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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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HCMV spread in fibroblast and EC cultures.(A) HFF, TIME cells and HUVEC were infected with VR1814, TB40/E and vTB40-BAC4 as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 0.1). The initial infection (day 2) as well as virus spread (day 8) were monitored by staining for HCMV ie1 protein expression. (B) Growth curves of vTB40-BAC4 on HFF, TIME cells and HUVEC. Cells were infected as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 1). Cell culture supernatants were harvested at the indicated time points post infection and virus titers determined by a TCID50 assay performed on HFF. (C) Infection capacities of supernatant-derived vTB40-BAC4. HFF, TIME cells and HUVEC were infected at an m.o.i. of 1 with day 8 supernatants obtained from the growth curves under (B). Infection capacities were monitored by staining for ie1 protein expression 48 hours post infection. Except where indicated, supernatants used for infection were titrated by a TCID50 assay on HFF.
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ppat-1001256-g001: HCMV spread in fibroblast and EC cultures.(A) HFF, TIME cells and HUVEC were infected with VR1814, TB40/E and vTB40-BAC4 as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 0.1). The initial infection (day 2) as well as virus spread (day 8) were monitored by staining for HCMV ie1 protein expression. (B) Growth curves of vTB40-BAC4 on HFF, TIME cells and HUVEC. Cells were infected as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 1). Cell culture supernatants were harvested at the indicated time points post infection and virus titers determined by a TCID50 assay performed on HFF. (C) Infection capacities of supernatant-derived vTB40-BAC4. HFF, TIME cells and HUVEC were infected at an m.o.i. of 1 with day 8 supernatants obtained from the growth curves under (B). Infection capacities were monitored by staining for ie1 protein expression 48 hours post infection. Except where indicated, supernatants used for infection were titrated by a TCID50 assay on HFF.

Mentions: When fibroblasts and EC are infected with HCMV in vitro, virus homogeneously spreads in fibroblast cultures whereas spread in endothelial cell cultures stays focal [17], [43]. Here, we infected fibroblasts and EC with the HCMV strains VR1814 and TB40/E, two clinical isolates passaged on endothelial cells, and vTB40-BAC4, a virus derived from TB40/E and cloned as a bacterial artificial chromosome (BAC). Infections were performed at a low multiplicity of infection (m.o.i.), and 2 or 8 days after infection cells were stained for HCMV immediate early 1 (ie1) protein expression. Numbers of initially infected HFF or EC were comparable (Fig. 1A, VR1814, day 2 and data not shown). When fibroblasts were infected, HCMV homogeneously spread throughout the culture indicating release of virus from infected cells and infection via free supernatant virus (Fig. 1A, day 8). In contrast, EC infection remained focal indicating virus transmission which delivers virus particles from cell-to-cell, without releasing it. This spread pattern in EC cultures was comparable for all HCMV strains tested and independent of whether a microvascular cell line (TIME) or primary macrovascular endothelial cells (HUVEC) were infected (Fig. 1A, day 8, lower panels). Focal spread in EC cultures could be completely inhibited by neutralizing anti-HCMV antibodies in human serum or anti-pUL131A antibodies (Fig. S1), indicating that virus spread in EC cultures was not due to direct cell-to-cell spread. Spread in fibroblast cultures was restricted from supernatant-driven spread to focal spread by human antiserum and not inhibited at all by anti-pUL131A antibodies (Fig. S1).


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

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

HCMV spread in fibroblast and EC cultures.(A) HFF, TIME cells and HUVEC were infected with VR1814, TB40/E and vTB40-BAC4 as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 0.1). The initial infection (day 2) as well as virus spread (day 8) were monitored by staining for HCMV ie1 protein expression. (B) Growth curves of vTB40-BAC4 on HFF, TIME cells and HUVEC. Cells were infected as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 1). Cell culture supernatants were harvested at the indicated time points post infection and virus titers determined by a TCID50 assay performed on HFF. (C) Infection capacities of supernatant-derived vTB40-BAC4. HFF, TIME cells and HUVEC were infected at an m.o.i. of 1 with day 8 supernatants obtained from the growth curves under (B). Infection capacities were monitored by staining for ie1 protein expression 48 hours post infection. Except where indicated, supernatants used for infection were titrated by a TCID50 assay on HFF.
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ppat-1001256-g001: HCMV spread in fibroblast and EC cultures.(A) HFF, TIME cells and HUVEC were infected with VR1814, TB40/E and vTB40-BAC4 as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 0.1). The initial infection (day 2) as well as virus spread (day 8) were monitored by staining for HCMV ie1 protein expression. (B) Growth curves of vTB40-BAC4 on HFF, TIME cells and HUVEC. Cells were infected as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 1). Cell culture supernatants were harvested at the indicated time points post infection and virus titers determined by a TCID50 assay performed on HFF. (C) Infection capacities of supernatant-derived vTB40-BAC4. HFF, TIME cells and HUVEC were infected at an m.o.i. of 1 with day 8 supernatants obtained from the growth curves under (B). Infection capacities were monitored by staining for ie1 protein expression 48 hours post infection. Except where indicated, supernatants used for infection were titrated by a TCID50 assay on HFF.
Mentions: When fibroblasts and EC are infected with HCMV in vitro, virus homogeneously spreads in fibroblast cultures whereas spread in endothelial cell cultures stays focal [17], [43]. Here, we infected fibroblasts and EC with the HCMV strains VR1814 and TB40/E, two clinical isolates passaged on endothelial cells, and vTB40-BAC4, a virus derived from TB40/E and cloned as a bacterial artificial chromosome (BAC). Infections were performed at a low multiplicity of infection (m.o.i.), and 2 or 8 days after infection cells were stained for HCMV immediate early 1 (ie1) protein expression. Numbers of initially infected HFF or EC were comparable (Fig. 1A, VR1814, day 2 and data not shown). When fibroblasts were infected, HCMV homogeneously spread throughout the culture indicating release of virus from infected cells and infection via free supernatant virus (Fig. 1A, day 8). In contrast, EC infection remained focal indicating virus transmission which delivers virus particles from cell-to-cell, without releasing it. This spread pattern in EC cultures was comparable for all HCMV strains tested and independent of whether a microvascular cell line (TIME) or primary macrovascular endothelial cells (HUVEC) were infected (Fig. 1A, day 8, lower panels). Focal spread in EC cultures could be completely inhibited by neutralizing anti-HCMV antibodies in human serum or anti-pUL131A antibodies (Fig. S1), indicating that virus spread in EC cultures was not due to direct cell-to-cell spread. Spread in fibroblast cultures was restricted from supernatant-driven spread to focal spread by human antiserum and not inhibited at all by anti-pUL131A antibodies (Fig. S1).

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