Nuclear expression of the lower matrix protein of human cytomegalovirus in peripheral blood leukocytes of immunocompromised viraemic patients

Revello, Maria Grazia; Percivalle, Elena; Matteo, Angela Di; Morini, Fulvia; Gerna, Giuseppe

doi:10.1099/0022-1317-73-2-437

Cited by 89 publications

(50 citation statements)

References 29 publications

(15 reference statements)

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“…was most likely due to translocation of pp65 after virus entry. It has previously been shown that virion pp65 accumulates in the nucleus before immediate early proteins, during early stages of the HCMV replication cycle (Revello et al, 1992;Grefte et al, 1992). The appearance of pp65 in the nucleus within 1 h p.i.…”

Section: Discussionmentioning

confidence: 98%

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Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32)

Hensel

Meyer²,

Gärtner³

et al. 1995

Journal of General Virology

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The human cytomegalovirus (HCMV) basic phosphoprotein pp 150, encoded by the UL32 gene, together with the two other major phosphoproteins, pp65 (ppUL83) and pp71 (ppUL82) and several minor structural proteins, form the tegument around the viral nucleocapsid. Experiments were undertaken to locate the area of assembly of tegument proteins ppl50 and pp65 and nucleocapsids in fibroblasts, in order to assess the functional role of these two structural proteins in HCMV morphogenesis. Whereas ppl50 expression starts during the cytoplasmic maturation of HCMV, pp65 is expressed in the early and late phases of HCMV gene transcription. Western blot analysis of isolated cell fractions showed that ppl50 is initially (48 h postinfection) localized in the nucleus, associated either with the nuclear membrane or with viral assembly regions, and later (72 h post-infection) in the cytoplasm. By indirect immunofluorescence, ppl50 and pp65 could be detected in nuclear subcompartments and were strongly associated with the nuclear membrane. Using immunogold analysis by electron microscopy, pp65 was exclusively detected within the matrix of cytoplasmic and extracellular dense bodies and of dense body-like structures in the nucleoplasm. These were localized in close contact with hypertrophic nucleoli, in the proximity of developing nucleocapsids and in special patches at the inner nuclear membrane L Positive immunostaining of ppl50 was observed at the surface of developing nucleocapsids concentrated within viral assembly regions in the nucleoplasm. Additionally, the tegument of cytoplasmic and extracellular virions was stained, whereas dense bodies or nuclear dense body-like structures did not react. Thus, the acquisition of the tegument protein pp 150 seems to start in special nuclear subcompartments of the HCMV-infected fibroblasts.

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Section: Discussionmentioning

confidence: 98%

“…Protein kinase activity may also be associated with pp65 itself (Somogyi et al, 1990). The pp65 associated with virions and dense bodies immediately appears in the nucleus after infection and also occurs in the cytoplasm after synthesis during the later stages of replication (Weiner et al, 1985;Revello et at., 1992).…”

Section: Introductionmentioning

confidence: 99%

Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32)

Hensel

Meyer²,

Gärtner³

et al. 1995

Journal of General Virology

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“…However, while initially the most represented viral antigen inside nuclei of PMNLs was thought to be the major immediate early (MIE) protein p72, subsequently it was shown that the most represented viral protein is pp65 ( Fig. 1) early late structural phosphoprotein [13]. This protein is abundantly detected within 1 h post-infection (p.i.)…”

Section: Hcmv Leukocyte and Endothelial Cell Tropismmentioning

confidence: 99%

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

Gerna

Revello

Baldanti

et al. 2017

Journal of General Virology

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Between the 1980s and 1990s, three assays were developed for diagnosis of human cytomegalovirus (HCMV) infections: leuko (L)-antigenemia, L-viremia and L-DNAemia, detecting viral protein pp65, infectious virus and viral DNA, respectively, in circulating leukocytes Repeated initial attempts to reproduce the three assays in vitro using laboratory-adapted strains and infected cell cultures were consistently unsuccessful. Results were totally reversed when wild-type HCMV strains were used to infect either fibroblasts or endothelial cells. Careful analysis and sequencing of plaque-purified viruses from recent clinical isolates drew attention to the ULb¢ region of the HCMV genome. Using bacterial artificial chromosome technology, it was shown by both gain-of-function and loss-of-function experiments that UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. In addition, a number of clinical isolates passaged in human fibroblasts had lost both properties (leuko-tropism and endothelial cell-tropism) when displaying a mutation in the UL131-128 locus

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“…The automated screening is finished when the whole cell area (400 and 1,600 microscopic fields, respectively, for preparations with a cell volume of [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] pl and 150-200 pl) has been analyzed or when 320 alarms have been found. The program then displays the number of screened fields, the number of counted cells and the number of alarms.…”

Section: Data Storage and Evaluationmentioning

confidence: 99%

“…tibody technique for the recognition of human cytomegalovirus (HCMV)-antigen (3,8,12,21). In addition to detection of cells, LEYTAS offers the possibility of quantitation by counting both virus-positive and virusnegative cells.…”

mentioning

confidence: 99%

Automated image cytometry for detection of rare, viral antigen‐positive cells in peripheral blood

Ploem-Zaaijer

Mesker

Boland³

et al. 1994

Cytometry

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A cell detection method based upon automated screening is described for recognition of low frequencies (1 in 100,000) of immuno‐enzymatically labelled white blood cells in human peripheral blood. The used image cytometry instrumentation (LEYTAS) includes a wide‐field, fully automated microscope (Autoplan) and a modular image analysis computer (MIAC), both from Leica, Wetzlar, Germany. The MIAC contains image boards for optimum use of mathematical morphology algorithms. Communication with the MIAC is via a personal computer. Programs for automated cell analysis have been written in C language. Main features of the system are fast analysis of, large microscope fields including a count of all cells, selection of objects of interest (alarms), and display of digitally stored images of these alarms. We tested this system for the detection of white blood cells expressing antigen of cytomegalovirus (pp65) in 50 human blood smears from kidney transplant recipients. Immuno‐enzymatic (peroxidase) staining was performed with DAB and counterstaining with hematoxylin. For determination of the sensitivity, a series of dilutions of a positive sample with a negative sample was performed. The lowest frequency detected was 1 antigenpositive cell / 3 × 105 antigen‐negative cells. Screening time was about 60 min for one million cells. © 1994 Wiley‐Liss, Inc.

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Nuclear expression of the lower matrix protein of human cytomegalovirus in peripheral blood leukocytes of immunocompromised viraemic patients

Cited by 89 publications

References 29 publications

Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32)

Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32)

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

Automated image cytometry for detection of rare, viral antigen‐positive cells in peripheral blood

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