Limits...
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.

Show MeSH

Related in: MedlinePlus

Generation of the Bo10 MuDir and Spliced BoHV-4 mutant.A. Schematic representation of the strategy followed to produce the recombinant BoHV-4 strains. We modified the BoHV-4 V.test Bo10 coding sequence (genomic coordinates 65844 to 66743) either by introducing a point mutation in the splicing donor site (Bo10 MuDir strain) or by replacing the entire Bo10 ORF by a sequence devoid of the intron (Bo10 Spliced strain). Splicing donor and acceptor sites are in red with splicing essential nucleotides in upper cases. The mutated nucleotide is in green. B. Verification of the molecular structure. Viral DNA was digested with BamHI, resolved by agarose gel electrophoresis, and hybridized with a 32P-labeled probe, corresponding to nucleotides 65900–66370 of the BoHV-4 V.test strain genome (coding for Bo10 Exon 1). The 7,137-bp wild-type (WT) band becomes 7060-bp (open arrow) for the Bo10 spliced mutant. Marker sizes in Kbp are indicated on the left. C. RT-PCR analysis of BoHV-4 Bo10 expression by the different mutants. MDBK cells were mock infected or infected with the different BoHV-4 mutant strains at a MOI of 1. Twenty-four hours p.i., expression of Bo10 was studied using different pairs of primers specific for the spliced and/or unspliced Bo10 cDNA. Uninfected cell samples (UI), reactions without reverse transcriptase (Neg. control) and amplification of viral genomic DNA are provided as controls. Sizes in bp are indicated on the right. D. Detection of the Bo10 encoded gp180 protein by the anti-Bo10-c15 serum. Purified virions (5*105 virions per lane) were subjected to western blotting with anti-Bo10-c15 serum as described in the Methods. The position of a molecular mass (MM) standard (in kDa) is shown on the right.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814654&req=5

ppat-1003753-g002: Generation of the Bo10 MuDir and Spliced BoHV-4 mutant.A. Schematic representation of the strategy followed to produce the recombinant BoHV-4 strains. We modified the BoHV-4 V.test Bo10 coding sequence (genomic coordinates 65844 to 66743) either by introducing a point mutation in the splicing donor site (Bo10 MuDir strain) or by replacing the entire Bo10 ORF by a sequence devoid of the intron (Bo10 Spliced strain). Splicing donor and acceptor sites are in red with splicing essential nucleotides in upper cases. The mutated nucleotide is in green. B. Verification of the molecular structure. Viral DNA was digested with BamHI, resolved by agarose gel electrophoresis, and hybridized with a 32P-labeled probe, corresponding to nucleotides 65900–66370 of the BoHV-4 V.test strain genome (coding for Bo10 Exon 1). The 7,137-bp wild-type (WT) band becomes 7060-bp (open arrow) for the Bo10 spliced mutant. Marker sizes in Kbp are indicated on the left. C. RT-PCR analysis of BoHV-4 Bo10 expression by the different mutants. MDBK cells were mock infected or infected with the different BoHV-4 mutant strains at a MOI of 1. Twenty-four hours p.i., expression of Bo10 was studied using different pairs of primers specific for the spliced and/or unspliced Bo10 cDNA. Uninfected cell samples (UI), reactions without reverse transcriptase (Neg. control) and amplification of viral genomic DNA are provided as controls. Sizes in bp are indicated on the right. D. Detection of the Bo10 encoded gp180 protein by the anti-Bo10-c15 serum. Purified virions (5*105 virions per lane) were subjected to western blotting with anti-Bo10-c15 serum as described in the Methods. The position of a molecular mass (MM) standard (in kDa) is shown on the right.

Mentions: We previously described two BoHV-4 Bo10 knockout strains [22], [30]. To unravel the function of both the spliced and the unspliced Bo10 expression products, we generated two additional Bo10 mutant viruses. In the Bo10 MuDir mutant, we punctually mutated the Bo10 splicing donor site (T to G) to only express the unspliced form. In contrast, the Bo10 Spliced strain only expresses the Bo10 spliced product (Figure 2A). Southern blots of viral DNA (Figure 2B) confirmed the expected genomic structures of the two mutants and their associated revertant strains. The expected mutations were further confirmed by DNA sequencing (data not shown). As expected, RT-PCR analysis showed that the Bo10 MuDir and the Bo10 Spliced strains express the unspliced or the spliced messenger RNA respectively (Figure 2C). Finally, immunoblotting with an anti-Bo10-c15 rabbit polyserum confirmed that only the Bo10 MuDir mutant virions lacked gp180, encoded by the spliced Bo10 product (Figure 2D) while content of other proteins such as gB appeared to be normal (Figure S1).


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)

Generation of the Bo10 MuDir and Spliced BoHV-4 mutant.A. Schematic representation of the strategy followed to produce the recombinant BoHV-4 strains. We modified the BoHV-4 V.test Bo10 coding sequence (genomic coordinates 65844 to 66743) either by introducing a point mutation in the splicing donor site (Bo10 MuDir strain) or by replacing the entire Bo10 ORF by a sequence devoid of the intron (Bo10 Spliced strain). Splicing donor and acceptor sites are in red with splicing essential nucleotides in upper cases. The mutated nucleotide is in green. B. Verification of the molecular structure. Viral DNA was digested with BamHI, resolved by agarose gel electrophoresis, and hybridized with a 32P-labeled probe, corresponding to nucleotides 65900–66370 of the BoHV-4 V.test strain genome (coding for Bo10 Exon 1). The 7,137-bp wild-type (WT) band becomes 7060-bp (open arrow) for the Bo10 spliced mutant. Marker sizes in Kbp are indicated on the left. C. RT-PCR analysis of BoHV-4 Bo10 expression by the different mutants. MDBK cells were mock infected or infected with the different BoHV-4 mutant strains at a MOI of 1. Twenty-four hours p.i., expression of Bo10 was studied using different pairs of primers specific for the spliced and/or unspliced Bo10 cDNA. Uninfected cell samples (UI), reactions without reverse transcriptase (Neg. control) and amplification of viral genomic DNA are provided as controls. Sizes in bp are indicated on the right. D. Detection of the Bo10 encoded gp180 protein by the anti-Bo10-c15 serum. Purified virions (5*105 virions per lane) were subjected to western blotting with anti-Bo10-c15 serum as described in the Methods. The position of a molecular mass (MM) standard (in kDa) is shown on the right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814654&req=5

ppat-1003753-g002: Generation of the Bo10 MuDir and Spliced BoHV-4 mutant.A. Schematic representation of the strategy followed to produce the recombinant BoHV-4 strains. We modified the BoHV-4 V.test Bo10 coding sequence (genomic coordinates 65844 to 66743) either by introducing a point mutation in the splicing donor site (Bo10 MuDir strain) or by replacing the entire Bo10 ORF by a sequence devoid of the intron (Bo10 Spliced strain). Splicing donor and acceptor sites are in red with splicing essential nucleotides in upper cases. The mutated nucleotide is in green. B. Verification of the molecular structure. Viral DNA was digested with BamHI, resolved by agarose gel electrophoresis, and hybridized with a 32P-labeled probe, corresponding to nucleotides 65900–66370 of the BoHV-4 V.test strain genome (coding for Bo10 Exon 1). The 7,137-bp wild-type (WT) band becomes 7060-bp (open arrow) for the Bo10 spliced mutant. Marker sizes in Kbp are indicated on the left. C. RT-PCR analysis of BoHV-4 Bo10 expression by the different mutants. MDBK cells were mock infected or infected with the different BoHV-4 mutant strains at a MOI of 1. Twenty-four hours p.i., expression of Bo10 was studied using different pairs of primers specific for the spliced and/or unspliced Bo10 cDNA. Uninfected cell samples (UI), reactions without reverse transcriptase (Neg. control) and amplification of viral genomic DNA are provided as controls. Sizes in bp are indicated on the right. D. Detection of the Bo10 encoded gp180 protein by the anti-Bo10-c15 serum. Purified virions (5*105 virions per lane) were subjected to western blotting with anti-Bo10-c15 serum as described in the Methods. The position of a molecular mass (MM) standard (in kDa) is shown on the right.
Mentions: We previously described two BoHV-4 Bo10 knockout strains [22], [30]. To unravel the function of both the spliced and the unspliced Bo10 expression products, we generated two additional Bo10 mutant viruses. In the Bo10 MuDir mutant, we punctually mutated the Bo10 splicing donor site (T to G) to only express the unspliced form. In contrast, the Bo10 Spliced strain only expresses the Bo10 spliced product (Figure 2A). Southern blots of viral DNA (Figure 2B) confirmed the expected genomic structures of the two mutants and their associated revertant strains. The expected mutations were further confirmed by DNA sequencing (data not shown). As expected, RT-PCR analysis showed that the Bo10 MuDir and the Bo10 Spliced strains express the unspliced or the spliced messenger RNA respectively (Figure 2C). Finally, immunoblotting with an anti-Bo10-c15 rabbit polyserum confirmed that only the Bo10 MuDir mutant virions lacked gp180, encoded by the spliced Bo10 product (Figure 2D) while content of other proteins such as gB appeared to be normal (Figure S1).

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