Coding potential of laboratory and clinical strains of human cytomegalovirus

Murphy, Eain A.; Yü, Dong; Grimwood, Jane; Schmutz, Jeremy; Dickson, Mark; Jarvis, Michael A.; Hahn, Gabriele; Nelson, Jay A.; Myers, R; Shenk, Thomas

doi:10.1073/pnas.2136652100

Cited by 466 publications

(466 citation statements)

References 49 publications

(33 reference statements)

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“…The reason for this is unknown, but it seems likely that it was caused by an illegitimate recombination event during the insertion of the BAC vector by homologous recombination in fibroblasts. Interestingly, a similar unanticipated deletion was found in the HCMV FIXBAC (Murphy et al, 2003b), which was constructed using the same BAC vector and flanking homologous arms (Hahn et al, 2002). With regard to previously reported TB40/E-variants, TB40-BAC4 resembles the Bart strain in that UL141 has a frameshift insertion at codon 63 (Tomasec et al, 2005); however, unlike strain Bart, UL144 and UL145 are intact and thus TB40-BAC4 is identical to the TB40/E sequence published by Dolan et al (2004) in all three genes.…”

Section: Sequence Alignment Of Tb40-bac4 With Various Hcmv Genomesmentioning

confidence: 59%

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: Sequence Alignment Of Tb40-bac4 With Various Hcmv Genomesmentioning

confidence: 59%

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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“…The reasons for these conflicting results remain unresolved. In contrast to endotheliotropic/clinical HCMV strains, such as Towne/E and TB40/E, laboratory/ fibroblast-adapted strains, such as AD169, Towne, and TB40/F, have reduced infectivity of endothelial/epithelial cells and monocytes because of losses or mutations in the U L BЈ region of the viral genome (35)(36)(37). Expression of distinct viral glycoproteins from the U L BЈ region allows viral infection of endothelial/epithelial cells but not of fibroblasts, indicating the existence of cell type-specific receptors for different strains of HCMV (38).…”

Section: Discussionmentioning

confidence: 99%

Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility

Chan

Nogalski²,

Yurochko³

2009

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

179

291

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Human cytomegalovirus (HCMV) rapidly induces a mobile and functionally unique proinflammatory monocyte following infection that is proposed to mediate viral spread. The cellular pathways used by HCMV to initiate these biological changes remain unknown. Here, we document the expression of the epidermal growth factor receptor (EGFR) on the surface of human peripheral blood monocytes but not on other blood leukocyte populations. Inhibition of EGFR signaling abrogated viral entry into monocytes, indicating that EGFR can serve as a cellular tropism receptor. Moreover, HCMV-activated EGFR was required for the induction of monocyte motility and transendothelial migration, two biological events required for monocyte extravasation into peripheral tissue, and thus viral spread. Transcriptome analysis revealed that HCMV-mediated EGFR signaling up-regulated neural Wiskott-Aldrich syndrome protein (N-WASP), an actin nucleator whose expression and function are normally limited in leukocytes. Knockdown of N-WASP expression blocked HCMV-induced but not phorbol 12-myristate 13-acetate (PMA)-induced monocyte motility, suggesting that a switch to and/or the distinct use of a new actin nucleator controlling motility occurs during HCMV infection of monocytes. Together, these data provide evidence that EGFR plays an essential role in the immunopathobiology of HCMV by mediating viral entry into monocytes and stimulating the aberrant biological activity that promotes hematogenous dissemination.T he wide range of pathological complications associated with human cytomegalovirus (HCMV) infection is a direct consequence of viral spread to peripheral organ sites and the broad cellular tropism of the virus (1). Monocytes are primary in vivo targets for HCMV and are believed to be responsible for hematogenous dissemination of HCMV to multiple organ systems (2). We previously showed that HCMV infection of monocytes polarized the infected cell toward a distinct proinflammatory phenotype that possessed the distinct biological changes necessary to promote viral spread (3-5). Specifically, HCMV infection induced polarization of infected monocytes toward an M1 proinflammatory cell type that simultaneously exhibited characteristics associated with an M2 antiinflammatory macrophage (6). The infected monocytes also displayed a high level of chemokinesis when compared with monocytes activated by LPS or phorbol 12-myristate 13-acetate (PMA) (4, 5). Moreover, an accelerated rate of differentiation from short-lived monocytes (nonpermissive for HCMV replication) into long-lived macrophages (permissive for HCMV replication) was observed following infection with HCMV (4). Based on our studies, we suggest that during primary infection, newly infected peripheral monocytes acquire a motile phenotype that promotes exit of the infected cell from the circulating blood into multiple organ tissue despite the absence of a chemotactic gradient. Once in the surrounding tissue, differentiation into HCMV replication-competent macrophages occurs, resulting in viral spread...

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“…BADwt (47) is derived from a BAC clone of the AD169 HCMV strain; BADrUL131 (19,22) is a derivative of BADwt in which the UL131 ORF has been repaired; BFXwt (48,49) is derived from a BAC clone of the VR1814 clinical HCMV isolate. Viruses were prepared by electroporation of BAC DNAs into HFFs, and the resulting virus preparation was amplified once in ARPE-19 cells or HFFs, unless otherwise specified.…”

Section: Methodsmentioning

confidence: 99%

Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells

Wang

Schröer

et al. 2007

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

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Human cytomegalovirus infects multiple cell types, including fibroblasts and epithelial cells. It penetrates fibroblasts by fusion at the cell surface but is endocytosed into epithelial cells. In this report, we demonstrate by electron microscopy that the virus uses two different routes to enter retinal pigmented epithelial cells, depending on the cell type in which the infecting virus was produced. Virus produced in epithelial cells preferentially fuses with the plasma membrane, whereas fibroblast-derived virus mostly enters by receptor-mediated endocytosis. Treatment of epithelial cells with agents that block endosome acidification inhibited infection by virus produced in fibroblasts but had only a modest effect on infection by virus from epithelial cells. Epithelial cell-generated virions had higher intrinsic ''fusion-from-without'' activity than fibroblast-generated particles, and the two virus preparations triggered different cellular signaling responses, as evidenced by markedly different transcriptional profiles. We propose that the cell type in which a human cytomegalovirus particle is produced likely influences its subsequent spread and its contribution to pathogenesis.host cell transcriptome ͉ pathogenesis ͉ virus spread

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Coding potential of laboratory and clinical strains of human cytomegalovirus

Cited by 466 publications

References 49 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

Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility

Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells

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