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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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Endothelial cells retain EC-tropic virus particles.HFF were infected with vBAC4-luc as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 0.2). Six days after infection supernatants were harvested, cells homogenized as described in Materials and Methods and different fractions of the homogenates tested on HFF and TIME cells for their infection capacities by luciferase assay. (A) shows the TIME/HFF infection ratios and (B) the absolute luciferase activities of the different cell preparations on HFF and TIME cells. Shown are means +/− SD of three independent experiments assayed in triplicates. For HFF all homogenate preparations were significantly less EC-tropic than the cell culture supernatants. For HUVEC the total homogenates and the resuspended pellets were significantly more EC-tropic than the cell culture supernatants (Student's t test).
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ppat-1001256-g005: Endothelial cells retain EC-tropic virus particles.HFF were infected with vBAC4-luc as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 0.2). Six days after infection supernatants were harvested, cells homogenized as described in Materials and Methods and different fractions of the homogenates tested on HFF and TIME cells for their infection capacities by luciferase assay. (A) shows the TIME/HFF infection ratios and (B) the absolute luciferase activities of the different cell preparations on HFF and TIME cells. Shown are means +/− SD of three independent experiments assayed in triplicates. For HFF all homogenate preparations were significantly less EC-tropic than the cell culture supernatants. For HUVEC the total homogenates and the resuspended pellets were significantly more EC-tropic than the cell culture supernatants (Student's t test).

Mentions: Whereas the virus released by EC is low in gH/gL/pUL(128,130,13A) complexes, focal spread in EC cultures was highly efficient. Like EC infection by supernatant virus, it can be blocked by anti-pUL128, anti-pUL130 and anti-pUL131A antibodies [16], [17], [44], [45]. This indicates that pUL(128,130,131A) are accessible to antibodies and promote infection of neighboring cells. We tested different cellular preparations for the presence of cell-associated EC-tropic virus. HUVEC and as a control HFF were infected with vBAC4-luc, and 6 days after infection supernatants were harvested. Cells were washed to remove loosely bound virus and then homogenized using cell douncers. Aliquots of the total homogenates, containing the disrupted cells and virus freed by cell disruption, were saved. Homogenates were then cleared by centrifugation at 3,500×g to separate supernatants containing virus, which can be released by physical disruption. The pellets of cell debris, containing virus which is not released from cells, were also resuspended. These four preparations were then tested on HFF and TIME cells by the luciferase assay. Virus supernatants from HFF and HUVEC showed a high and a low EC infection capacity, respectively (Fig. 5A). All three homogenate preparations from HFF showed a reduced EC infection capacity, when compared to HFF supernatant virus. Notably, the two HUVEC preparations, which contained cell debris showed an about tenfold higher EC infection capacity than the HUVEC supernatants (Fig. 5A). Thus, the progeny able to infect EC is released by HFF, but remained tightly associated with cellular structures in the case of EC. The differences observed are not due to different quantities of virus in the different preparations, because all HFF- and HUVEC-derived preparations showed high luciferase values, when tested on HFF (Fig. 5B). The tenfold differences between HUVEC-derived preparations, containing broken cells, and those without cells are due to high and low luciferase values on TIME cells, respectively (Fig. 5B). Highly EC-tropic virus could neither be released from EC by sonication nor by several rounds of freezing and thawing (data not shown). Taken together, the data show that HFF readily release, whereas HUVEC tightly retain EC-tropic virus.


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

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

Endothelial cells retain EC-tropic virus particles.HFF were infected with vBAC4-luc as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 0.2). Six days after infection supernatants were harvested, cells homogenized as described in Materials and Methods and different fractions of the homogenates tested on HFF and TIME cells for their infection capacities by luciferase assay. (A) shows the TIME/HFF infection ratios and (B) the absolute luciferase activities of the different cell preparations on HFF and TIME cells. Shown are means +/− SD of three independent experiments assayed in triplicates. For HFF all homogenate preparations were significantly less EC-tropic than the cell culture supernatants. For HUVEC the total homogenates and the resuspended pellets were significantly more EC-tropic than the cell culture supernatants (Student's t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020925&req=5

ppat-1001256-g005: Endothelial cells retain EC-tropic virus particles.HFF were infected with vBAC4-luc as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 0.2). Six days after infection supernatants were harvested, cells homogenized as described in Materials and Methods and different fractions of the homogenates tested on HFF and TIME cells for their infection capacities by luciferase assay. (A) shows the TIME/HFF infection ratios and (B) the absolute luciferase activities of the different cell preparations on HFF and TIME cells. Shown are means +/− SD of three independent experiments assayed in triplicates. For HFF all homogenate preparations were significantly less EC-tropic than the cell culture supernatants. For HUVEC the total homogenates and the resuspended pellets were significantly more EC-tropic than the cell culture supernatants (Student's t test).
Mentions: Whereas the virus released by EC is low in gH/gL/pUL(128,130,13A) complexes, focal spread in EC cultures was highly efficient. Like EC infection by supernatant virus, it can be blocked by anti-pUL128, anti-pUL130 and anti-pUL131A antibodies [16], [17], [44], [45]. This indicates that pUL(128,130,131A) are accessible to antibodies and promote infection of neighboring cells. We tested different cellular preparations for the presence of cell-associated EC-tropic virus. HUVEC and as a control HFF were infected with vBAC4-luc, and 6 days after infection supernatants were harvested. Cells were washed to remove loosely bound virus and then homogenized using cell douncers. Aliquots of the total homogenates, containing the disrupted cells and virus freed by cell disruption, were saved. Homogenates were then cleared by centrifugation at 3,500×g to separate supernatants containing virus, which can be released by physical disruption. The pellets of cell debris, containing virus which is not released from cells, were also resuspended. These four preparations were then tested on HFF and TIME cells by the luciferase assay. Virus supernatants from HFF and HUVEC showed a high and a low EC infection capacity, respectively (Fig. 5A). All three homogenate preparations from HFF showed a reduced EC infection capacity, when compared to HFF supernatant virus. Notably, the two HUVEC preparations, which contained cell debris showed an about tenfold higher EC infection capacity than the HUVEC supernatants (Fig. 5A). Thus, the progeny able to infect EC is released by HFF, but remained tightly associated with cellular structures in the case of EC. The differences observed are not due to different quantities of virus in the different preparations, because all HFF- and HUVEC-derived preparations showed high luciferase values, when tested on HFF (Fig. 5B). The tenfold differences between HUVEC-derived preparations, containing broken cells, and those without cells are due to high and low luciferase values on TIME cells, respectively (Fig. 5B). Highly EC-tropic virus could neither be released from EC by sonication nor by several rounds of freezing and thawing (data not shown). Taken together, the data show that HFF readily release, whereas HUVEC tightly retain EC-tropic virus.

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