Human Cytomegalovirus Glycoprotein gO Complexes with gH/gL, Promoting Interference with Viral Entry into Human Fibroblasts but Not Entry into Epithelial Cells

AbstractThe advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

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A Novel Human Cytomegalovirus Glycoprotein, gpUS9, Which Promotes Cell-to-Cell Spread in Polarized Epithelial Cells, Colocalizes with the Cytoskeletal Proteins E-Cadherin and F-Actin

Journal of Virology ◽

10.1128/jvi.72.7.5717-5727.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5717-5727 ◽

Cited By ~ 17

Author(s):

Ekaterina Maidji ◽

Sharof Tugizov ◽

Gerardo Abenes ◽

Thomas Jones ◽

Lenore Pereira

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Retinal Pigment ◽

Human Fibroblasts ◽

Basolateral Membrane ◽

Cytoskeletal Proteins ◽

Retinal Pigment Epithelial Cells ◽

Cell Junctions ◽

Polarized Epithelial Cells ◽

Cell Spread

ABSTRACT Processes by which human herpesviruses penetrate and are released from polarized epithelial cells, which have distinct apical and basolateral membrane domains differing in protein and lipid content, are poorly understood. We recently reported that human cytomegalovirus (CMV) mutants with deletions of the gene US9 formed wild-type plaques in cultures of human fibroblasts but were impaired in the capacity for cell-to-cell spread in polarized human retinal pigment epithelial cells. Unlike the glycoproteins that are required for infection, the protein encoded by CMV US9 plays an accessory role by promoting dissemination of virus across cell-cell junctions of polarized epithelial cells. To identify the product and investigate its specialized functions, we selected Madine-Darby canine kidney II (MDCK) epithelial cells that constitutively express CMV US9 or, as a control, US8. The gene products, designated gpUS9 and gpUS8, were glycosylated proteins of comparable molecular masses but differed considerably in intracellular distribution and solubility. Immunofluorescence laser scanning confocal microscopy indicated that, like gpUS8, gpUS9 was present in the endoplasmic reticulum and Golgi compartments of nonpolarized cells. In polarized epithelial cells, gpUS9 also accumulated along lateral membranes, colocalizing with cadherin and actin, and was insoluble in Triton X-100, a property shared with proteins that associate with the cytoskeleton. We hypothesize that gpUS9 may enhance the dissemination of CMV in infected epithelial tissues by associating with the cytoskeletal matrix.

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An Acidic Cluster in the Cytosolic Domain of Human Cytomegalovirus Glycoprotein B Is a Signal for Endocytosis from the Plasma Membrane

Journal of Virology ◽

10.1128/jvi.73.10.8677-8688.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8677-8688 ◽

Cited By ~ 42

Author(s):

Sharof Tugizov ◽

Ekaterina Maidji ◽

Jianqiao Xiao ◽

Lenore Pereira

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Human Cytomegalovirus ◽

Human Fibroblasts ◽

Endocytic Pathway ◽

Glycoprotein B ◽

Endocytic Recycling ◽

Cytosolic Domain ◽

Recycling Pathway ◽

Apical Membranes

ABSTRACT We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway.

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Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells

10.21203/rs.3.rs-380905/v1 ◽

2021 ◽

Author(s):

Ahmed Al Qaffas ◽

Salvatore Camiolo ◽

Mai Vo ◽

Alexis Aguiar ◽

Amine Ourahmane ◽

...

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Viral Entry ◽

Sequence Data ◽

Laboratory Strain ◽

Serial Passage ◽

Wild Type Virus ◽

Protein Coding ◽

Genetic Changes ◽

Long Read

Abstract The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

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Human Cytomegalovirus Entry into Epithelial and Endothelial Cells Depends on Genes UL128 to UL150 and Occurs by Endocytosis and Low-pH Fusion

Journal of Virology ◽

10.1128/jvi.80.2.710-722.2006 ◽

2006 ◽

Vol 80 (2) ◽

pp. 710-722 ◽

Cited By ~ 229

Author(s):

Brent J. Ryckman ◽

Michael A Jarvis ◽

Derek D. Drummond ◽

Jay A. Nelson ◽

David C. Johnson

Keyword(s):

Endothelial Cells ◽

Epithelial Cells ◽

Human Cytomegalovirus ◽

Early Stage ◽

Human Fibroblasts ◽

Cell Types ◽

Low Ph ◽

Virus Particles ◽

Peg Treatment ◽

Infection Processes

ABSTRACT Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRΔ4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRΔ4 displayed a number of defects in early infection processes. Adsorption and entry of TRΔ4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRΔ4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

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Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Journal of Biological Chemistry ◽

10.1074/jbc.m115.652230 ◽

2015 ◽

Vol 290 (26) ◽

pp. 15985-15995 ◽

Cited By ~ 29

Author(s):

John W. Loughney ◽

Richard R. Rustandi ◽

Dai Wang ◽

Matthew C. Troutman ◽

Lawrence W. Dick ◽

...

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Viral Entry ◽

Neutralizing Epitopes

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Human Cytomegalovirus Infection of Caco-2 Cells Occurs at the Basolateral Membrane and Is Differentiation State Dependent

Journal of Virology ◽

10.1128/jvi.73.6.4552-4560.1999 ◽

1999 ◽

Vol 73 (6) ◽

pp. 4552-4560 ◽

Cited By ~ 36

Author(s):

Michael A. Jarvis ◽

C. Edward Wang ◽

Heather L. Meyers ◽

Patricia P. Smith ◽

Christopher L. Corless ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Human Cytomegalovirus ◽

Viral Entry ◽

Hcmv Infection ◽

Cell Model ◽

Basolateral Membrane ◽

In Vitro Models

ABSTRACT Epithelial cells are known to be a major target for human cytomegalovirus (HCMV) infection; however, the analysis of virus-cell interactions has been difficult to approach due to the lack of in vitro models. In this study, we established a polarized epithelial cell model using a colon epithelial cell-derived cell line (Caco-2) that is susceptible to HCMV infection at early stages of cellular differentiation. Infection of polarized cells was restricted to the basolateral surface whereas virus was released apically, which was consistent with the apical and not basolateral surface localization of two essential viral glycoproteins, gB and gH. HCMV infection resulted in the development of a cytopathology characteristic of HCMV infection of colon epithelium in vivo, and infection did not spread from cell to cell. The inability of HCMV to infect Caco-2 cells at late stages of differentiation was due to a restriction at the level of viral entry and was consistent with the sequestration of a cellular receptor for HCMV. These observations provide the first evidence that restriction of HCMV replication in epithelial cells is due to a receptor-mediated phenomenon.

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Human cytomegalovirus serum neutralizing antibodies block virus infection of endothelial/epithelial cells, but not fibroblasts, early during primary infection

Journal of General Virology ◽

10.1099/vir.0.83523-0 ◽

2008 ◽

Vol 89 (4) ◽

pp. 853-865 ◽

Cited By ~ 137

Author(s):

Giuseppe Gerna ◽

Antonella Sarasini ◽

Marco Patrone ◽

Elena Percivalle ◽

Loretta Fiorina ◽

...

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Neutralizing Antibodies ◽

Geometric Mean ◽

Human Fibroblasts ◽

Cell Infection ◽

Neutralizing Activity ◽

Hyperimmune Globulin ◽

Human Sera ◽

Infected Cells

A panel of human sera exhibited a ≥128-fold higher neutralizing potency against a human cytomegalovirus (HCMV) clinical isolate propagated and tested in endothelial (or epithelial) cells than against the same virus infecting human fibroblasts. In a group of 18 primary infections, the reverse geometric mean titre was in the range of 10–15 in human fibroblasts within the first 3 months after the onset of infection, whereas the endothelial cell infection-neutralizing activity was already present within the first 10 days, reaching median levels of 122, 320 and 545 at respectively 30, 60 and 90 days after onset, then declining slowly. This difference was also confirmed in the majority of reactivated and remote HCMV infections, as well as in a hyperimmune globulin preparation. The antibody response to HCMV pUL131A, pUL130 and pUL128 locus products, which are required for endothelial/epithelial cell infection, provided a potential molecular basis for such a differential neutralizing activity. In addition, monoclonal/monospecific antibodies raised against the pUL131A, pUL130 and pUL128 proteins were found to display an inhibitory activity on HCMV plaque formation and HCMV leukocyte transfer from HCMV-infected cells. Hence, conventional determination of the neutralizing activity of human sera in fibroblasts is misleading. Antibodies to pUL131A, pUL130 and pUL128 appear to display a major HCMV-neutralizing and dissemination-inhibiting activity.

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