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Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection.

Flori L, Rogel-Gaillard C, Cochet M, Lemonnier G, Hugot K, Chardon P, Robin S, Lefèvre F - BMC Genomics (2008)

Bottom Line: We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry.The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff.Our results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, DGA, UMR 314, Laboratoire de Radiobiologie et d'Etude du Génome, Jouy-en-Josas, F-78350 France. laurence.flori@jouy.inra.fr

ABSTRACT

Background: Transcriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. To identify as completely as possible, viral and cellular genes regulated during infection, we simultaneously analyzed PrV and cellular transcriptome modifications using two microarrays i.e. a laboratory-made combined SLA/PrV microarray, consisting of probes for all PrV genes and for porcine genes contained in the Swine Leukocyte Antigen (SLA) complex, and the porcine generic Qiagen-NRSP8 oligonucleotide microarray. We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry.

Results: An increase in the number of differentially expressed cellular genes and PrV genes especially from 4 h post-infection (pi) was observed concomitantly with the onset of viral progeny while no early global cellular shutoff was recorded. Many cellular genes were down-regulated from 4 h pi and their number increased until 12 h pi. UL41 transcripts encoding the virion host shutoff protein were first detected as differentially expressed at 8 h pi. The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff. We found that many biological processes are altered during PrV infection. Indeed, several genes involved in the SLA class I antigenic presentation pathway (SLA-Ia, TAP1, TAP2, PSMB8 and PSMB9), were down-regulated, thus contributing to viral immune escape from this pathway and other genes involved in apoptosis, nucleic acid metabolism, cytoskeleton signaling as well as interferon-mediated antiviral response were also modulated during PrV infection.

Conclusion: Our results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression.

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SLA I cell surface decrease on PrV infected PK15 cells. Histogram overlays of MHC class I expression detected by the mAb PT85A are shown. The number in red represents the percent mean fluorescence intensity [(mean channel fluorescence of the infected sample at 8 h pi/mean channel fluorescence of the mock-infected cells at 8 h pi) × 100]. The results represent one of three representative experiments.
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Figure 5: SLA I cell surface decrease on PrV infected PK15 cells. Histogram overlays of MHC class I expression detected by the mAb PT85A are shown. The number in red represents the percent mean fluorescence intensity [(mean channel fluorescence of the infected sample at 8 h pi/mean channel fluorescence of the mock-infected cells at 8 h pi) × 100]. The results represent one of three representative experiments.

Mentions: Since our experiments, as well as other studies [6,7], have clearly indicated a down-regulation of the MHC class I genes during PrV infection, we checked, by flow cytometry, for a down-regulation of surface MHC class I molecules on PrV infected PK15 cells at 8 h pi. To visualize infected cells, we used, in the same experimental conditions, a recombinant PrV strain (derived from NIA3) expressing the green fluorescent protein (GFP). Ninety percent of the cells appeared infected and 73% of these infected cells expressed surface MHC class I molecules on their surface while 89.1% and 83.9% of the mock-infected cells expressed MHC class I molecules at 0 and 8 h pi, respectively (data not shown). The MHC class I mean fluorescence intensity of infected cells at 8 h pi was 50.9% of that of mock-infected cells (mean of three experiments) thus confirming a clear decrease of MHC class I molecules expression on the surface of infected cells (Figure 5). As a control, we observed that the expression of tubulin, detected by Western blot, remains unchanged even 8 h pi in PK15 cells (data not shown).


Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection.

Flori L, Rogel-Gaillard C, Cochet M, Lemonnier G, Hugot K, Chardon P, Robin S, Lefèvre F - BMC Genomics (2008)

SLA I cell surface decrease on PrV infected PK15 cells. Histogram overlays of MHC class I expression detected by the mAb PT85A are shown. The number in red represents the percent mean fluorescence intensity [(mean channel fluorescence of the infected sample at 8 h pi/mean channel fluorescence of the mock-infected cells at 8 h pi) × 100]. The results represent one of three representative experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335119&req=5

Figure 5: SLA I cell surface decrease on PrV infected PK15 cells. Histogram overlays of MHC class I expression detected by the mAb PT85A are shown. The number in red represents the percent mean fluorescence intensity [(mean channel fluorescence of the infected sample at 8 h pi/mean channel fluorescence of the mock-infected cells at 8 h pi) × 100]. The results represent one of three representative experiments.
Mentions: Since our experiments, as well as other studies [6,7], have clearly indicated a down-regulation of the MHC class I genes during PrV infection, we checked, by flow cytometry, for a down-regulation of surface MHC class I molecules on PrV infected PK15 cells at 8 h pi. To visualize infected cells, we used, in the same experimental conditions, a recombinant PrV strain (derived from NIA3) expressing the green fluorescent protein (GFP). Ninety percent of the cells appeared infected and 73% of these infected cells expressed surface MHC class I molecules on their surface while 89.1% and 83.9% of the mock-infected cells expressed MHC class I molecules at 0 and 8 h pi, respectively (data not shown). The MHC class I mean fluorescence intensity of infected cells at 8 h pi was 50.9% of that of mock-infected cells (mean of three experiments) thus confirming a clear decrease of MHC class I molecules expression on the surface of infected cells (Figure 5). As a control, we observed that the expression of tubulin, detected by Western blot, remains unchanged even 8 h pi in PK15 cells (data not shown).

Bottom Line: We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry.The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff.Our results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, DGA, UMR 314, Laboratoire de Radiobiologie et d'Etude du Génome, Jouy-en-Josas, F-78350 France. laurence.flori@jouy.inra.fr

ABSTRACT

Background: Transcriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. To identify as completely as possible, viral and cellular genes regulated during infection, we simultaneously analyzed PrV and cellular transcriptome modifications using two microarrays i.e. a laboratory-made combined SLA/PrV microarray, consisting of probes for all PrV genes and for porcine genes contained in the Swine Leukocyte Antigen (SLA) complex, and the porcine generic Qiagen-NRSP8 oligonucleotide microarray. We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry.

Results: An increase in the number of differentially expressed cellular genes and PrV genes especially from 4 h post-infection (pi) was observed concomitantly with the onset of viral progeny while no early global cellular shutoff was recorded. Many cellular genes were down-regulated from 4 h pi and their number increased until 12 h pi. UL41 transcripts encoding the virion host shutoff protein were first detected as differentially expressed at 8 h pi. The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff. We found that many biological processes are altered during PrV infection. Indeed, several genes involved in the SLA class I antigenic presentation pathway (SLA-Ia, TAP1, TAP2, PSMB8 and PSMB9), were down-regulated, thus contributing to viral immune escape from this pathway and other genes involved in apoptosis, nucleic acid metabolism, cytoskeleton signaling as well as interferon-mediated antiviral response were also modulated during PrV infection.

Conclusion: Our results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression.

Show MeSH
Related in: MedlinePlus