Mutational Mapping of pUL131A of Human Cytomegalovirus Emphasizes Its Central Role for Endothelial Cell Tropism

In the 1970s–1980s, a striking increase in the number of disseminated human cytomegalovirus (HCMV) infections occurred in immunosuppressed patient populations. Autopsy findings documented the in vivo disseminated infection (besides fibroblasts) of epithelial cells, endothelial cells, and polymorphonuclear leukocytes. As a result, multiple diagnostic assays, such as quantification of HCMV antigenemia (pp65), viremia (infectious virus), and DNAemia (HCMV DNA) in patient blood, were developed. In vitro experiments showed that only low passage or endothelial cell-passaged clinical isolates, and not laboratory-adapted strains, could reproduce both HCMV leuko- and endothelial cell-tropism, which were found through genetic analysis to require the three viral genes UL128, UL130, and UL131 of the HCMV UL128 locus (UL128L). Products of this locus, together with gH/gL, were shown to form the gH/gL/pUL128L pentamer complex (PC) required for infection of epithelial cells/endothelial cells, whereas gH/gL and gO form the gH/gL/gO trimer complex (TC) required for infection of all cell types. In 2016, following previous work, a receptor for the TC that mediates entry into fibroblasts was identified as PDGFRα, while in 2018, a receptor for the PC that mediates entry into endothelial/epithelial cells was identified as neuropilin2 (Nrp2). Furthermore, the olfactory receptor family member OR14I1 was recently identified as a possible additional receptor for the PC in epithelial cells. Thus, current data support two models of viral entry: (i) in fibroblasts, following interaction of PDGFRα with TC, the latter activates gB to fuse the virus envelope with the cell membrane, whereas (ii) in epithelial cells/endothelial cells, interaction of Nrp2 (and OR14I1) with PC promotes endocytosis of virus particles, followed by gB activation by gH/gL/gO (or gH/gL) and final low-pH entry into the cell.

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Mapping the viral genetic determinants of endothelial cell tropism in human cytomegalovirus

Journal of Clinical Virology ◽

10.1016/s1386-6532(02)00089-6 ◽

2002 ◽

Vol 25 ◽

pp. 97-109 ◽

Cited By ~ 9

Author(s):

Cynthia A Bolovan-Fritts ◽

Jean A Wiedeman

Keyword(s):

Endothelial Cell ◽

Human Cytomegalovirus ◽

Cell Tropism ◽

Genetic Determinants

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

Journal of General Virology ◽

10.1099/vir.0.83286-0 ◽

2008 ◽

Vol 89 (2) ◽

pp. 359-368 ◽

Cited By ~ 245

Author(s):

Christian Sinzger ◽

Gabriele Hahn ◽

Margarete Digel ◽

Ruth Katona ◽

Kerstin Laib Sampaio ◽

...

Keyword(s):

Endothelial Cell ◽

Human Cytomegalovirus ◽

Restriction Fragment ◽

Artificial Chromosome ◽

High Titre ◽

Cell Tropism ◽

Bac Clone ◽

Hcmv Strain ◽

A Cell ◽

Selection Of

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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Prediction of endothelial cell tropism of human cytomegalovirus strains

Journal of Clinical Virology ◽

10.1016/j.jcv.2005.11.011 ◽

2006 ◽

Vol 35 (4) ◽

pp. 470-473 ◽

Cited By ~ 4

Author(s):

G GERNA ◽

A SARASINI ◽

E GENINI ◽

E PERCIVALLE ◽

M REVELLO

Keyword(s):

Endothelial Cell ◽

Human Cytomegalovirus ◽

Cell Tropism

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Charge Cluster-to-Alanine Scanning of UL128 for Fine Tuning of the Endothelial Cell Tropism of Human Cytomegalovirus

Journal of Virology ◽

10.1128/jvi.01069-08 ◽

2008 ◽

Vol 82 (22) ◽

pp. 11239-11246 ◽

Cited By ~ 17

Author(s):

Andrea Schuessler ◽

Kerstin Laib Sampaio ◽

Christian Sinzger

Keyword(s):

Amino Acids ◽

Endothelial Cell ◽

Human Cytomegalovirus ◽

Fine Tuning ◽

Intermediate Phenotype ◽

Rational Approach ◽

Cell Tropism ◽

Functional Sites ◽

Charge Cluster ◽

Terminal Cluster

ABSTRACT The viral genes UL128, UL130, and UL131A have been identified as major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV), with deletion of either gene causing a null phenotype. We hypothesized that a functional scanning of these genes by minor genetic modifications would allow for the generation of mutants with an intermediate phenotype. By combining charge cluster-to-alanine (CCTA) mutagenesis with markerless mutagenesis of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain, we analyzed UL128 in order to identify functional sites and hence enable targeted modulation of the EC tropism of HCMV. A total of nine mutations in eight charge clusters were tested. Three of the CCTA mutations severely reduced EC tropism, three were irrelevant, two had a weak effect on cell tropism, and one mutation in the most C-terminal cluster caused an intermediate phenotype. All of the highly effective mutations were located in a core region (amino acids 72 to 106) which appears to be particularly crucial for EC tropism. The intermediate effect of mutations in the C-terminal cluster could be modulated by varying the number of amino acids replaced with alanine. This study provides a rational approach for targeted modulation of HCMV cell tropism, which may aid in the development of HCMV strains with a desired degree of attenuation.

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The attenuated Towne strain of human cytomegalovirus may revert to both endothelial cell tropism and leuko- (neutrophil- and monocyte-) tropism in vitro

Journal of General Virology ◽

10.1099/0022-1317-83-8-1993 ◽

2002 ◽

Vol 83 (8) ◽

pp. 1993-2000 ◽

Cited By ~ 25

Author(s):

Giuseppe Gerna ◽

Elena Percivalle ◽

Antonella Sarasini ◽

Fausto Baldanti ◽

M. Grazia Revello

Keyword(s):

Endothelial Cell ◽

Cell Cultures ◽

Human Cytomegalovirus ◽

Biological Properties ◽

Clinical Isolates ◽

Viral Gene ◽

Cell Tropism ◽

Human Embryonic Lung ◽

Healthy Human

The Towne strain of human cytomegalovirus (HCMV), originally recovered from the urine of a congenitally infected newborn, was attenuated through 125 passages in human embryonic lung fibroblast cell cultures. Although reliable markers of attenuation were not identified, the virus was shown to be attenuated by inoculation of both healthy human volunteers and immunocompromised patients. More recently, Towne (like other laboratory-adapted strains) was shown not to have two biological properties typical of recent clinical isolates: endothelial cell tropism and polymorphonuclear leukocyte tropism. These markers of attenuation are lost by all clinical isolates on extensive propagation in cell cultures and are apparently associated with one another. Here, we show that Towne may reacquire both endothelial cell tropism and leuko- (polymorphonuclear- and monocyte-) tropism on adaptation to growth in endothelial cell cultures. However, reversion to endothelial cell tropism is dissociated from reversion to leukotropism, since the latter was reacquired 10–20 passages later. Thus, these two biological properties, which were considered to be encoded by the same viral gene(s), appear to be distinct. Both restriction fragment length polymorphism and Southern blot analysis demonstrated the identity of the attenuated and endothelial cell tropic variants of Towne, thus suggesting that only minor variations (mutations) of the viral genome may be responsible for loss or reacquisition of the two biological properties. Viral genes involved in endothelial cell tropism and leukotropism remain to be identified. However, reversion of attenuated strains to pathogenicity in vivo cannot be excluded a priori.

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Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0509201102 ◽

2005 ◽

Vol 102 (50) ◽

pp. 18153-18158 ◽

Cited By ~ 344

Author(s):

D. Wang ◽

T. Shenk

Keyword(s):

Endothelial Cell ◽

Human Cytomegalovirus ◽

Protein Complex ◽

Cell Tropism ◽

Virion Protein

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Role of human cytomegalovirus UL131A in cell type-specific virus entry and release

Journal of General Virology ◽

10.1099/vir.0.81921-0 ◽

2006 ◽

Vol 87 (9) ◽

pp. 2451-2460 ◽

Cited By ~ 132

Author(s):

Barbara Adler ◽

Laura Scrivano ◽

Zsolt Ruzcics ◽

Brigitte Rupp ◽

Christian Sinzger ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Human Cytomegalovirus ◽

Virus Entry ◽

Cell Tropism ◽

Virus Release ◽

Wild Type ◽

Cell Infection ◽

Infected Cells ◽

In Cis

The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169 with wild-type UL131A in cis in an ectopic position restored endothelial cell tropism. The UL131A protein was found in virions in a complex with gH. Coinfection of fibroblasts with UL131A-negative and -positive viruses restored the endothelial cell tropism of UL131A-negative virions by complementing the virions with UL131A protein. Virus entry into endothelial cells, but not into fibroblasts, was blocked by an antipeptide antiserum to pUL131A. AD169, cis-complemented with wild-type UL131A, showed an impaired release of infectious particles from fibroblasts. A comparable defect in virus release was observed when UL131A was expressed ectopically in a virus background already expressing an intact copy of UL131A. In contrast, virus release from infected endothelial cells was not affected by UL131A. These data suggest a dual role for pUL131A in virus entry and virus exit from infected cells.

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Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

Journal of Virology ◽

10.1128/jvi.00572-10 ◽

2010 ◽

Vol 84 (18) ◽

pp. 9019-9026 ◽

Cited By ~ 13

Author(s):

Andrea Schuessler ◽

Kerstin Laib Sampaio ◽

Laura Scrivano ◽

Christian Sinzger

Keyword(s):

Endothelial Cell ◽

Human Cytomegalovirus ◽

Artificial Chromosome ◽

Protein Domain ◽

Cell Infection ◽

Peptide Motifs ◽

Charged Amino Acids ◽

C Terminus ◽

Charge Cluster ◽

A Charge

ABSTRACT The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.

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