Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Townelong as BACs and site-directed mutagenesis using a PCR-based technique

Hahn, Gabriele; Rose, Dietlind; Wagner, Markus; Rhiel, Sylvia; McVoy, Michael A.

doi:10.1016/s0042-6822(02)00061-2

Cited by 42 publications

(34 citation statements)

References 46 publications

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“…The cloning of an EC-tropic clinical HCMV isolate (VR1814) as a bacterial artificial chromosome (BAC) in Escherichia coli (FIX-BAC) by adapting a previously reported method (23) provided standard genetic material with preserved wild-type characteristics for rapid mutagenesis in E. coli (14,15,24). Site-directed mutagenesis of FIX-BAC has been used to show that, unlike the murine cytomegalovirus (MCMV) (4), the HCMV-encoded ribonucleotide reductase homolog (UL45) is not the genetic determinant of EC tropism of HCMV (14) and is dispensable for growth in vitro (14,26).…”

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confidence: 99%

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

Hahn

Revello

Patrone

et al. 2004

J Virol

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448

336

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Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium-leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by trans-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection.

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confidence: 99%

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

Hahn

Revello

Patrone

et al. 2004

J Virol

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448

336

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“…ATCC Towne (VR-977) has been demonstrated to contain a mixture of two variants, i.e., Towne short , which lacks ULbЈ sequence, and Towne long , which includes this genomic fragment (11,12,20,29). Thus, in principle, AD169-varT and AD160-varATCC somehow reflect the situation found in Towne.…”

Section: Discussionmentioning

confidence: 99%

The Latency-Associated UL138 Gene Product of Human Cytomegalovirus Sensitizes Cells to Tumor Necrosis Factor Alpha (TNF-α) Signaling by Upregulating TNF-α Receptor 1 Cell Surface Expression

Montag

Wagner

Gruska

et al. 2011

J Virol

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Many viruses antagonize tumor necrosis factor alpha (TNF-␣) signaling in order to counteract its antiviral properties. One way viruses achieve this goal is to reduce TNF-␣ receptor 1 (TNFR1) on the surface of infected cells. Such a mechanism is also employed by human cytomegalovirus (HCMV), as recently reported by others and us. On the other hand, TNF-␣ has also been shown to foster reactivation of HCMV from latency. By characterizing a new variant of HCMV AD169, we show here that TNFR1 downregulation by HCMV only becomes apparent upon infection of cells with HCMV strains lacking the so-called ULb region. This region contains genes involved in regulating viral immune escape, cell tropism, or latency and is typically lost from laboratory strains but present in low-passage strains and clinical isolates. We further show that although ULb-positive viruses also contain the TNFR1-antagonizing function, this activity is masked by a dominant TNFR1 upregulation mediated by the ULb gene product UL138. Isolated expression of UL138 in the absence of viral infection upregulates TNFR1 surface expression and can rescue both TNFR1 reexpression and TNF-␣ responsiveness of cells infected with an HCMV mutant lacking the UL138-containing transcription unit. Given that the UL138 gene product is one of the few genes recognized to be expressed during HCMV latency and the known positive effects of TNF-␣ on viral reactivation, we suggest that via upregulating TNFR1 surface expression UL138 may sensitize latently infected cells to TNF-␣-mediated reactivation of HCMV.

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“…The genomes of several HCMV strains have been captured as BACs, including a number based on low-passage viruses, and these are proving to be an invaluable resource for research (49)(50)(51)(52)(53)(54). However, none contains the full complement of HCMV genes, both because of mutations that occurred in cell culture prior to being cloned and, in most cases, because sequences were deleted in order to accommodate the BAC vector.…”

Section: Figurementioning

confidence: 99%

Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication

Stanton¹,

Baluchova²,

Dargan³

et al. 2010

J. Clin. Invest.

239

375

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Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.

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Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Townelong as BACs and site-directed mutagenesis using a PCR-based technique

Cited by 42 publications

References 46 publications

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

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

The Latency-Associated UL138 Gene Product of Human Cytomegalovirus Sensitizes Cells to Tumor Necrosis Factor Alpha (TNF-α) Signaling by Upregulating TNF-α Receptor 1 Cell Surface Expression

Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication

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