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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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Clusters of PK15 gene expression levels identified by the k-means method. The expression levels of genes that are differentially expressed between infected and mock-infected cells at each time point are analyzed by the k-means method (three clusters). Averages of normalized intensities for all mock-infected conditions and for each infected condition were centered (median) by genes. For each cluster, one graph and one clustering picture are represented. The graph shows the mean of the expression levels of all genes (x = Time; y = mean of levels of expression) and the clustering picture depicts the mean of each gene expression level for all mock-infected time points and for each infected time point (x = Time; y = level of expression).
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Figure 4: Clusters of PK15 gene expression levels identified by the k-means method. The expression levels of genes that are differentially expressed between infected and mock-infected cells at each time point are analyzed by the k-means method (three clusters). Averages of normalized intensities for all mock-infected conditions and for each infected condition were centered (median) by genes. For each cluster, one graph and one clustering picture are represented. The graph shows the mean of the expression levels of all genes (x = Time; y = mean of levels of expression) and the clustering picture depicts the mean of each gene expression level for all mock-infected time points and for each infected time point (x = Time; y = level of expression).

Mentions: The number of differentially expressed cellular probes increased with time in parallel to viral gene expression (Table 1). Between 0 and 2 h pi most of the SLA/immune probes showed no change and few differentially expressed genes were detected from 1 h pi with the Qiagen-NRPS8 microarray (Table 1). As shown in Table 1, the SLA/PrV microarray identified 1, 0, 2, 36, 107 and 107 differentially expressed probes at 0, 1, 2, 4, 8 and 12 h pi, respectively [see Additional file 1] and the Qiagen-NRSP8 microarray identified 31, 181, 2756, 6693 differentially expressed probes at 1, 2, 4 and 8 h pi [see Additional file 2], respectively. The SLA/PrV microarray data show that 86 (31/36), 82 (88/107), and 87 % (93/107) of the differentially expressed probes were down-regulated at 4, 8 and 12 h pi, respectively and the Qiagen-NRSP8 microarray data show that 13 (4/31), 26 (47/181), 54 (1494/2756) and 52 % (3509/6693) of the differentially expressed probes explored in this case were down-regulated at 1, 2, 4 and 8 h pi, respectively (Table 1). With the k-means method, the expression levels for each condition (time and infection status) of the SLA/PrV differentially expressed probes set were clustered in three groups (Figure 4). Eighty-eight probes with a small decrease in expression levels from 4 h pi were found in the first cluster and 45 probes with a stronger decrease in expression levels from 2 h pi in the second cluster. The third cluster contained 27 up-regulated probes at 8 h pi. The results obtained with both microarrays show that many cellular genes were down-regulated during the time course of the experiment especially between 4 and 12 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)

Clusters of PK15 gene expression levels identified by the k-means method. The expression levels of genes that are differentially expressed between infected and mock-infected cells at each time point are analyzed by the k-means method (three clusters). Averages of normalized intensities for all mock-infected conditions and for each infected condition were centered (median) by genes. For each cluster, one graph and one clustering picture are represented. The graph shows the mean of the expression levels of all genes (x = Time; y = mean of levels of expression) and the clustering picture depicts the mean of each gene expression level for all mock-infected time points and for each infected time point (x = Time; y = level of expression).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Clusters of PK15 gene expression levels identified by the k-means method. The expression levels of genes that are differentially expressed between infected and mock-infected cells at each time point are analyzed by the k-means method (three clusters). Averages of normalized intensities for all mock-infected conditions and for each infected condition were centered (median) by genes. For each cluster, one graph and one clustering picture are represented. The graph shows the mean of the expression levels of all genes (x = Time; y = mean of levels of expression) and the clustering picture depicts the mean of each gene expression level for all mock-infected time points and for each infected time point (x = Time; y = level of expression).
Mentions: The number of differentially expressed cellular probes increased with time in parallel to viral gene expression (Table 1). Between 0 and 2 h pi most of the SLA/immune probes showed no change and few differentially expressed genes were detected from 1 h pi with the Qiagen-NRPS8 microarray (Table 1). As shown in Table 1, the SLA/PrV microarray identified 1, 0, 2, 36, 107 and 107 differentially expressed probes at 0, 1, 2, 4, 8 and 12 h pi, respectively [see Additional file 1] and the Qiagen-NRSP8 microarray identified 31, 181, 2756, 6693 differentially expressed probes at 1, 2, 4 and 8 h pi [see Additional file 2], respectively. The SLA/PrV microarray data show that 86 (31/36), 82 (88/107), and 87 % (93/107) of the differentially expressed probes were down-regulated at 4, 8 and 12 h pi, respectively and the Qiagen-NRSP8 microarray data show that 13 (4/31), 26 (47/181), 54 (1494/2756) and 52 % (3509/6693) of the differentially expressed probes explored in this case were down-regulated at 1, 2, 4 and 8 h pi, respectively (Table 1). With the k-means method, the expression levels for each condition (time and infection status) of the SLA/PrV differentially expressed probes set were clustered in three groups (Figure 4). Eighty-eight probes with a small decrease in expression levels from 4 h pi were found in the first cluster and 45 probes with a stronger decrease in expression levels from 2 h pi in the second cluster. The third cluster contained 27 up-regulated probes at 8 h pi. The results obtained with both microarrays show that many cellular genes were down-regulated during the time course of the experiment especially between 4 and 12 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