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HCMV spread and cell tropism are determined by distinct virus populations.

Scrivano L, Sinzger C, Nitschko H, Koszinowski UH, Adler B - PLoS Pathog. (2011)

Bottom Line: Human cytomegalovirus (HCMV) can infect many different cell types in vivo.Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts.Our findings offer completely new views on how HCMV spread may be controlled by its host cells.

View Article: PubMed Central - PubMed

Affiliation: Max von Pettenkofer-Institut für Virologie, Ludwig-Maximilians-Universität München, München, Germany.

ABSTRACT
Human cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells.

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vBAC4-luc as a tool to study infection capacities.(A) HFF and in parallel TIME cells were infected on 96 well plates at an m.o.i. of 0.3 with a cell culture supernatant derived from HFF infected with vBAC4-luc. After infection, PAA was added and 48 hours later cells either stained for HCMV ie1 protein expression or lysed and subjected to a luciferase assay. The experiment was performed in triplicates. For ie1 staining, three independent wells were infected and one microscopic field per well was counted. For the luciferase assay, lysates from three wells were analysed. Shown are means +/− SD of these triplicates. The TIME cell infection capacity was related to the HFF infection capacity which was set to 100%, and the ratio expressed in percent. (B) HFF and TIME cells were infected with serial 5-fold dilutions of a supernatant derived from HFF infected with vBAC4-luc starting at an m.o.i of 0.5 and 48 hours later analysed by a luciferase assay performed in triplicates. The background level of the luciferase assay is indicated by the dotted line. (C) Two independent luciferase assays using different batches of HFF and TIME cells. Three TIME cell and three HFF supernatants from three independent infections with vBAC4-luc were assayed. For the luciferase assay, cells were infected at an m.o.i. of 0.1 and analysed in triplicates 48 hours after infection. Shown are means +/− SD of three supernatants tested in triplicates. (D) HFF, TIME cells and HUVEC were infected at an m.o.i. of 0.02 (centrifugal enhancement) with supernatants from vBAC4-luc, vBAC4-luc/UL131Astop and vBAC4-luc/ΔgO infections of HFF. 48 hours after infection cells were subjected to a luciferase assay. Shown are means +/− SD of luciferase activities determined in triplicates.
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ppat-1001256-g002: vBAC4-luc as a tool to study infection capacities.(A) HFF and in parallel TIME cells were infected on 96 well plates at an m.o.i. of 0.3 with a cell culture supernatant derived from HFF infected with vBAC4-luc. After infection, PAA was added and 48 hours later cells either stained for HCMV ie1 protein expression or lysed and subjected to a luciferase assay. The experiment was performed in triplicates. For ie1 staining, three independent wells were infected and one microscopic field per well was counted. For the luciferase assay, lysates from three wells were analysed. Shown are means +/− SD of these triplicates. The TIME cell infection capacity was related to the HFF infection capacity which was set to 100%, and the ratio expressed in percent. (B) HFF and TIME cells were infected with serial 5-fold dilutions of a supernatant derived from HFF infected with vBAC4-luc starting at an m.o.i of 0.5 and 48 hours later analysed by a luciferase assay performed in triplicates. The background level of the luciferase assay is indicated by the dotted line. (C) Two independent luciferase assays using different batches of HFF and TIME cells. Three TIME cell and three HFF supernatants from three independent infections with vBAC4-luc were assayed. For the luciferase assay, cells were infected at an m.o.i. of 0.1 and analysed in triplicates 48 hours after infection. Shown are means +/− SD of three supernatants tested in triplicates. (D) HFF, TIME cells and HUVEC were infected at an m.o.i. of 0.02 (centrifugal enhancement) with supernatants from vBAC4-luc, vBAC4-luc/UL131Astop and vBAC4-luc/ΔgO infections of HFF. 48 hours after infection cells were subjected to a luciferase assay. Shown are means +/− SD of luciferase activities determined in triplicates.

Mentions: We used vBAC4-luc to evaluate EC and fibroblast infection capacities of virus preparations on HFF and TIME cells. The luciferase signals obtained from HFF and TIME cell infections were related to each other and expressed as TIME/HFF infection ratios, and thus, represent relative EC infection capacities. After infection, phosphono acetic acid (PAA) was added to block the viral DNA replication and the further amplification of the luciferase signal. Thus, the luciferase activity evaluates infection of cells in a fashion analogous to staining cells for HCMV ie1 protein expression. Indeed, when infection with one and the same virus preparation was evaluated either by counting ie1-positive cells or by measuring the luciferase activity in cell lysates, both methods always gave comparable results (Fig. 2A and data not shown). The assay proved to be linear over a wide range of m.o.i and highly sensitive (Fig. 2B).


HCMV spread and cell tropism are determined by distinct virus populations.

Scrivano L, Sinzger C, Nitschko H, Koszinowski UH, Adler B - PLoS Pathog. (2011)

vBAC4-luc as a tool to study infection capacities.(A) HFF and in parallel TIME cells were infected on 96 well plates at an m.o.i. of 0.3 with a cell culture supernatant derived from HFF infected with vBAC4-luc. After infection, PAA was added and 48 hours later cells either stained for HCMV ie1 protein expression or lysed and subjected to a luciferase assay. The experiment was performed in triplicates. For ie1 staining, three independent wells were infected and one microscopic field per well was counted. For the luciferase assay, lysates from three wells were analysed. Shown are means +/− SD of these triplicates. The TIME cell infection capacity was related to the HFF infection capacity which was set to 100%, and the ratio expressed in percent. (B) HFF and TIME cells were infected with serial 5-fold dilutions of a supernatant derived from HFF infected with vBAC4-luc starting at an m.o.i of 0.5 and 48 hours later analysed by a luciferase assay performed in triplicates. The background level of the luciferase assay is indicated by the dotted line. (C) Two independent luciferase assays using different batches of HFF and TIME cells. Three TIME cell and three HFF supernatants from three independent infections with vBAC4-luc were assayed. For the luciferase assay, cells were infected at an m.o.i. of 0.1 and analysed in triplicates 48 hours after infection. Shown are means +/− SD of three supernatants tested in triplicates. (D) HFF, TIME cells and HUVEC were infected at an m.o.i. of 0.02 (centrifugal enhancement) with supernatants from vBAC4-luc, vBAC4-luc/UL131Astop and vBAC4-luc/ΔgO infections of HFF. 48 hours after infection cells were subjected to a luciferase assay. Shown are means +/− SD of luciferase activities determined in triplicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020925&req=5

ppat-1001256-g002: vBAC4-luc as a tool to study infection capacities.(A) HFF and in parallel TIME cells were infected on 96 well plates at an m.o.i. of 0.3 with a cell culture supernatant derived from HFF infected with vBAC4-luc. After infection, PAA was added and 48 hours later cells either stained for HCMV ie1 protein expression or lysed and subjected to a luciferase assay. The experiment was performed in triplicates. For ie1 staining, three independent wells were infected and one microscopic field per well was counted. For the luciferase assay, lysates from three wells were analysed. Shown are means +/− SD of these triplicates. The TIME cell infection capacity was related to the HFF infection capacity which was set to 100%, and the ratio expressed in percent. (B) HFF and TIME cells were infected with serial 5-fold dilutions of a supernatant derived from HFF infected with vBAC4-luc starting at an m.o.i of 0.5 and 48 hours later analysed by a luciferase assay performed in triplicates. The background level of the luciferase assay is indicated by the dotted line. (C) Two independent luciferase assays using different batches of HFF and TIME cells. Three TIME cell and three HFF supernatants from three independent infections with vBAC4-luc were assayed. For the luciferase assay, cells were infected at an m.o.i. of 0.1 and analysed in triplicates 48 hours after infection. Shown are means +/− SD of three supernatants tested in triplicates. (D) HFF, TIME cells and HUVEC were infected at an m.o.i. of 0.02 (centrifugal enhancement) with supernatants from vBAC4-luc, vBAC4-luc/UL131Astop and vBAC4-luc/ΔgO infections of HFF. 48 hours after infection cells were subjected to a luciferase assay. Shown are means +/− SD of luciferase activities determined in triplicates.
Mentions: We used vBAC4-luc to evaluate EC and fibroblast infection capacities of virus preparations on HFF and TIME cells. The luciferase signals obtained from HFF and TIME cell infections were related to each other and expressed as TIME/HFF infection ratios, and thus, represent relative EC infection capacities. After infection, phosphono acetic acid (PAA) was added to block the viral DNA replication and the further amplification of the luciferase signal. Thus, the luciferase activity evaluates infection of cells in a fashion analogous to staining cells for HCMV ie1 protein expression. Indeed, when infection with one and the same virus preparation was evaluated either by counting ie1-positive cells or by measuring the luciferase activity in cell lysates, both methods always gave comparable results (Fig. 2A and data not shown). The assay proved to be linear over a wide range of m.o.i and highly sensitive (Fig. 2B).

Bottom Line: Human cytomegalovirus (HCMV) can infect many different cell types in vivo.Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts.Our findings offer completely new views on how HCMV spread may be controlled by its host cells.

View Article: PubMed Central - PubMed

Affiliation: Max von Pettenkofer-Institut für Virologie, Ludwig-Maximilians-Universität München, München, Germany.

ABSTRACT
Human cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells.

Show MeSH
Related in: MedlinePlus