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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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BoMac myeloid cells express relatively less gp180 glycoprotein in comparison with epithelial cells.A–B. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., relative expressions of Bo10 spliced vs unspliced transcripts (A) and of Bo10 spliced vs ORF8 transcripts (B) were estimated as described in the Methods. The data presented are the average ± SEMs for 3 measurements and were analyzed by Student's t-test, ** p<0.01. C. Detection of the gp180, encoded by the Bo10 spliced transcript, and gB glycoproteins in bovine epithelial and myeloid cells. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., these cells were subjected to western blotting with anti-Bo10-c15 serum (recognizing gp180) and mAb 35 (recognizing gB) as described in the Methods. On the gp180 blot, open and filled triangles indicate respectively the specific 180 kDa protein and a background band. On the gB blot, open and filled arrow heads indicate uncleaved gB and furin-cleaved gB C-terminus respectively. The position of a molecular mass (MM) standard (in kDa) is shown on the left.
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ppat-1003753-g004: BoMac myeloid cells express relatively less gp180 glycoprotein in comparison with epithelial cells.A–B. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., relative expressions of Bo10 spliced vs unspliced transcripts (A) and of Bo10 spliced vs ORF8 transcripts (B) were estimated as described in the Methods. The data presented are the average ± SEMs for 3 measurements and were analyzed by Student's t-test, ** p<0.01. C. Detection of the gp180, encoded by the Bo10 spliced transcript, and gB glycoproteins in bovine epithelial and myeloid cells. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., these cells were subjected to western blotting with anti-Bo10-c15 serum (recognizing gp180) and mAb 35 (recognizing gB) as described in the Methods. On the gp180 blot, open and filled triangles indicate respectively the specific 180 kDa protein and a background band. On the gB blot, open and filled arrow heads indicate uncleaved gB and furin-cleaved gB C-terminus respectively. The position of a molecular mass (MM) standard (in kDa) is shown on the left.

Mentions: Different bovine cell lines were then investigated for expression of Bo10 spliced and unspliced transcripts by quantitative PCR. In order to be able to compare the numbers of both transcripts, a plasmid encoding both Bo10 spliced and unspliced specific sequences was constructed and used as a unique standard curve for both reactions. Interestingly, while the infected epithelial MDBK cells displayed around 1000 times more Bo10 spliced transcript than unspliced transcript, this ratio was more than 10 times lower in the BoMac myeloid cells (Figure 4A). We then compared the number of Bo10 spliced transcripts, encoding gp180, to the number of ORF8 transcripts, encoding gB, as we have shown that one of the glycoproteins shielded by gp180 is gB (Figure 3C) [30]. While the infected epithelial MDBK cells displayed around 10 times more Bo10 spliced transcripts than ORF8 transcripts, this ratio was around 10 times lower in the BoMac myeloid cells (Figure 4B), suggesting that in these cells, there could be less expression of gp180 glycoprotein in comparison to gB. Similar results were obtained when the amount of Bo10 spliced transcripts was compared to the number of ORF47 mRNA encoding gL (Figure S4), as gL is another viral glycoprotein shielded by gp180. Finally, these results were confirmed by western blotting on MDBK or BoMac cells (Figure 4C). Interestingly, while gB glycoprotein was readily detectable in both cell types, almost no gp180 glycoprotein was observed in BoHV-4 WT infected BoMac cells.


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)

BoMac myeloid cells express relatively less gp180 glycoprotein in comparison with epithelial cells.A–B. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., relative expressions of Bo10 spliced vs unspliced transcripts (A) and of Bo10 spliced vs ORF8 transcripts (B) were estimated as described in the Methods. The data presented are the average ± SEMs for 3 measurements and were analyzed by Student's t-test, ** p<0.01. C. Detection of the gp180, encoded by the Bo10 spliced transcript, and gB glycoproteins in bovine epithelial and myeloid cells. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., these cells were subjected to western blotting with anti-Bo10-c15 serum (recognizing gp180) and mAb 35 (recognizing gB) as described in the Methods. On the gp180 blot, open and filled triangles indicate respectively the specific 180 kDa protein and a background band. On the gB blot, open and filled arrow heads indicate uncleaved gB and furin-cleaved gB C-terminus respectively. The position of a molecular mass (MM) standard (in kDa) is shown on the left.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814654&req=5

ppat-1003753-g004: BoMac myeloid cells express relatively less gp180 glycoprotein in comparison with epithelial cells.A–B. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., relative expressions of Bo10 spliced vs unspliced transcripts (A) and of Bo10 spliced vs ORF8 transcripts (B) were estimated as described in the Methods. The data presented are the average ± SEMs for 3 measurements and were analyzed by Student's t-test, ** p<0.01. C. Detection of the gp180, encoded by the Bo10 spliced transcript, and gB glycoproteins in bovine epithelial and myeloid cells. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., these cells were subjected to western blotting with anti-Bo10-c15 serum (recognizing gp180) and mAb 35 (recognizing gB) as described in the Methods. On the gp180 blot, open and filled triangles indicate respectively the specific 180 kDa protein and a background band. On the gB blot, open and filled arrow heads indicate uncleaved gB and furin-cleaved gB C-terminus respectively. The position of a molecular mass (MM) standard (in kDa) is shown on the left.
Mentions: Different bovine cell lines were then investigated for expression of Bo10 spliced and unspliced transcripts by quantitative PCR. In order to be able to compare the numbers of both transcripts, a plasmid encoding both Bo10 spliced and unspliced specific sequences was constructed and used as a unique standard curve for both reactions. Interestingly, while the infected epithelial MDBK cells displayed around 1000 times more Bo10 spliced transcript than unspliced transcript, this ratio was more than 10 times lower in the BoMac myeloid cells (Figure 4A). We then compared the number of Bo10 spliced transcripts, encoding gp180, to the number of ORF8 transcripts, encoding gB, as we have shown that one of the glycoproteins shielded by gp180 is gB (Figure 3C) [30]. While the infected epithelial MDBK cells displayed around 10 times more Bo10 spliced transcripts than ORF8 transcripts, this ratio was around 10 times lower in the BoMac myeloid cells (Figure 4B), suggesting that in these cells, there could be less expression of gp180 glycoprotein in comparison to gB. Similar results were obtained when the amount of Bo10 spliced transcripts was compared to the number of ORF47 mRNA encoding gL (Figure S4), as gL is another viral glycoprotein shielded by gp180. Finally, these results were confirmed by western blotting on MDBK or BoMac cells (Figure 4C). Interestingly, while gB glycoprotein was readily detectable in both cell types, almost no gp180 glycoprotein was observed in BoHV-4 WT infected BoMac cells.

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