Podcasts about mcmv

Play Episode Listen Later Jan 1, 1994

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.

action dna phase factor thursday night football medizin gamma icp replication inhibition tumour synergistic interferon necrosis ifn cytomegalovirus cd4 t murine mcmv ie1 mcmv dna

Expression of the murine cytomegalovirus glycoprotein H by recombinant vaccinia virus

Play Episode Listen Later Jan 1, 1994

The sequence of the gene encoding glycoprotein H (gH) of murine cytomegalovirus (MCMV) strain Smith was determined and compared with the sequence of the gH of MCMV strain K181. Transcriptional analysis showed that gH is encoded by a large mRNA of 5.0 kb, which is synthesized late in infection. A recombinant vaccinia virus expressing the MCMV gH open reading frame was constructed (Vac-gH). Anti-MCMV serum precipitated a protein of 87K from Vac-gH-infected cells. Reactivity with a monoclonal antibody showed the identity of the MCMV gH with a 87K envelope glycoprotein described previously by Loh and Qualtiere. Immunization of mice with the Vac-gH recombinant gave rise to an anti-gH serum, which neutralized MCMV without complement in vitro.

expression medizin mrna gh immunization reactivity loh cytomegalovirus recombinant transcriptional murine 87k glycoprotein mcmv vaccinia

Restoration of cytomegalovirus antigen presentation by gamma interferon combats viral escape.

Play Episode Listen Later Jan 1, 1994

An immediate-early protein of murine cytomegalovirus (MCMV), pp89, elicits an immunodominant and protective major histocompatibility complex (MHC) class I Ld-restricted CD8+ T-lymphocyte response. Remarkably, presentation of the naturally processed peptide of pp89, the nonapeptide YPHFMPTNL, is abolished during permissive MCMV infection in vitro. This defect in pp89 presentation is due to the expression of MCMV early gene functions that specifically block the transport of peptide-charged MHC class I complexes to the cell surface (M. Del Val, H. Hengel, H. Häcker, U. Hartlaub, T. Ruppert, P. Lucin, and U. H. Koszinowski, J. Exp. Med. 176:729-738, 1992). Here, we demonstrate that MCMV-specific CD8+ T lymphocytes can reconstitute pp89 presentation in a parakrine fashion. The lymphocytes mediate the restoration of antigen presentation by MCMV-infected cells by releasing gamma interferon (IFN-gamma). IFN-gamma has no effect on synthesis and stability of the viral antigen pp89 nor does it interfere with the expression of viral early genes and their inhibitory effect on MHC class I molecular maturation. IFN-gamma results in a 25-fold increase in the synthesis of MHC class I molecules and a similar increase in the abundance of pp89-derived peptide. Many of the MHC molecules remain retained by the viral effect, but a surplus of MHC molecules escapes the effect and provides the effective surface presentation of the peptide. Adoptive cell transfer studies demonstrate the IFN-gamma dependence of CD8+ T-lymphocyte function in vivo. Altogether, these data reconcile the paradoxical findings of an impaired pp89 presentation in vitro in parallel with pp89-specific CD8+ T-cell protection in vivo. The results also imply a role of IFN-gamma in the T-lymphocyte-mediated control of cytomegalovirus infection. The known propensity of cytomegalovirus to cause serious disease in the immunocompromised host is discussed in the light of these findings

escape restoration viral presentation med medizin gamma exp combats antigens adoptive ruppert mhc interferon ifn cytomegalovirus hengel cd8 t mcmv

Identification and expression of a murine cytomegalovirus early gene coding for an Fc receptor

Play Episode Listen Later Jan 1, 1994

Several herpesviruses, including cytomegalovirus, induce receptors for the Fc domain of murine immunoglobulin G (IgG) molecules. Viral genes coding for these receptors have been characterized only for alphaherpesviruses. In this report, we describe a new approach that led to the identification of an Fc receptor (FcR) of murine cytomegalovirus (MCMV). The Fc fragment of IgG precipitated glycoproteins (gp) of 86 to 88 and 105 kDa from MCMV-infected cells. Deglycosylation by endoglycosidase F resulted in a protein with a molecular mass of 64 kDa. Injection of complete MCMV DNA or of DNA fragments, and the subsequent testing of cytoplasmic binding of IgG by immunofluorescence microscopy, was used to search for the coding region in the MCMV genome. The gene was located in the HindIII J fragment, map units 0.838 to 0.846, where an open reading frame of 1,707 nucleotides predicts a gp of 569 amino acids with a calculated molecular mass of 65 kDa. The sequence of this gp is related to those of the gE proteins of herpes simplex virus type 1 and varicella-zoster virus. The defined length of the mRNA, 1,838 nucleotides, was in agreement with that of a 1.9-kb RNA expressed throughout the replication cycle, starting at the early stages of infection. Expression of the gene fcr1 by recombinant vaccinia virus resulted in the synthesis of gp86/88 and gp105, each with FcR properties, and the correct identification of the gene encoding the FcR was confirmed by the DNA injection method.

dna viral ge expression coding medizin mrna identification rna injection receptor igg kda cytomegalovirus fcr murine mcmv g igg mcmv dna

Interferon y Stimulation Modulates the Proteolytic Activity and Cleavage Site Preference of 20S Mouse Proteasomes

Medizin - Open Access LMU - Teil 10/22

Play Episode Listen Later Jan 1, 1994

The proteasome is a 700-kD multisubunit enzyme complex with several proteolytically active sites. The enzyme complex is involved in both ubiquitin-dependent and -independent protein degradation and may contribute to the processing of antigens presented by major histocompatibility complex (MHC) class I molecules. Here we demonstrate that treatment of mouse fibroblast cells with 20 U interferon qr (IFN-y) for 3 d induces a change in the proteasome subunit composition and that the B-type subunit LMP2, which is encoded in the MHC class II region, is incorporated into the enzyme complex. This is paralleled by reduction of the homologous 6-subunit. IFN-3' stimulation results in a downregulation of the chymotrypsin-like Suc-LLVY-MCA peptide hydrolyzing activity of 20S proteasomes whereas the trypsin-like activity remains unaffected. When tested as a substrate a synthetic 25-mer polypeptide whose sequence covers the antigenic nonapeptide YPHFMPTNL of the MCMV pp89, 20S proteasomes of IFN-3'-induced cells exhibit altered chymotrypsin-like cleavage site preferences. In the absence of IFN-qr induction, the naturally processed nonamer peptide that is presented by MHC class.I molecules appears as a minor cleavage product. IFN-'y activation does not result in an increase of the final peptide but results in a different set of peptides. We hypothesize that these peptides represent precursor peptides that can be trimmed to final peptide size.

activity mouse kd medizin preference stimulation cleavage 20s mhc interferon ifn lmp2 mcmv proteolytic

Alloreactive cytotoxic T lymphocytes generated in the presence of viral- derived peptides show exquisite peptide and MHC specificity

Play Episode Listen Later Jan 1, 1993

The nature of alloreactivity to MHC molecules has been enigmatic, primarily because of the observation that allogeneic responses are considerably stronger than syngeneic responses. To better determine the specificity potential of allogeneic responses, we have generated alloreactive CTL specific for exogenous, viral-derived peptide ligands. This approach allowed us to critically evaluate both the peptide- and MHC-specificity of these alloreactive T cells. Exploiting the accessibility of the H-2Ld class I molecule for exogenous peptide ligands, alloreactive CTL were generated that are specific for either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis virus (LCMV) peptides bound by Ld alloantigens. Peptide specificity was initially observed in bulk cultures of alloreactive CTL only when tested on peptide-sensitized T2.Ld target cells that have defective presentation of endogenous peptides. Subsequent cloning of bulk alloreactive CTL lines generated to MCMV yielded CTL clones that had exquisitely specific MCMV peptide recognition requirement. All of the MCMV/Ld alloreactive CTL clones were also exquisitely MHC-specific in that none of the CTL clones lysed targets expressing MCMV/Lq complexes, even though Lq differs from Ld by only six amino acid residues and Lq also binds the MCMV peptide. This observation clearly demonstrates that alloreactive CTL are capable of the same degree of specificity for target cell recognition as are syngeneic CTL in MHC-restricted responses.

presence viral medizin generated subsequent ld t2 peptides exploiting specificity derived exquisite mhc ctl lq lymphocytes mcmv cytotoxic

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

Medizin - Open Access LMU - Teil 10/22

Play Episode Listen Later Jan 1, 1993

Interferon gamma (IFN) represents an essential cytokine involved in murine cytomegalovirus (MCMV) clearance from the salivary gland and the control of horizontal transmission. Because IFN 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) in the antiviral defence. Neutralization of endogenous TNF 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 and TNF. Depletion of endogenous TNF in adoptive cell transfer recipients diminished the antiviral function of CD8 T lymphocytes suggesting that TNF 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 and IFN could not limit virus replication in vivo. The results suggest that, similar to IFN, TNF is an integral part of the protective mechanisms involved in cytomegalovirus clearance.

resistance factor infection thursday night football participation medizin depletion tumour interferon cd4 endogenous necrosis ifn cytomegalovirus cd8 t cd8 cd4 t neutralization mcmv

An acidic region of the 89K murine cytomegalovirus immediate early protein interacts with DNA

Play Episode Listen Later Jan 1, 1992

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.

dna region protein medizin binding acidic cytomegalovirus murine mcmv ssdna sds page ie1

Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3.

Play Episode Listen Later Jan 1, 1992

We have previously defined ie3 as a coding region located downstream of the ie1 gene which gives rise to a 2.75-kb immediate-early (IE) transcript. Here we describe the structural organization of the ie3 gene, the amino acid sequence of the gene product, and some of the functional properties of the protein. The 2.75-kb ie3 mRNA is generated by splicing and is composed of four exons. The first three exons, of 300, 111, and 191 nucleotides (nt), are shared with the ie1 mRNA and are spliced to exon 5, which is located downstream of the fourth exon used by the ie1 mRNA. Exon 5 starts 28 nt downstream of the 3' end of the ie1 mRNA and has a length of 1,701 nt. The IE3 protein contains 611 amino acids, the first 99 of which are shared with the ie1 product pp89. The IE3 protein expressed at IE times has a relative mobility of 88 kDa in gels, and a mobility shift to 90 kDa during the early phase is indicative of posttranslational modification. Sequence comparison reveals significant homology of the exon 5-encoded amino acid sequence with the respective sequence of UL 122, a component of the IE1-IE2 complex of human cytomegalovirus (HCMV). This homology is also apparent at the functional level. The IE3 protein is a strong transcriptional activator of the murine cytomegalovirus (MCMV) e1 promoter and shows an autoregulatory function by repression of the MCMV ie1/ie3 promoter. The high degree of conservation between the MCMV ie3 and HCMV IE2 genes and their products with regard to gene structure, amino acid sequence, and protein functions suggests that these genes play a comparable role in the transcriptional control of the two cytomegaloviruses.

expression functional medizin mrna sequence structural ul characterization kda exon cytomegalovirus murine mcmv hcmv

Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment

Play Episode Listen Later Jan 1, 1992

Selective expression of murine cytomegalovirus (MCMV) immediate-early (IE) genes leads to the presentation by the major histocompatibility complex (MHC) class I molecule L a of a peptide derived from MCMV IE protein pp89 (Reddehase, M. J., J. B. Rothbard, and U. H. Koszinowski. 1989. Nature (Lond.). 337:651). Characterization of endogenous antigenic peptides identified the pp89 peptide as the nonapeptide msYPHFMFFNLt76 (del Val, M., H.-J. Schlicht, T. Ruppert, M. J. Reddehase, and U. H. Koszinowski. 1991. Cell. 66:1145). Subsequent expression of MCMV early genes prevents presentation of pp89 (del Val, M., K. Mfinch, M. J. Reddehase, and U. H. Koszinowski. 1989. Cell. 58:305). We report on the mechanism by which MCMV early genes interfere with antigen presentation. Expression of the IE promoter-driven bacterial gene lacZ by recombinant MCMV subjected antigen presentation of B-galactosidase to the same control and excluded antigen specificity. The La-dependent presence of naturally processed antigenic peptides also in nonpresenting cells located the inhibitory function subsequent to the step of antigen processing. The finding that during the E phase of MCMV gene expression the MHC class I heavy chain glycosylation remained in an Endo H-sensitive form suggested a block within the endoplasmic reticulum/c/s-Golgi compartment. The failure to present antigenic peptides was explained by a general retention of nascent assembled trimolecular MHC class I complexes. Accordingly, at later stages of infection a significant decrease of surface MHC class I expression was seen, whereas other membrane glycoproteins remained unaffected. Thus, MCMV E genes endow this virus with an effective immune evasion potential. These results also indicate that the formation of the trimolecular complex of MHC dass I heavy chain, ~2-microglobulin, and the finally trimmed peptide is completed before entering the medial-Golgi compartment.

Characterization of the murine cytomegalovirus genes encoding the major DNA binding protein and the ICP18.5 homolog

Play Episode Listen Later Jan 1, 1992

In several herpesviruses the genes for the major DNA binding protein (MDBP), a putative assembly protein, the glycoprotein B (gB), and the viral DNA polymerase (pol) coliocate. In murine cytomegalovirus (MCMV), two members of this gene block, pol (Elliott, Clark, Jaquish, and Spector, 1991, Virology 185, 169-186) and gB (Rapp, Messerle, BOhler, Tannheimer, Keil, and Koszinowski, 1992, J. Virol., 66,4399-4406) have been characterized. Here the two other MCMV genes are characterized, the gene encoding the MDBP and the ICP18.5 homolog encoding a putative assembly protein. Like in human cytomegalovirus (HCMV) the genes order is pol, gB, ICP18.5, and MDBP. The 4.2-kb MDBP mRNA is expressed first in the early phase, whereas the 3.0-kb ICP18.5 mRNA is a late transcript. The open reading frame of the MDBP gene has the capacity of encoding a protein of 1191 amino acids with a predicted molecular mass of 131.7 kDa. The MCMV ICP18.5 ORF is translated into a polypeptide of 798 amino acids with a calculated molecular mass of 89.1 kDa. Comparison of the amino acid sequences of the predicted proteins of MCMV with the respective proteins of HCMV, Epstein-Barr virus (EBV), and herpes simplex virus type-1 (HSV-1) reveals a striking homology ranging from 72% (HCMV), 50% (EBV), to 45% (HSV-1) for the MDBP sequence and from 74% (HCMV), 51 % (EBV), to 49% (HSV-1) for the ICP18.5 sequence. These results establish the elose relationship of the two cytomegaloviruses, and underline the usefulness of the murine model for studies on the biology of the CMV infection.

Identification of the murine cytomegalovirus glycoprotein B gene and its expression by recombinant vaccinia virus

Medizin - Open Access LMU - Teil 07/22

Play Episode Listen Later Jan 1, 1992

The gene encoding glycoprotein B (gB) of murine cytomegalovirus (MCMV) strain Smith was identified, sequenced, and expressed by recombinant vaccinia virus. The gB gene was found adjacent to the polymerase gene, as it is in the genome of human cytomegalovirus (HCMV). The open reading frame consists of 2,784 nucleotides capable of encoding a protein of 928 amino acids. Comparison with gB homologs of other herpesviruses revealed a high degree of homology. The similarity between the MCMV gB and the HCMV gB is most prominent, since 45% of the amino acids are identical. In addition, all cysteine residues are at homologous positions, indicating a similar tertiary structure of the two proteins. In contrast to HCMV, the MCMV gB mRNA is a true late transcript. A recombinant vaccinia virus expressing the MCMV gB gene has been constructed (Vac-gB). Antibodies raised against the Vac-gB recombinant precipitated proteins of 130, 105, and 52 kDa from MCMV-infected cells. The identity of the MCMV gB with the major envelope glycoprotein of MCMV described by Loh et al. was shown (L. C. Loh, N. Balachandran, and L. F. Qualtiere, Virology 166:206-216, 1988). Immunization of mice with the Vac-gB recombinant gave rise to neutralizing antibodies.

comparison expression gb medizin identification antibodies virology immunization kda loh bgb cytomegalovirus recombinant balachandran murine glycoprotein mcmv vaccinia hcmv

Predominant utilization of V beta 8+ T cell receptor genes in the H-2Ld- restricted cytotoxic T cell response against the immediate-early protein pp89 of the murine cytomegalovirus

Play Episode Listen Later Jan 1, 1989

Cytotoxic T cell responses to the murine Cytomegalovirus (MCMV) were elicited in BALB/c mice (H-2d) by infectious virus. Eight days after infection, MCMV-primed local lymph node T cells were either depleted for T cells expressing a V beta 8+ TCR or separated into V beta 8+ and V beta 8- subpopulations by a cell sorter using the mAb F23.1. T cells were then expanded in vitro under limiting dilution conditions in the presence of IL-2 and in the absence of viral Ag to avoid selection by Ag in vitro. Frequencies of CTL precursors specific for the Immediate- Early-Ag 1 of MCMV and restricted to H-2Ld were determined. L cells of the endogenous haplotype H-2k cotransfected with the genes for MCMV-IE 1 and H-2Ld were used as target cells. Detection of a CTL response required previous priming of the animals by infection in vivo (less than 1/10(6) for nonimmunized animals). In primed animals CTL precursors of this specificity and restriction were three to fivefold more frequent in the V beta 8+ population (1/9.900 to 1/22.300) than in the V beta 8- population (1/57.000 to 1/87.200). Control experiments showed that frequencies were not influenced by the treatment with the anti-V beta 8-antibody and the fluorescein-labeled anti-Ig itself. V beta 8+ and V beta 8- T cells did not reveal any frequency differences when several other responses were determined (TNP-specific self- restricted CTL precursor; Th cells specific for keyhole limpet hemocyanin or Listeria monocytogenes).

beta protein genes medizin detection frequencies restricted utilization receptor listeria tcr ctl balb cytomegalovirus predominant tnp murine mcmv cytotoxic cytotoxic t mcmv ie

The 89,000-Mr murine cytomegalovirus immediate-early protein stimulates c-fos expression and cellular DNA synthesis

Medizin - Open Access LMU - Teil 06/22

Play Episode Listen Later Jan 1, 1988

The immediate-early (IE) genes of murine cytomegalovirus (MCMV) are expressed in the absence of prior viral protein synthesis and regulate the transcription of MCMV early genes. The effect of MCMV IE genes on growth induction was studied. Different plasmids containing MCMV IE genes were microinjected into arrested NIH 3T3 mouse fibroblasts. Plasmids containing the ieI gene coding for the 89,000-Mr major IE protein pp89 were found to stimulate the expression of the c-fos protooncogene. Synthesis of pp89 and its transport to the nucleus appeared to be required for c-fos expression. DNA synthesis occurred in cells that were injected with MCMV IE genes and in neighboring cells that were not injected. The results suggest that the phosphoprotein pp89 stimulates cells to enter the cell cycle.

dna protein expression medizin cellular synthesis stimulates cytomegalovirus murine mcmv iei plasmids mcmv ie

A nonstructural viral protein expressed by a recombinant vaccinia virus protects against lethal cytomegalovirus infection

Medizin - Open Access LMU - Teil 06/22

Play Episode Listen Later Jan 1, 1988

The nonstructural immediate-early protein pp89 of murine cytomegalovirus (MCMV) is the first viral protein synthesized after infection and has a regulatory function in viral gene expression. Despite its localization in the nucleus of infected cells, pp89 is also the dominant antigen recognized by MCMV-specific cytolytic T lymphocytes. The recombinant vaccinia virus MCMV-ieI-VAC, which expresses pp89, was used to study the capacity of this protein to induce protective immunity in BALB/c mice. Vaccination with MCMV-ieI-VAC induced a long-lasting immunity that protected mice against challenge with a lethal dose of MCMV but did not prevent infection and morbidity. In vivo depletion of CD8+ T lymphocytes before challenge completely abrogated the protective immunity. CD8+ T lymphocytes derived from MCMV-ieI-VAC-primed donors and adoptively transferred into sublethally irradiated and MCMV-infected recipients were found to limit viral replication in host tissues, whereas CD4+ T lymphocytes and pp89-specific antiserum had no protective effect. The data demonstrate for the first time that a single nonstructural viral protein can confer protection against a lethal cytolytic infection and that this immunity is entirely mediated by the CD8+ subpopulation of T lymphocytes.

viral vaccination protein infection medizin protects lethal expressed balb cytomegalovirus recombinant cd8 t cd8 cd4 t mcmv vaccinia

Interaction of the 89K murine cytomegalovirus immediate-early protein with core histones

Medizin - Open Access LMU - Teil 06/22

Play Episode Listen Later Jan 1, 1988

The conditions that permit the interaction of immediate-early proteins of murine cytornegalovirus (MCMV) with DNA were studied. Chromatography of extracts from infected cells on MCMV DNA cellulose and calf thymus DNA cellulose showed that pp89, the regulatory major immediate-early protein, interacts with DNA and dissociates at salt concentrations between 0.3 and 0.6 M NaCl. pp76, a cleavage product of pp89, and additional minor ie1 proteins eluted already at low ionic strength. Cellular DNA-binding factors were required for association of pp89 with DNA. These factors were identified as core histones. Chromatography of IE proteins on histone-Sepharose in the absence of DNA revealed a high-binding affinity that was resistant to 2 M NaCl. These results suggest that pp89 has no direct DNA-binding activity. A role for an amino acid sequence homology in the N-terminal region of pp89 with histone H2B in the pp89-histone-DNA Interaction is discussed.

dna protein medizin interaction h2b cytomegalovirus chromatography murine mcmv histones sepharose mcmv dna

Location, transcripts, and translation products

Play Episode Listen Later Jan 1, 1987

Cloned genomic fragments from the region (0.769 to 0.818 map units) coding for immediate-early (IE) transcripts of murine cytomegalovirus (MCMV) were used to analyze the physical organization of this region, the direction of transcription, and the proteins synthesized in vitro. Three IE transcription units could be identified. From IE coding region 1 (ie1; 0.781 to 0.796 map units) a dominant 2.75-kilobase (kb) RNA was transcribed from right to left on the prototype arrangement of the MCMV genome which directed the synthesis of an 89,000-molecular-weight polypeptide (89K polypeptide), the major IE protein. This phosphoprotein (pp89) has been shown to be active in the regulation of transcription. Upstream of ie1 and separated by the MCMV enhancer sequence was a second IE coding region, ie2, which was mapped at 0.803 to 0.817 map units. From ie2 a 1.75-kb RNA of moderate abundance was transcribed in the direction opposite to that of the ie1 RNA. After hybrid selection of the ie2 transcript, a 43,000-molecular-weight translation product was detected. A third coding region, ie3, was located directly downstream of ie1 (0.773 to 0.781 map units). The series of RNAs with low abundance, terminating in ie3, probably used the ie1 transcription start site and ranged from 1.0 to 5.1 kb in size. The 5.1-kb RNA apparently represents the nonspliced transcript from both coding regions ie1 and ie3. A 15K polypeptide was translated in vitro from RNA that was hybrid selected by ie3 sequences. Immunoprecipitation with monoclonal antibody revealed that 31K to 67K polypeptides were related to pp89. Some of these proteins were translated from RNAs that were smaller than 2.75 kb. Polypeptides related to pp89 were also synthesized in vivo. Because polypeptides unrelated to pp89 that were translated from RNA that was selected by ie2 and ie3 sequences were not immunoprecipitated by murine antisera, we assumed that the amount of these proteins synthesized in vivo during infection was probably very low

products location translation medizin rna upstream 15k cloned rnas 31k mcmv polypeptides

Host immune response to cytomegalovirus

Play Episode Listen Later Jan 1, 1987

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.

medizin ld immune response ctl cytomegalovirus mcmv ie1 mcmv dna mcmv ie

Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus

Play Episode Listen Later Jan 1, 1987

The murine immediate-early (IE) protein pp89 is a nonstructural virus- encoded phosphoprotein residing in the nucleus of infected cells, where it acts as transcriptional activator. Frequency analysis has shown that in BALB/c mice the majority of virus-specific CTL recognize IE antigens. The present study was performed to assess whether pp89 causes membrane antigen expression detected by IE-specific CTL. Site-directed mutagenesis has been used to delete the introns from gene ieI, encoding pp89, for subsequent integration of the continuous coding sequence into the vaccinia virus genome. After infection with the vaccinia recombinant, the authentic pp89 was expressed in cells that became susceptible to lysis by an IE-specific CTL clone. Priming of mice with the vaccinia recombinant sensitized polyclonal CTL that recognized MCMV- infected cells and transfected cells expressing pp89. Thus, a herpesviral IE polypeptide with essential function in viral transcriptional regulation can also serve as a dominant antigen for the specific CTL response of the host.

protein frequency recognition medizin expressed priming ctl lymphocytes balb cytomegalovirus recombinant murine mcmv iei vaccinia

A nonstructural polypeptide encoded by immediate-early transcription unit 1 of murine cytomegalovirus is recognized by cytolytic T lymphocytes

Play Episode Listen Later Jan 1, 1987

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

data recognized medizin ld transcription mhc encoded ctl lymphocytes cytomegalovirus murine mcmv polypeptide ie1 mcmv dna

CD8-positive T lymphocytes specific for murine cytomegalovirus immediate-early antigens mediate protective immunity

Play Episode Listen Later Jan 1, 1987

We have shown in a murine model system for acute, lethal cytomegalovirus (CMV) disease in the immunocompromised natural host that control of virus multiplication in tissues, protection from virus-caused tissue destruction, and survival are mediated by virus-specific CD8+ CD4-T lymphocytes. Protection from a lethal course of disease did not result in a rapid establishment of virus latency, but led to a long-lasting, persistent state of infection. The CD8- CD4+ subset of T lymphocytes was not effective by itself in controlling murine CMV (MCMV) multiplication in tissue or essential for the protective function of the CD8+ CD4- effector cells. The antiviral efficacy of the purified CD8+ CD4- subset was not impaired by preincubation with fibroblasts that presented viral structural antigens, but was significantly reduced after depletion of effector cells specific for the nonstructural immediate-early antigens of MCMV, which are specified by the first among a multitude of viral genes expressed during MCMV replication in permissive cells. Thus, MCMV disease provides the first example of a role for nonstructural herpesvirus immediate-early antigens in protective immunity.

positive medizin antigens mediate cmv lymphocytes cytomegalovirus murine mcmv protective immunity

The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription

Play Episode Listen Later Jan 1, 1986

To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters. The regulatory function of the ie1 product was confirmed by transient expression assays in which MCMV IE genes were cotransfected into L cells together with recombinant constructs of the CAT gene. For CAT activation by the ie1 product, a promoter region was required, but there was no preferential activation of a herpes simplex virus type 1 delayed-early promoter. All plasmid constructs that contained the intact coding sequences for pp89 induced gene expression in trans. The MCMV enhancer region was not essential for the expression of a functional IE gene product, and testing of the cis-regulatory activity of the MCMV enhancer revealed a low activity in L cells. Another region transcribed at IE times of infection, IE coding region 2, was unable to induce CAT expression and also did not augment the functional activity of ie1 after cotransfection.

cat protein medizin transcription activates cytomegalovirus murine mcmv mcmv ie

Cloned long-term cytolytic T-lymphocyte line with specificity for an immediate-early membrane antigen of murine cytomegalovirus

Play Episode Listen Later Jan 1, 1986

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.

long term medizin lyt specificity cloned membrane antigens ctl lymphocytes balb cytomegalovirus murine mcmv ie1

Studies on the Morphogenesis of Murine Cytomegalovirus