Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus

Rüger, Barbara; Klages, Sabine; Walla, B; Albrecht, Jens; Fleckenstein, Bernhard; Tomlinson, Peter; Barrell, Barclay G.

doi:10.1128/jvi.61.2.446-453.1987

Cited by 137 publications

(61 citation statements)

References 33 publications

(31 reference statements)

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“…Our data are consistent with that study and extend those results by characterizing the UL98 gene region and its protein product. This multiple use of 3Ј ends by RNAs of different kinetic classes has been demonstrated previously and appears to be a common means for HCMV to utilize genetic information (2,6,7,9,10,19,21,28,41). These data indirectly imply that HCMV genes and RNAs may be regulated mostly at the level of their promoters and 5Ј sequences, since RNAs of different kinetic classes share 3Ј ends.…”

supporting

confidence: 61%

The human cytomegalovirus UL98 gene transcription unit overlaps with the pp28 true late gene (UL99) and encodes a 58-kilodalton early protein

Adam

Jervey

Kohler

et al. 1995

J Virol

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A murine monoclonal antibody (I2) reacts strongly with the nucleus of human cytomegalovirus (HCMV)infected human fibroblasts. Western blot (immunoblot) analysis using I2 demonstrated that a protein with an apparent molecular mass of 58-kDa (E58) was expressed at 5 h after infection, and levels increased through 72 h. Immunoblot screening of an early cDNA expression library resulted in a positive clone which hybridized to the right end of the XbaI C fragment of the HCMV Towne strain. Further analysis demonstrated that the E58-specific clone was homologous to the putative UL98 open reading frame, which has been proposed to encode the viral alkaline exonuclease homolog. RNA analysis demonstrated a 3.0-kb RNA which is expressed at early times after infection, as well as in the absence of viral DNA replication, and which is 3 coterminal with the pp28 (UL99) gene region. Insertion of the UL98 genomic sequence into a eucaryotic expression vector and subsequent Western blot analysis using I2 demonstrated that the expressed protein comigrated with E58 from infected cells. E58 also reacts specifically with a previously described antibody, anti-P 2-1 , which was proposed to recognize a putative late 58-kDa protein. E58 comigrates with the putative late 58-kDa protein, indicating that these two proteins are likely the same. Analysis of the UL98 promoter revealed a TATATAA sequence located at nucleotide 142525. Insertion of the putative promoter 5 to a reporter gene demonstrated that the UL98 promoter was activated in cotransfection experiments with IE1 and IE2 proteins. These studies demonstrate that UL98 is a bona fide early gene, which is consistent with its probable role as the viral alkaline exonuclease gene.

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supporting

confidence: 61%

The human cytomegalovirus UL98 gene transcription unit overlaps with the pp28 true late gene (UL99) and encodes a 58-kilodalton early protein

Adam

Jervey

Kohler

et al. 1995

J Virol

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“…While neither of these properties clearly identifies this protein as being the MCMV homolog of the HCMV UL82 protein, they are both properties of matrix proteins of HCMV. Additionally, M83 is a true late protein, as are all known HCMV matrix components other than UL83 (10,16,26,40,44,63). These properties of M83 support its putative identification.…”

Section: Discussionmentioning

confidence: 74%

“…A third oligonucleotide probe, designated 5ЈM83, was located at the 3Ј end of the M84 (11,13,14). This program selected the following residues for alignment: in HCMV, 268 to 374 for UL82, 257 to 360 for UL83, and 409 to 510 for UL84; in MCMV, 272 to 373 for M82, 259 to 361 for M83, and 253 to 356 for M84 (23,63). Gap penalty was set at a value of 8, and the PAM250 scoring matrix was used.…”

Section: Analysis Of M82 M83 and M84 Orf Homologies And Evolutionarmentioning

confidence: 99%

“…On the basis of the colinearity of recently identified MCMV genes with their HCMV counterparts, we selected subgenomic fragments of MCMV for sequencing (8,9,12,41). In this way, we were able to identify a region of MCMV HindIII fragment C containing ORFs which displayed colinearity and significant homology with the UL86 major capsid protein, the UL85 putative capsid protein, and the UL82 and UL83 matrix phosphoproteins of HCMV (7,8,27,39,63,65). Additionally, one ORF was identified in a position colinear with and possessing weak homology to the nonstructural HCMV UL84 protein (23).…”

mentioning

confidence: 94%

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Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins

Cranmer

Clark

Morello

et al. 1996

J Virol

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We have identified three open reading frames (ORFs) in murine cytomegalovirus (MCMV), designated M82, M83, and M84, which likely encode homologs of the human cytomegalovirus (HCMV) UL82 and UL83 matrix phosphoproteins. These ORFs, in the HindIII C fragment of MCMV, are colinear with the UL82, UL83, and UL84 ORFs of HCMV. M82 encodes a 598-amino-acid (aa) protein with homology to UL82, M83 encodes an 809-aa protein with homology to UL82 and UL83, and M84 encodes a 587-aa protein with homology to UL83 and UL84. Analysis of transcription by Northern (RNA) blotting indicated that the M82 and M83 ORFs are transcribed as 2.2-and 5-kb mRNAs, respectively, at 24 to 48 h postinfection (p.i.), while M84 is transcribed as a 6.9-kb mRNA only at 8 h p.i. All transcripts appear to terminate at the same position 3 of the M82 ORF. Of the products of the three ORFs, only M83 is strongly recognized by hyperimmune mouse serum. The M83 protein is a virion-associated phosphoprotein with an apparent molecular mass of 125 kDa. In MCMV-infected cells, it is detectable by Western blotting (immunoblotting) only at 48 h p.i. in the absence of phosphonoacetic acid, consistent with late gene expression. The M83 ORF is also expressed at high levels in cells infected by a recombinant vaccinia virus and yields a protein which is serologically cross-reactive and comigrates with the authentic MCMV protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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“…Herein, we present a series of in vitro and in vivo experiments designed to demonstrate proof-of-concept for high throughput identification of antigens recognised by T cell responses in human or murine systems, using IVTT products unpurified, affinity purified through nickel resin or magnetic beads, or absorbed in beads to enhance the cell mediated immunogenicity by promoting dendritic cell uptake [20,21]. IVTT produced antigens of FluM and FluHA from Influenza A virus [22], CMVpp65 from Cytomegalovirus [23] and EBNA3A from Epstein-Barr virus [24] were assayed using bulk human PBMC for T cell recognition. Additionally, Plasmodium yoelii circumsporozoite protein (PyCSP) [25] IVTT products were assayed for antigenicity in vitro using splenocytes from PyCSP-immunized mice, and for immunogenicity in vivo as assessed by capacity to boost a PyCSP-specific immune response primed by plasmid DNA.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Approaches to Identify the Targets of Cellular Immunity on a Proteome-Wide Scale

et al. 2011

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BackgroundVaccine development against malaria and other complex diseases remains a challenge for the scientific community. The recent elucidation of the genome, proteome and transcriptome of many of these complex pathogens provides the basis for rational vaccine design by identifying, on a proteome-wide scale, novel target antigens that are recognized by T cells and antibodies from exposed individuals. However, there is currently no algorithm to effectively identify important target antigens from genome sequence data; this is especially challenging for T cell targets. Furthermore, for some of these pathogens, such as Plasmodium, protein expression using conventional platforms has been problematic but cell-free in vitro transcription translation (IVTT) strategies have recently proved successful. Herein, we report a novel approach for proteome-wide scale identification of the antigenic targets of T cell responses using IVTT products.Principal FindingsWe conducted a series of in vitro and in vivo experiments using IVTT proteins either unpurified, absorbed to carboxylated polybeads, or affinity purified through nickel resin or magnetic beads. In vitro studies in humans using CMV, EBV, and Influenza A virus proteins showed antigen-specific cytokine production in ELIspot and Cytometric Bead Array assays with cells stimulated with purified or unpurified IVTT antigens. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost showed antigen-specific cytokine production using purified IVTT antigens only. Overall, the nickel resin method of IVTT antigen purification proved optimal in both human and murine systems.ConclusionsThis work provides proof of concept for the potential of high-throughput approaches to identify T cell targets of complex parasitic, viral or bacterial pathogens from genomic sequence data, for rational vaccine development against emerging and re-emerging diseases that pose a threat to public health.

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Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus

Cited by 137 publications

References 33 publications

The human cytomegalovirus UL98 gene transcription unit overlaps with the pp28 true late gene (UL99) and encodes a 58-kilodalton early protein

The human cytomegalovirus UL98 gene transcription unit overlaps with the pp28 true late gene (UL99) and encodes a 58-kilodalton early protein

Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins

Evaluation of Approaches to Identify the Targets of Cellular Immunity on a Proteome-Wide Scale

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