epitope were constructed using as a vector the hepatitis B virus core antigen encoded in recombinant vaccinia virus. In infected cells expressing the chimeric proteins, only IE1 epitope sequences that were recognized as synthetic peptides at concentrations lower than 10(-6) M were presented to CTL. Vaccination of mice with the recombinant vaccinia virus that encoded a chimeric protein carrying the optimal 9-amino-acid IE1 epitope sequence elicited CD8+ T lymphocytes with antiviral activity and, furthermore, protected against lethal disease. The results thus show for the first time that recombinant vaccines containing a single foreign nonameric CTL epitope can induce T-lymphocyte-mediated protective immunity.

We have constructed target cells by cotransfection of the MHC gene Ld and fragments of murine cytomegalovirus (MCMV) DNA coding for nonstructural immediate-early (IE) proteins. Transfectants were tested by using CTL clone IE1 with specificity for an IE epitope presented in association with Ld. Data show that clone IE1 recognizes a product of the ie1 transcription unit of MCMV, and that its specificity is shared by approximately 25% of polyclonal IE-specific CTL. The results provide the first definite evidence that expression of a herpes virus IE gene encoding a regulatory protein gives rise to antigen expression detectable by specific CTL

To confirm that immediate-early (IE) genes of murine cytomegalovirus (MCMV) give rise to antigens recognized by specific cytolytic T lymphocytes (CTL), a 10.8-kilobase fragment of MCMV DNA which is abundantly transcribed at IE times was transfected into L cells expressing the Ld class I major histocompatibility glycoprotein. The viral genome fragment contains sequences of the three IE transcription units of MCMV: ie1, ie2, and ie3. In the transfected cell lines, only the predominant 2.75-kilobase transcript of ie1 and its translation product pp89 could be detected. The transfectants were analyzed for membrane expression of an IE antigen by employing clone IE1, an IE-specific CTL clone, as the probe. Only cells that expressed both the MCMV IE gene(s) and the Ld gene were recognized by the CTL clone.

Long-term cytolytic T-lymphocyte (CTL) lines that are specific for distinct antigens associated with different phases of the replicative cycle of the murine cytomegalovirus (MCMV) were established by cloning of CTL lines derived from lymph nodes of latently infected BALB/c mice. Two CTL clones were characterized in detail. Both displayed the Lyt-2+, L3T4- surface phenotype, and the recognition of their respective target antigens was class I (DLd) major histocompatibility complex antigen restricted. Clone S1 was specific for a structural antigen of MCMV, and clone IE1 detected an MCMV-specified immediate-early (IE) membrane antigen. Clone IE1 retained lytic activity, antigen specificity, and self-restriction after prolonged propagation in the presence of recombinant human interleukin-2 without restimulation by antigen. This interleukin-2-dependent line of the clone IE1, line IE1-IL, can serve as a reference line for the definition of the antigenic determinant IE1 of an IE membrane antigen.