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A gammaherpesvirus uses alternative splicing to regulate its tropism and its sensitivity to neutralization.

Machiels B, Stevenson PG, Vanderplasschen A, Gillet L - PLoS Pathog. (2013)

Bottom Line: Human gammaherpesviruses are associated with the development of lymphomas and epithelial malignancies.While epithelial cells produce virions with high levels of the accessory envelope protein gp180, encoded by a Bo10 spliced product, myeloid cells express reduced levels of gp180.This molecular switch could therefore allow BoHV-4 to promote either, on the one hand, its dissemination into the organism, or, on the other hand, its transmission between hosts.

View Article: PubMed Central - PubMed

Affiliation: Immunology-Vaccinology Laboratory, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.

ABSTRACT
Human gammaherpesviruses are associated with the development of lymphomas and epithelial malignancies. The heterogeneity of these tumors reflects the ability of these viruses to route infection to different cell types at various stages of their lifecycle. While the Epstein Barr virus uses gp42--human leukocyte antigen class II interaction as a switch of cell tropism, the molecular mechanism that orientates tropism of rhadinoviruses is still poorly defined. Here, we used bovine herpesvirus 4 (BoHV-4) to further elucidate how rhadinoviruses regulate their infectivity. In the absence of any gp42 homolog, BoHV-4 exploits the alternative splicing of its Bo10 gene to produce distinct viral populations that behave differently based on the originating cell. While epithelial cells produce virions with high levels of the accessory envelope protein gp180, encoded by a Bo10 spliced product, myeloid cells express reduced levels of gp180. As a consequence, virions grown in epithelial cells are hardly infectious for CD14+ circulating cells, but are relatively resistant to antibody neutralization due to the shielding property of gp180 for vulnerable entry epitopes. In contrast, myeloid virions readily infect CD14+ circulating cells but are easily neutralized. This molecular switch could therefore allow BoHV-4 to promote either, on the one hand, its dissemination into the organism, or, on the other hand, its transmission between hosts.

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Myeloid virions are more infectious for CD14+ PBMCs but are more sensitive to antibody neutralization.A. Infection by co-cultures. MDBK or BoMac cells previously stained with the membrane dye PKH26 were infected with BoHV-4 WT BAC virions (MOI of 1). Three hours p.i., cells were washed with acidic solution (PBS pH 3) in order to inactivate and remove the inoculum, and 12 hours p.i., freshly isolated PBMCs or MDBK controls were added. After 24 hours of co-cultivation, cells were collected and the proportion of eGFP expressing cells was measured in PKH26+ and PKH26− cells. For PBMC co-cultures, the proportion of eGFP expressing cells was measured in PKH26− CD14+ cells. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. B. BoHV-4 WT BAC cell-free virions propagated on MDBK or BoMac cells were added to bovine PBMCs at a MOI of 1 according to titers measured on MDBK cells. 24 hours later, percentages of eGFP positive cells were measured in CD14+ PBMCs by flow cytometry. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. C. BoHV-4 WT BAC virions propagated on MDBK or BoMac cells were incubated with sera of 3 different rabbits infected with BoHV-4 V.test strain (propagated on MDBK cells). After incubation (2 h, 37°C), the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 3 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, * p<0.05, ** p<0.01, *** p<0.001.
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ppat-1003753-g006: Myeloid virions are more infectious for CD14+ PBMCs but are more sensitive to antibody neutralization.A. Infection by co-cultures. MDBK or BoMac cells previously stained with the membrane dye PKH26 were infected with BoHV-4 WT BAC virions (MOI of 1). Three hours p.i., cells were washed with acidic solution (PBS pH 3) in order to inactivate and remove the inoculum, and 12 hours p.i., freshly isolated PBMCs or MDBK controls were added. After 24 hours of co-cultivation, cells were collected and the proportion of eGFP expressing cells was measured in PKH26+ and PKH26− cells. For PBMC co-cultures, the proportion of eGFP expressing cells was measured in PKH26− CD14+ cells. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. B. BoHV-4 WT BAC cell-free virions propagated on MDBK or BoMac cells were added to bovine PBMCs at a MOI of 1 according to titers measured on MDBK cells. 24 hours later, percentages of eGFP positive cells were measured in CD14+ PBMCs by flow cytometry. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. C. BoHV-4 WT BAC virions propagated on MDBK or BoMac cells were incubated with sera of 3 different rabbits infected with BoHV-4 V.test strain (propagated on MDBK cells). After incubation (2 h, 37°C), the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 3 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, * p<0.05, ** p<0.01, *** p<0.001.

Mentions: We first compared phenotypes of MDBK and BoMac derived virions by co-culture experiments (Figure 6A). While naïve MDBK cells co-cultured overnight with BoHV-4 WT BAC infected MDBK cells became >80% eGFP+, CD14+ PBMCs cultured under the same conditions remained nearly entirely uninfected (<1% eGFP+) (Figure 6A). In contrast, BoHV-4 WT BAC infected BoMac cells produced lower amount of virions, as demonstrated by the fact that only ∼20% of co-cultured MDBK cells became eGFP+. However, these few virions were much more infectious for CD14+ PBMCs (>10% eGFP+) in comparison with MDBK derived virions (Figure 6A). Therefore direct contact with infected myeloid BoMac cells allowed efficient BoHV-4 infection of circulating CD14+ PBMCs.


A gammaherpesvirus uses alternative splicing to regulate its tropism and its sensitivity to neutralization.

Machiels B, Stevenson PG, Vanderplasschen A, Gillet L - PLoS Pathog. (2013)

Myeloid virions are more infectious for CD14+ PBMCs but are more sensitive to antibody neutralization.A. Infection by co-cultures. MDBK or BoMac cells previously stained with the membrane dye PKH26 were infected with BoHV-4 WT BAC virions (MOI of 1). Three hours p.i., cells were washed with acidic solution (PBS pH 3) in order to inactivate and remove the inoculum, and 12 hours p.i., freshly isolated PBMCs or MDBK controls were added. After 24 hours of co-cultivation, cells were collected and the proportion of eGFP expressing cells was measured in PKH26+ and PKH26− cells. For PBMC co-cultures, the proportion of eGFP expressing cells was measured in PKH26− CD14+ cells. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. B. BoHV-4 WT BAC cell-free virions propagated on MDBK or BoMac cells were added to bovine PBMCs at a MOI of 1 according to titers measured on MDBK cells. 24 hours later, percentages of eGFP positive cells were measured in CD14+ PBMCs by flow cytometry. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. C. BoHV-4 WT BAC virions propagated on MDBK or BoMac cells were incubated with sera of 3 different rabbits infected with BoHV-4 V.test strain (propagated on MDBK cells). After incubation (2 h, 37°C), the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 3 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, * p<0.05, ** p<0.01, *** p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814654&req=5

ppat-1003753-g006: Myeloid virions are more infectious for CD14+ PBMCs but are more sensitive to antibody neutralization.A. Infection by co-cultures. MDBK or BoMac cells previously stained with the membrane dye PKH26 were infected with BoHV-4 WT BAC virions (MOI of 1). Three hours p.i., cells were washed with acidic solution (PBS pH 3) in order to inactivate and remove the inoculum, and 12 hours p.i., freshly isolated PBMCs or MDBK controls were added. After 24 hours of co-cultivation, cells were collected and the proportion of eGFP expressing cells was measured in PKH26+ and PKH26− cells. For PBMC co-cultures, the proportion of eGFP expressing cells was measured in PKH26− CD14+ cells. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. B. BoHV-4 WT BAC cell-free virions propagated on MDBK or BoMac cells were added to bovine PBMCs at a MOI of 1 according to titers measured on MDBK cells. 24 hours later, percentages of eGFP positive cells were measured in CD14+ PBMCs by flow cytometry. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. C. BoHV-4 WT BAC virions propagated on MDBK or BoMac cells were incubated with sera of 3 different rabbits infected with BoHV-4 V.test strain (propagated on MDBK cells). After incubation (2 h, 37°C), the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 3 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, * p<0.05, ** p<0.01, *** p<0.001.
Mentions: We first compared phenotypes of MDBK and BoMac derived virions by co-culture experiments (Figure 6A). While naïve MDBK cells co-cultured overnight with BoHV-4 WT BAC infected MDBK cells became >80% eGFP+, CD14+ PBMCs cultured under the same conditions remained nearly entirely uninfected (<1% eGFP+) (Figure 6A). In contrast, BoHV-4 WT BAC infected BoMac cells produced lower amount of virions, as demonstrated by the fact that only ∼20% of co-cultured MDBK cells became eGFP+. However, these few virions were much more infectious for CD14+ PBMCs (>10% eGFP+) in comparison with MDBK derived virions (Figure 6A). Therefore direct contact with infected myeloid BoMac cells allowed efficient BoHV-4 infection of circulating CD14+ PBMCs.

Bottom Line: Human gammaherpesviruses are associated with the development of lymphomas and epithelial malignancies.While epithelial cells produce virions with high levels of the accessory envelope protein gp180, encoded by a Bo10 spliced product, myeloid cells express reduced levels of gp180.This molecular switch could therefore allow BoHV-4 to promote either, on the one hand, its dissemination into the organism, or, on the other hand, its transmission between hosts.

View Article: PubMed Central - PubMed

Affiliation: Immunology-Vaccinology Laboratory, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.

ABSTRACT
Human gammaherpesviruses are associated with the development of lymphomas and epithelial malignancies. The heterogeneity of these tumors reflects the ability of these viruses to route infection to different cell types at various stages of their lifecycle. While the Epstein Barr virus uses gp42--human leukocyte antigen class II interaction as a switch of cell tropism, the molecular mechanism that orientates tropism of rhadinoviruses is still poorly defined. Here, we used bovine herpesvirus 4 (BoHV-4) to further elucidate how rhadinoviruses regulate their infectivity. In the absence of any gp42 homolog, BoHV-4 exploits the alternative splicing of its Bo10 gene to produce distinct viral populations that behave differently based on the originating cell. While epithelial cells produce virions with high levels of the accessory envelope protein gp180, encoded by a Bo10 spliced product, myeloid cells express reduced levels of gp180. As a consequence, virions grown in epithelial cells are hardly infectious for CD14+ circulating cells, but are relatively resistant to antibody neutralization due to the shielding property of gp180 for vulnerable entry epitopes. In contrast, myeloid virions readily infect CD14+ circulating cells but are easily neutralized. This molecular switch could therefore allow BoHV-4 to promote either, on the one hand, its dissemination into the organism, or, on the other hand, its transmission between hosts.

Show MeSH
Related in: MedlinePlus