Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during Culture
            <i>In Vitro</i>

Murrell, Isa; Wilkie, Gavin S.; Davison, Andrew J.; Statkute, Evelina; Fielding, Ceri Alan; Tomašec, Peter; Wilkinson, Gavin W. G.; Stanton, Richard J.

doi:10.1128/jvi.02858-15

Cited by 65 publications

(66 citation statements)

References 81 publications

(135 reference statements)

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“…The virus was then propagated in vitro with RL13 and UL128-131A under the control of a tetracycline repressor (tetR). This strategy prevented the acquisition of mutations in culture (20). The pentameric complex and gpRL13 were subsequently restored to virions during a single infectious cycle in fibroblasts lacking tetR (Fig.…”

Section: Resultsmentioning

confidence: 99%

The pentameric complex drives immunologically covert cell–cell transmission of wild-type human cytomegalovirus

Murrell

Bedford

Ladell

et al. 2017

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

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110

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Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell-cell transmission. This process of cell-cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128-131A complex, a feature of WT but not passaged strains of HCMV.virology | immune evasion | herpesvirus | HCMV | cell-cell spread H uman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in the immunocompromised and the leading infectious cause of congenital malformation. As a result, a vaccine has been designated as a priority. However, basic studies of clinically relevant isolates that inform our understanding of the disease process are limited due to the rapid accumulation of genetic mutations during in vitro passage of HCMV. The same three genetic sites are reproducibly affected: the RL13 gene; the UL128 locus (UL128L), which comprises UL128, UL30, and UL131A; and the ∼15-kb U L /b′ gene region (1). Deletions in the U L /b′ region can affect tropism [UL148 (2)], latency [UL136 and UL138 (3-7)], and resistance to NK cells [UL141 (8-10), UL142 (11, 12), and UL135 (13)]. Disabling mutations in RL13 and UL128L independently contribute to the release of high-titer cell-free virus, whereas loss of UL128L restricts virus entry to fibroblasts alone by preventing assembly of a pentameric glycoprotein complex (gH/gL/pUL128/ pUL130/pUL131A) in the virion envelope (1,14,15).The rapid selection of mutations is a major obstacle to the propagation of genetically intact "clinical" strains of HCMV, which in turn has led to significant gaps in our understanding of the affiliated disease processes (16). In particular, HCMV is largely cell-associated in vivo (17), and clinical isolates exhibit a similar phenotype in vitro (17, 18), yet most studies in the field are based on laboratory strains that produce high titers of cell-free virus (19). As a consequence, little is known about the fundamental processes involved in the infectious spread of cell-associated HCMV.In this study, we used a system tha...

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Section: Resultsmentioning

confidence: 99%

The pentameric complex drives immunologically covert cell–cell transmission of wild-type human cytomegalovirus

Murrell

Bedford

Ladell

et al. 2017

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

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110

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“…The RL13 ORF encodes an envelope glycoprotein that has been described as an inhibitor of HCMV replication in culture and mutations in RL13 have been documented during cell-free passage in fibroblasts and epithelial cells (40, 54, 55). While the ME recombinant used in the present studies harbors such a RL13 mutation, the RL13 expression status of the TB and TR BAC-clones is unclear.…”

Section: Resultsmentioning

confidence: 99%

Specialization for cell-free or cell-to-cell spread of BAC-cloned HCMV strains is determined by factors beyond the UL128-131 and RL13 loci

Schultz

Lanchy

Day

et al. 2019

Preprint

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ABSTRACTIt is widely held that clinical isolates of human cytomegalovirus (HCMV) are highly cell-associated, and mutations affecting the UL128-131 and RL13 loci that arise in culture lead to the appearance of a cell-free spread phenotype. The BAC-clone Merlin (ME), expresses abundant UL128-131, is RL13-impaired and produces low infectivity virions in fibroblasts, whereas TB40/e (TB) and TR are low in UL128-131, RL13-intact and produce virions of much higher infectivity. Despite these differences, quantification of spread by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts. In epithelial cells, ME spread more efficiently, consistent with robust UL128-131 expression. Strikingly, ME spread far better than TB or TR in the presence of neutralizing antibodies on both cell types, indicating that ME is not simply deficient at cell-free spread, but is particularly efficient at cell-to-cell spread, whereas TB and TR cell-to-cell spread is poor. Sonically disrupted ME-infected cells contained scant infectivity, suggesting that the efficient cell-to-cell spread mechanism of ME depends on features of the intact cells such as junctions or intracellular trafficking processes. Even when UL128-131 was transcriptional repressed, cell-to-cell spread of ME was still more efficient than TB or TR. Moreover, RL13 expression comparably reduced both cell-free and cell-to-cell spread of all three strains, suggesting that it acts at a stage of assembly and/or egress common to both routes of spread. Thus, HCMV strains can be highly specialized for either for cell-free or cell-to-cell spread and these phenotypes are determined by factors beyond the UL128-131 or RL13 loci.IMPORTANCEBoth cell-free and cell-to-cell spread are likely important for the natural biology of HCMV. In culture, strains clearly differ in their capacity for cell-free spread as a result of differences in the quantity and infectivity of extracellular released progeny. However, it has been unclear whether “cell-associated” phenotypes are simply the result of poor cell-free spread, or are indicative of particularly efficient cell-to-cell spread mechanisms. By measuring the kinetics of spread at early time points, we were able to show that HCMV strains can be highly specialized to either cell-free or cell-to-cell mechanisms, and this was not strictly linked the efficiency of cell-free spread. Our results provide a conceptual approach to evaluating intervention strategies for their ability to limit cell-free or cell-to-cell spread as independent processes.

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“…Strain Merlin contains the complete genetic complement of HCMV, and is frame shifted in two genes (RL13 – , UL128 – ). Alterations to the BAC were monitored by local PCR and Sanger sequencing, and the entire genomes of the viruses were confirmed by Illumina sequencing as described previously (Murrell et al, 2016). Details of the viruses are provided in Table 1 and a list of primers used in their construction and local sequencing of the BACs are provided in Tables 2 and 3 respectively.…”

Section: Methodsmentioning

confidence: 99%

Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation

Fielding

Weekes

Nobre

et al. 2017

eLife

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The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation.DOI: http://dx.doi.org/10.7554/eLife.22206.001

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Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during Culture In Vitro

Cited by 65 publications

References 81 publications

The pentameric complex drives immunologically covert cell–cell transmission of wild-type human cytomegalovirus

The pentameric complex drives immunologically covert cell–cell transmission of wild-type human cytomegalovirus

Specialization for cell-free or cell-to-cell spread of BAC-cloned HCMV strains is determined by factors beyond the UL128-131 and RL13 loci

Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation

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