Human Cytomegalovirus UL131-128 Genes Are Indispensable for Virus Growth in Endothelial Cells and Virus Transfer to Leukocytes

Hahn, Gabriele; Revello, Maria Grazia; Patrone, Marco; Percivalle, Elena; Campanini, Giulia; Sarasini, Antonella; Wagner, Markus; Gallina, Andrea; Milanesi, G.; Koszinowski, Ulrich H.; Baldanti, Fausto; Gerna, Giuseppe

doi:10.1128/jvi.00695-09

Cited by 81 publications

(180 citation statements)

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“…This technique (i) greatly facilitates genetic manipulation of viral genes in the context of the viral genome, (ii) enables amplification of the genome in the absence of selective pressure, and (iii) yields clonal viral genomes without the need of plaque purifications (Brune et al, 1999;Messerle et al, 2000). Here, we report generation of BACs derived from TB40/E in order to yield genetically pure and highly endotheliotropic clones of this virus and to test the hypothesis that the phenotype of variants contained within TB40/E is determined by the UL128-UL131A gene region previously linked to loss of EC tropism during extended fibroblast adaptation (Adler et al, 2006;Hahn et al, 2004;Wang & Shenk, 2005b).…”

Section: Introductionmentioning

confidence: 99%

Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E

Sinzger

Hahn

Digel

et al. 2008

Journal of General Virology

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362

355

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Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

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Section: Introductionmentioning

confidence: 99%

Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E

Sinzger

Hahn

Digel

et al. 2008

Journal of General Virology

Self Cite

362

355

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“…Mutations in the UL131A-UL128 gene locus are sufficient to eliminate epithelial/endothelial tropism and occur spontaneously within only a few passages of wild-type (WT) HCMV in fibroblasts (15,16). In addition, Pentamer cell surface overexpression interferes with HCMV entry into epithelial cells, but not into fibroblasts, suggesting the presence of a cell-typespecific Pentamer receptor (17).…”

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confidence: 99%

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Ciferri

Chandramouli

Donnarumma

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

141

189

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Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/ gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/ gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.human cytomegalovirus | HCMV | Pentamer complex | gH/gL/gO | virus entry

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“…The IgG antibody responses to the glycoprotein complexes representing the pentamer (gH/gL/ pUL128L), the dimer gH/gL (Hahn et al, 2004;Ryckman et al, 2008) and gB were investigated. In addition, the neutralizing antibody response was determined in parallel by inoculating virus-serum mixtures onto ARPE-19 epithelial cells and HELF cells.…”

Section: Discussionmentioning

confidence: 99%

“…In 2004, in collaboration with the group of U. Koszinowksi (Munchen, Germany), it was documented for the first time that HCMV UL128-131 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes (Hahn et al, 2004). Subsequently, it was shown that the UL128, UL130 and UL131 locus (thus referred to as UL128L) gene products formed a pentameric complex with gH/gL (gH/gL/pUL128/pUL130/pUL131), which was required for infection of both endothelial and epithelial cells (Wang & Shenk, 2005;Ryckman et al, 2008;.…”

Section: Introductionmentioning

confidence: 99%

Differential kinetics of human cytomegalovirus load and antibody responses in primary infection of the immunocompetent and immunocompromised host

Gerna

Lilleri²,

Fornara

et al. 2015

Journal of General Virology

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The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. In total, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4 + and CD8 + HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, with the two antibody levels then overlapping from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting for up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titres in the immunocompetent over the entire one-year followup. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4 + T cells. In this setting, the elevated antibody response occurred in the presence of differentiated follicular helper T cells in the blood, which decreased in number as did antibody titres upon reappearance of HCMV-specific CD4 + T cells.

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Human Cytomegalovirus UL131-128 Genes Are Indispensable for Virus Growth in Endothelial Cells and Virus Transfer to Leukocytes

Cited by 81 publications

References 0 publications

Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E

Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Differential kinetics of human cytomegalovirus load and antibody responses in primary infection of the immunocompetent and immunocompromised host

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