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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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PrV growth kinetics in PK15 cells. The PK15 cells were infected with PrV NIA3 at 20 MOI in HMS-M medium. PrV was titrated in the medium by plaque assay at different times pi (0, 4, 8, 12, 22, 26, 32 and 50 h pi). PrV titer was expressed as plaque forming units per ml (pfu/ml).
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Figure 2: PrV growth kinetics in PK15 cells. The PK15 cells were infected with PrV NIA3 at 20 MOI in HMS-M medium. PrV was titrated in the medium by plaque assay at different times pi (0, 4, 8, 12, 22, 26, 32 and 50 h pi). PrV titer was expressed as plaque forming units per ml (pfu/ml).

Mentions: The six time points, which were studied in this experiment – i.e. 0, 1, 2, 4, 8 and 12 hours (h) post-infection (pi), were chosen according to viral growth kinetics observed in PK15 cells in our experimental conditions (Figure 2). The expression of viral genes was detected between 2 and 12 h pi and increased during time and most of the genes were expressed at 8 and 12 h pi. The hierarchical clustering (HCL) of viral gene expression levels according to all conditions (time and infection status) allowed us to distinguish two main groups: i) mock-infection at all time points and infection until 2 h pi ii) infection from 4 until 12 h (data not shown). With the k-means method, we identified three transcript clusters with similar expression profiles (Figure 3). The average expression levels for the first cluster (29 probes) showed little variation and only from 8 h pi. The second cluster contained 30 probes corresponding to genes, the expression level of which increased from 4 h pi. The last group (21 probes) displayed a higher increase of expression level from 2 to 8 h pi.


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)

PrV growth kinetics in PK15 cells. The PK15 cells were infected with PrV NIA3 at 20 MOI in HMS-M medium. PrV was titrated in the medium by plaque assay at different times pi (0, 4, 8, 12, 22, 26, 32 and 50 h pi). PrV titer was expressed as plaque forming units per ml (pfu/ml).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: PrV growth kinetics in PK15 cells. The PK15 cells were infected with PrV NIA3 at 20 MOI in HMS-M medium. PrV was titrated in the medium by plaque assay at different times pi (0, 4, 8, 12, 22, 26, 32 and 50 h pi). PrV titer was expressed as plaque forming units per ml (pfu/ml).
Mentions: The six time points, which were studied in this experiment – i.e. 0, 1, 2, 4, 8 and 12 hours (h) post-infection (pi), were chosen according to viral growth kinetics observed in PK15 cells in our experimental conditions (Figure 2). The expression of viral genes was detected between 2 and 12 h pi and increased during time and most of the genes were expressed at 8 and 12 h pi. The hierarchical clustering (HCL) of viral gene expression levels according to all conditions (time and infection status) allowed us to distinguish two main groups: i) mock-infection at all time points and infection until 2 h pi ii) infection from 4 until 12 h (data not shown). With the k-means method, we identified three transcript clusters with similar expression profiles (Figure 3). The average expression levels for the first cluster (29 probes) showed little variation and only from 8 h pi. The second cluster contained 30 probes corresponding to genes, the expression level of which increased from 4 h pi. The last group (21 probes) displayed a higher increase of expression level from 2 to 8 h pi.

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