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Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65).

ABSTRACT: CD8 T cells are the principal antiviral effectors controlling cytomegalovirus (CMV) infection. For human CMV, the virion tegument protein ppUL83 (pp65) has been identified as a source of immunodominant peptides and is regarded as a candidate for cytoimmunotherapy and vaccination. Two sequence homologs of ppUL83 are known for murine CMV, namely the virion protein ppM83 (pp105) expressed late in the viral replication cycle and the nonstructural protein pM84 (p65) expressed in the early phase. Here we show that ppM83, unlike ppUL83, is not delivered into the antigen presentation pathway after virus penetration before or in absence of viral gene expression, while other virion proteins of murine CMV are processed along this route. In cytokine secretion-based assays, ppM83 and pM84 appeared to barely contribute to the acute immune response and to immunological memory. Specifically, the frequencies of M83 and M84 peptide-specific CD8 T cells were low and undetectable, respectively. Nonetheless, in a murine model of cytoimmunotherapy of lethal CMV disease, M83 and M84 peptide-specific cytolytic T-cell lines proved to be highly efficient in resolving productive infection in multiple organs of cell transfer recipients. These findings demonstrate that proteins which fail to prime a quantitatively dominant immune response can nevertheless represent relevant antigens in the effector phase. We conclude that quantitative and qualitative immunodominance are not necessarily correlated. As a consequence of these findings, there is no longer a rationale for considering T-cell abundance as the key criterion for choosing specificities to be included in immunotherapy and immunoprophylaxis of CMV disease and of viral infections in general.

SUBMITTER: Holtappels R

PROVIDER: S-EPMC114382 | biostudies-literature | 2001 Jul

REPOSITORIES: biostudies-literature

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Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65).

Holtappels R R Podlech J J Grzimek N K NK Thomas D D Pahl-Seibert M F MF Reddehase M J MJ

Journal of virology 20010701 14

CD8 T cells are the principal antiviral effectors controlling cytomegalovirus (CMV) infection. For human CMV, the virion tegument protein ppUL83 (pp65) has been identified as a source of immunodominant peptides and is regarded as a candidate for cytoimmunotherapy and vaccination. Two sequence homologs of ppUL83 are known for murine CMV, namely the virion protein ppM83 (pp105) expressed late in the viral replication cycle and the nonstructural protein pM84 (p65) expressed in the early phase. Here ...[more]

PMID: 11413326

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Project description:We previously identified two open reading frames (ORFs) of murine cytomegalovirus (MCMV), M83 and M84, which are putative homologs of the human cytomegalovirus (HCMV) UL83 tegument phosphoprotein pp65 (L. D. Cranmer, C. L. Clark, C. S. Morello, H. E. Farrell, W. D. Rawlinson, and D. H. Spector, J. Virol. 70:7929-7939, 1996). In this report, we show that unlike the M83 gene product, the M84 protein is expressed at early times in the infection and cannot be detected in the virion. To elucidate the functional differences between the two pp65 homologs in acute and latent MCMV infections, we constructed two MCMV K181 mutants in which either the M83 or M84 ORF was deleted. The resultant viruses, designated DeltaM83 and DeltaM84, respectively, were found to replicate in NIH 3T3 cells with kinetics identical to those of the parent strain. Western blot analysis demonstrated that except for the absence of M83 or M84 protein expression in the respective mutants, no global perturbations of protein expression were detected. When DeltaM83 and DeltaM84 were inoculated intraperitoneally (i.p.) into BALB/c mice, both viruses showed similar attenuated growth in the spleen, liver, and kidney. However, only DeltaM83 was severely growth restricted in the salivary glands, a phenotype that was abolished upon restoration of the M83 ORF. DeltaM83's growth was similarly restricted in the salivary glands of the resistant C3H/HeN or highly sensitive 129/J strain, as well as in the lungs of all three strains following intranasal inoculation. Using a nested-PCR assay, we found that both DeltaM83 and DeltaM84 established latency in BALB/c mice, with slightly decreased levels of DeltaM83 and DeltaM84 genomic DNAs, relative to K181, observed in the salivary glands and lungs. Immunization of BALB/c mice with 10(5) PFU of K181, DeltaM83, or DeltaM84 i.p. provided similar levels of protection against lethal challenge. Although immunization with 200 PFU of DeltaM83 also provided complete protection, this dose allowed both the immunizing and challenge viruses to establish latency in the spleen. Our results show that the two MCMV pp65 homologs differ in their expression kinetics, virion association, and influence on viral tropism and/or dissemination.

Project description:UnlabelledThe mechanisms that lead to the tegumentation of herpesviral particles are only poorly defined. The phosphoprotein 65 (pp65) is the most abundant constituent of the virion tegument of human cytomegalovirus (HCMV). It is, however, nonessential for virion formation. This seeming discrepancy has not met with a satisfactory explanation regarding the role of pp65 in HCMV particle morphogenesis. Here, we addressed the question of how the overall tegument composition of the HCMV virion depended on pp65 and how the lack of pp65 influenced the packaging of particular tegument proteins. To investigate this, we analyzed the proteomes of pp65-positive (pp65pos) and pp65-negative (pp65neg) virions by label-free quantitative mass spectrometry and determined the relative abundances of tegument proteins. Surprisingly, only pUL35 was elevated in pp65neg virions. As the abundance of pUL35 in the HCMV tegument is low, it is unlikely that it replaced pp65 as a structural component in pp65neg virions. A subset of proteins, including the third most abundant tegument protein, pUL25, as well as pUL43, pUL45, and pUL71, were reduced in pp65neg or pp65low virions, indicating that the packaging of these proteins was related to pp65. The levels of tegument components, like pp28 and the capsid-associated tegument proteins pp150, pUL48, and pUL47, were unaffected by the lack of pp65. Our analyses demonstrate that deletion of pp65 is not compensated for by other viral proteins in the process of virion tegumentation. The results are concordant with a model of pp65 serving as an optional scaffold protein that facilitates protein upload into the outer tegument of HCMV particles.ImportanceThe assembly of the tegument of herpesviruses is only poorly understood. Particular proteins, like HCMV pp65, are abundant tegument constituents. pp65 is thus considered to play a major role in tegument assembly in the process of virion morphogenesis. We show here that deletion of the pp65 gene leads to reduced packaging of a subset of viral proteins, indicating that pp65 acts as an optional scaffold protein mediating protein upload into the tegument.

Project description:Abstract: We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration. Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response. The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3). In contrast, NF-{kappa}B is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus. pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells. pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state. Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response. By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response. Polyadenylated RNA from 107 confluent HFs was purified by using Oligotex (QIAGEN, Valencia, Calif.) from Trizol (Invitrogen)-extracted total RNA. This RNA was prepared, reverse transcribed, labeled with Cy3-dUTP or Cy5-dUTP (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by random primed synthesis with DNA pol I Klenow (Amersham Life Science, Inc., Cleveland, Ohio), and hybridized to spotted, human cDNA microarrays as previously described. Sequence-verified human cDNA microarrays (HE and HG series, 31,000 spots; HD51 series, 17,000 spots) were produced at Stanford. Images were collected by using a GenePix 4000B microarray scanner, manually flagged to eliminate poor spots and analyzed by GenePix Pro 2.0 (Axon, Union City, Calif.). A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Keywords: disease_state_design, pathogenicity_design

Dataset Information

Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65).

Publications

Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65).

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