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Expression of frog virus 3 genes is impaired in mammalian cell lines.

Eaton HE, Metcalf J, Brunetti CR - Virol. J. (2008)

Bottom Line: Following a FV3 infection in fathead minnow (FHM) cells 75L was found in vesicles throughout the cytoplasm as early as 3 hours post-infection.While 75L expressed strongly in FHM cells, our findings revealed no 75L expression in mammalian cells lines despite evidence of a FV3 infection.As a result, 75L was codon optimized and transfection of the codon optimized construct resulted in detectable expression in mammalian cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Trent University, Peterborough, ON, Canada. heathereaton@trentu.ca

ABSTRACT
Frog virus 3 (FV3) is a large DNA virus that is the prototypic member of the family Iridoviridae. To examine levels of FV3 gene expression we generated a polyclonal antibody against the FV3 protein 75L. Following a FV3 infection in fathead minnow (FHM) cells 75L was found in vesicles throughout the cytoplasm as early as 3 hours post-infection. While 75L expressed strongly in FHM cells, our findings revealed no 75L expression in mammalian cells lines despite evidence of a FV3 infection. One explanation for the lack of gene expression in mammalian cell lines may be inefficient codon usage. As a result, 75L was codon optimized and transfection of the codon optimized construct resulted in detectable expression in mammalian cells. Therefore, although FV3 can infect and replicate in mammalian cell lines, the virus may not express its full complement of genes due to inefficient codon usage in mammalian species.

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FV3 infected BGMK and HeLa cells do not express the gene 75L. FHM, HeLa, and BGMK cells were infected with FV3 at an MOI of 1. At 0, 3, 8, 16, and 32 hours post-infection, cells were fixed and a FV3 infection was detected using anti-FV3 antibodies (blue: A, C, E) and 75L was detected using anti-FV3-75L antibodies (green: B, D, F). No images of FHM cells at 32 hours were taken as the cells had succumbed to infection. Cells were visualized using DIC and indirect immunofluorescence images were captured on a laser scanning confocal microscope.
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Figure 1: FV3 infected BGMK and HeLa cells do not express the gene 75L. FHM, HeLa, and BGMK cells were infected with FV3 at an MOI of 1. At 0, 3, 8, 16, and 32 hours post-infection, cells were fixed and a FV3 infection was detected using anti-FV3 antibodies (blue: A, C, E) and 75L was detected using anti-FV3-75L antibodies (green: B, D, F). No images of FHM cells at 32 hours were taken as the cells had succumbed to infection. Cells were visualized using DIC and indirect immunofluorescence images were captured on a laser scanning confocal microscope.

Mentions: In FHM cells, the anti-FV3 serum was able to detect antigen as early as 3 hours post-infection (Figure 1:A). In addition, 75L expression was also detectable in FHM cells starting at 3 hours post-infection and expression increased as the infection progressed (Figure 1:B). In contrast, expression of FV3 in HeLa and BGMK cells was not detectable until 16 hours post-infection (Figure 1:C,E) and no detectable 75L expression was observed in these cell lines even as late as 32 hours post-infection (Figure 1:D,F). Therefore, although a FV3 infection was detected in all three cell lines, 75L, a non-essential gene only expressed in FHM cells, an ectothermic cell line.


Expression of frog virus 3 genes is impaired in mammalian cell lines.

Eaton HE, Metcalf J, Brunetti CR - Virol. J. (2008)

FV3 infected BGMK and HeLa cells do not express the gene 75L. FHM, HeLa, and BGMK cells were infected with FV3 at an MOI of 1. At 0, 3, 8, 16, and 32 hours post-infection, cells were fixed and a FV3 infection was detected using anti-FV3 antibodies (blue: A, C, E) and 75L was detected using anti-FV3-75L antibodies (green: B, D, F). No images of FHM cells at 32 hours were taken as the cells had succumbed to infection. Cells were visualized using DIC and indirect immunofluorescence images were captured on a laser scanning confocal microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2500002&req=5

Figure 1: FV3 infected BGMK and HeLa cells do not express the gene 75L. FHM, HeLa, and BGMK cells were infected with FV3 at an MOI of 1. At 0, 3, 8, 16, and 32 hours post-infection, cells were fixed and a FV3 infection was detected using anti-FV3 antibodies (blue: A, C, E) and 75L was detected using anti-FV3-75L antibodies (green: B, D, F). No images of FHM cells at 32 hours were taken as the cells had succumbed to infection. Cells were visualized using DIC and indirect immunofluorescence images were captured on a laser scanning confocal microscope.
Mentions: In FHM cells, the anti-FV3 serum was able to detect antigen as early as 3 hours post-infection (Figure 1:A). In addition, 75L expression was also detectable in FHM cells starting at 3 hours post-infection and expression increased as the infection progressed (Figure 1:B). In contrast, expression of FV3 in HeLa and BGMK cells was not detectable until 16 hours post-infection (Figure 1:C,E) and no detectable 75L expression was observed in these cell lines even as late as 32 hours post-infection (Figure 1:D,F). Therefore, although a FV3 infection was detected in all three cell lines, 75L, a non-essential gene only expressed in FHM cells, an ectothermic cell line.

Bottom Line: Following a FV3 infection in fathead minnow (FHM) cells 75L was found in vesicles throughout the cytoplasm as early as 3 hours post-infection.While 75L expressed strongly in FHM cells, our findings revealed no 75L expression in mammalian cells lines despite evidence of a FV3 infection.As a result, 75L was codon optimized and transfection of the codon optimized construct resulted in detectable expression in mammalian cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Trent University, Peterborough, ON, Canada. heathereaton@trentu.ca

ABSTRACT
Frog virus 3 (FV3) is a large DNA virus that is the prototypic member of the family Iridoviridae. To examine levels of FV3 gene expression we generated a polyclonal antibody against the FV3 protein 75L. Following a FV3 infection in fathead minnow (FHM) cells 75L was found in vesicles throughout the cytoplasm as early as 3 hours post-infection. While 75L expressed strongly in FHM cells, our findings revealed no 75L expression in mammalian cells lines despite evidence of a FV3 infection. One explanation for the lack of gene expression in mammalian cell lines may be inefficient codon usage. As a result, 75L was codon optimized and transfection of the codon optimized construct resulted in detectable expression in mammalian cells. Therefore, although FV3 can infect and replicate in mammalian cell lines, the virus may not express its full complement of genes due to inefficient codon usage in mammalian species.

Show MeSH
Related in: MedlinePlus