Pp65 Gene Research Articles

Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells.

The development of rapid, efficient, and safe methods for generating Ag-specific T cells is necessary for the clinical application of adoptive immunotherapy. We show that B cells stimulated with CD40 ligand and IL-4 (CD40-B cells) can be efficiently transduced with retroviral vectors encoding a model Ag, CMV tegument protein pp65 gene, and maintain high levels of costimulatory molecules after gene transfer. CTL lines specific for pp65 were readily generated in all four healthy CMV-seropositive donors by stimulating autologous CD8(+) T cells with these transduced CD40-B cells, both of which were derived from 10 ml peripheral blood. ELISPOT assays revealed that the CTL lines used multiple HLA alleles as restricting elements. Thus, CD40-B cells transduced retrovirally with Ag-encoding cDNA can be potent APC and facilitate to generate Ag-specific CTL in vitro.

Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8(+) T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay.

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are useful tools for studying the CTL responses exclusively among those who own the major histocompatibility complex (MHC) class I molecules that present the peptides. For widening the application, an efficient strategy to determine such epitopes in the context of a given MHC is highly desirable. A rapid and efficient strategy is presented for the determination of CTL epitopes in the context of given MHC molecules of interest through multiple screenings consisting of a computer-assisted algorithm and MHC stabilization and enzyme-linked immunospot assays. A major cytomegalovirus (CMV)-specific CTL epitope, QYDPVAALF, in the amino acid sequence of its lower matrix 65 kd phosphoprotein (pp65) presented by HLA-A*2402 molecules was identified from 83 candidate peptides. The results indicate that the CMV-specific CTL response is highly focused to pp65 in the context of HLA-A*2402. Endogenous processing and presentation was confirmed using a peptide-specific CD8(+) T-cell clone as the effectors and autologous fibroblast cells infected with recombinant vaccinia virus expressing pp65 gene or CMV as antigen-presenting cells. Flow cytometric analysis of intracellular interferon-gamma production revealed 0.04% to 0.27% of CD8(+) T cells in peripheral blood of HLA-A24(+) and CMV-seropositive donors to be specific for the peptide. The tetrameric MHC-peptide complexes specifically bound to the reactive T-cell clone and 0.79% of CD8(+) T cells in peripheral blood from a seropositive donor. The peptide could be a useful reagent to study CTL responses to CMV among populations positive for HLA-A*2402.

The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL.

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.

Human cytomegalovirus pp65 lower matrix phosphoprotein harbours two transplantable nuclear localization signals.

Human cytomegalovirus phosphoprotein pp65 is targeted to the cell nucleus immediately after infection. Deletion and point mutation analysis of the pp65 gene expressed in insect cells showed that two hydrophilic regions (HP1 and HP2) within the pp65 C-terminal 40% each harboured an independent nuclear localization signal (NLS); strong association to the nuclear stroma also requires the N-terminal domain. Either region, when fused to chloramphenicol acetyltransferase, localized the reporter protein to the nucleus in insect cells as well as in NIH 3T3 cells and human lung fibroblasts. In addition, HP1 was found to be the target of pp65 Ser/Thr phosphorylation in insect cells and a prokaryotically expressed HP1 was actively phosphorylated in vitro by casein kinase II, for which two site clusters map in HP1. These findings indicate that pp65 includes two NLSs, one of which has the potential to be modulated by phosphorylation.

The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture.

The phosphoprotein pp65 (ppUL83) of human cytomegalovirus (HCMV) is abundantly synthesized during lytic infection in cultured fibroblasts. As a major constituent of extracellular particles, it gains entry to infected cells immediately after adsorption and subsequently translocates to the cell nucleus. This efficient transport is mediated by unique nuclear localization signals. To study the function of pp65, a viral deletion mutant was constructed by replacing the pp65 gene with the bacterial neomycin phosphotransferase gene, driven by the simian virus 40 early promoter. The resulting virus, RVAd65, could be grown and selected on human fibroblasts without complementation. The deletion of the pp65 gene in RVAd65 was verified by using Southern blot and PCR analyses. The lack of expression from the gene was investigated by immunoblotting with pp65-specific monoclonal antibodies. Single-cycle growth analyses showed that RVAd65 grew to levels of infectivity comparable to those of the wild-type virus. Therefore, pp65 is nonessential for the growth of HCMV in human fibroblasts. Electron microscopy revealed no differences in the processes of virion morphogenesis, although the maturation appeared to be delayed. However, the kinetics of expression of the immediate-early genes UL122 and UL123, the early gene UL44, and the late gene UL32 were the same in RVAd65-infected cells as in wild-type virus-infected cells in immunoblot analyses. In vitro phosphorylation assays showed that some of the virion proteins were labelled to a markedly reduced extent by virion-associated kinases in RVAd65 compared with wild-type virus. We therefore conclude that although deletion of the pp65 gene does not abolish replication of HCMV, a recombinant virus lacking pp65 displays phenotypic alterations compared with wild-type virus during growth in cultured fibroblasts.

Growth kinetics of human cytomegalovirus are altered in monocyte-derived macrophages.

Stimulation of monocytes/macrophages with activated nonadherent cells allows productive nonlytic growth of human cytomegalovirus (HCMV), but the viral replication cycle is delayed relative to replication of HCMV in human fibroblasts. Analysis of infected monocyte-derived macrophage (MDM) mRNA for major immediate-early (MIE 86, 72, and 55) and late (pp65 and gB) gene expression by reverse transcription PCR indicates that transcription peaks at 3 and 7 days postinfection (dpi), respectively. In contrast, in human fibroblast controls, mRNA for MIE and late gene expression peaked at 5 and 48 h postinfection, respectively. Consistent with reverse transcription PCR experiments, double-label antibody experiments first detected MIE antigen expression at 12 h postinfection, peaking at 3 dpi, and late (pp65 or gB) antigen expression at 5 dpi, peaking at 7 dpi. MIE antigen was not detected between 3 and 7 dpi but reappeared and was coexpressed with pp65 in enlarged MDM nuclei at 7 dpi. After 7 dpi, macrophages with numerous vacuoles containing large amounts of pp65 and gB were observed in culture. These vacuoles were frequently seen at cellular contact points, suggesting that cell-to-cell transfer of virus was the major mode of viral transmission. Consistent with this observation, infectious virus was recovered from MDM cellular lysates but not culture supernatant. The delayed growth and compartmentalization of HCMV in macrophages may allow the cell to accommodate the viral replication cycle without cell lysis. In addition, the macrophage may function as a vehicle for cell-to-cell transmission of HCMV.

Human cytomegalovirus strain towne pp65 gene: Nucleotide sequence and expression in Escherichia coli

The human cytomegalovirus (HCMV) encodes a 65-kDa tegument protein (pp65), which has been reported to be a target of immune response during natural infection. We have cloned and sequenced the gene encoding pp65 of HCMV Towne strain (pp65 Towne), and have expressed this gene in Escherichia coli in order to study certain antigenic and structural properties of this polypeptide. The pp65 Towne gene had a 99% nucleotide similarity and 99.7% amino acid similarity to pp65 of HCMV AD169 strain (pp65 AD169). However, unlike the pp65 AD169 gene, the pp65 Towne gene was found to be incapable of undergoing RNA splicing due to a base substitution in the critical 3′ splice-acceptor site. Insertion of this protein coding sequence into the bacterial expression plasmids enabled synthesis in E. coli of an immunoreactive pp65-related polypeptide. The recombinant pp65 (rpp65) reacted strongly in immunoblot analysis with pp65-specific murine and human monoclonal antibodies as well as with anti-pp65 rabbit antiserum. In immunoblot analysis, the reactivity of rpp65 with a panel of human HCMV-immune sera indicated that some sera were reactive while other HCMV seropositive sera were nonreactive, a finding similar to that for native pp65.

Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products

To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, we examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. Analysis of promoter deletion mutants indicated that the 5' minimal sequence required for activation is -61 from the CAP site (+1) and that an 8-base-pair sequence located at -51 to -58 is necessary for activation of the pp65 promoter. This sequence is repeated once at +93 and is found as an inverted repeat at +67. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene.