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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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Map of the PrV transcriptome. The relative location of transcripts and designed amplicons are shown. UL and US regions of the PrV genome are represented in black while IR and TR regions are in pink. Transcripts corresponding to each gene are represented with arrows (coding region in orange and non-coding regions or introns in green). Amplicons are represented in blue on the genome. The name of each amplicon is written above the corresponding genome location. Size is measured in kb.
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Figure 1: Map of the PrV transcriptome. The relative location of transcripts and designed amplicons are shown. UL and US regions of the PrV genome are represented in black while IR and TR regions are in pink. Transcripts corresponding to each gene are represented with arrows (coding region in orange and non-coding regions or introns in green). Amplicons are represented in blue on the genome. The name of each amplicon is written above the corresponding genome location. Size is measured in kb.

Mentions: The 1789 DNA/cDNA probes spotted on the SLA/PrV microarray fall into four distinct probe sets: i) 420 probes localized on a segment of chromosome 7 between the loci PRL and PRIM2A (SSC7p1.1-q1.2), which includes the extended-SLA region and represents 272 unique sequences, 111 belonging to the strict SLA region between the loci UBD and RING1 [16]; ii) 73 probes specific to 73 genes encoding molecules involved in immunity and localized outside the SLA region [18]; iii) 80 PrV probes specific to the 70 viral genes (Figure 1) and iv) 1170 probes randomly chosen for data normalization from porcine cDNA AGENAE library [19,20]. The PrV/SLA microarray covers 72.5% of the annotated sequences of the strict SLA region (= 111/153) [16,21].


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)

Map of the PrV transcriptome. The relative location of transcripts and designed amplicons are shown. UL and US regions of the PrV genome are represented in black while IR and TR regions are in pink. Transcripts corresponding to each gene are represented with arrows (coding region in orange and non-coding regions or introns in green). Amplicons are represented in blue on the genome. The name of each amplicon is written above the corresponding genome location. Size is measured in kb.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Map of the PrV transcriptome. The relative location of transcripts and designed amplicons are shown. UL and US regions of the PrV genome are represented in black while IR and TR regions are in pink. Transcripts corresponding to each gene are represented with arrows (coding region in orange and non-coding regions or introns in green). Amplicons are represented in blue on the genome. The name of each amplicon is written above the corresponding genome location. Size is measured in kb.
Mentions: The 1789 DNA/cDNA probes spotted on the SLA/PrV microarray fall into four distinct probe sets: i) 420 probes localized on a segment of chromosome 7 between the loci PRL and PRIM2A (SSC7p1.1-q1.2), which includes the extended-SLA region and represents 272 unique sequences, 111 belonging to the strict SLA region between the loci UBD and RING1 [16]; ii) 73 probes specific to 73 genes encoding molecules involved in immunity and localized outside the SLA region [18]; iii) 80 PrV probes specific to the 70 viral genes (Figure 1) and iv) 1170 probes randomly chosen for data normalization from porcine cDNA AGENAE library [19,20]. The PrV/SLA microarray covers 72.5% of the annotated sequences of the strict SLA region (= 111/153) [16,21].

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