Page 37 - M. Immunology

The open reading frame 5 (ORF5) of porcine circovirus type 2 (PCV2) modulates host innate immunity by
suppressing transcription factors involved in type I interferon signaling pathway
Na Young Lee, Taehoon Chun
Department of Biotechnology, Major in Molecular Bioengineering,
Korea University Graduate School, Seoul, Republic of Korea

BACKGROUND & AIM
• PCV2 is a non-enveloped icosahedral virus which infects to pig causing porcine circovirus-associated disease (PCVAD) (Rodríguez-Arrioja et
al., 2002). The majority of PCVAD is a postweaning multisystemic wasting syndrome (PMWS) which is one of the factor for largest economic
losses in the swine industry (Alarcon et al., 2013). In PCV2, there are five major OFRs (Lv et al., 2015; Ren et al., 2016). Among these, ORF5
was recently identified but the functional role during pathogenesis after PCV2 infection is unknown (Lv et al.,2015).
• The aim of this study is to investigate the functional role of PCV2 ORF5 in PCV2-infected porcine epithelial cells.

METHODS
• Preparation of cDNA library and Illumina sequencing - In this study, we used PCV1 free PK15 cells (porcine kidney epithelial cells). We constructed
tandem dimers of the PCV2 genome (PCV2 type a) and ORF5 deletion mutant of PCV2 (ΔORF5 PCV2) and infected PCV2 into PK15 cells. Total RNAs from mock
(empty vector) and PCV2 ORF5 expressing PK15 cells were extracted using TRIzol. Then, One μg of total RNA from each cell was converted into cDNA libraries.
Final cDNA libraries were quantitated by Real-time PCR, and then sequenced using an Illumina HiSeq4000 sequence.
• Transcriptional analysis by quantitative real-time PCR - To monitor mRNA expression of genes encoding proteins involved in type I IFN production,
mock and PCV2 ORF5 expressing PK15 cells were treated without or with Poly I:C (10 μg/ml, Sigma). Before treatment, Poly I:C was dissolved in the distilled
water. Twenty-four h after treatment, cells were harvested to isolate mRNA. Then, cDNA from each mRNA sample was synthesized. After each cDNA synthesis,
Real-time PCR was conducted. Also, PK15 cells were infected with WT PCV2 or ΔORF5 PCV2. Twelve h after viral infection, mRNAs from these infected cells
were purified and their cDNAs were synthesized using the same method described above.
RESULTS

(a) (b) Fig. 1. Overview of RNA-seq and differential expression analysis
in PK15 cells expressing PCV2 ORF5 or an empty vector.
(a) Hierarchical clustering analysis of DEGs (differentially expressed
genes) between PK15 cells expressing PCV2 ORF5 protein (ORF5)
and those expressing an empty vector (Mock). ‘Yellow’ indicates
relatively higher expression and ‘blue’ indicates lower expression.
(b) GO functional enrichment analysis according to biological process
for DEGs. X axis represents DEG count while Y axis represents GO
terms. (c) Genes involved in type I IFN signaling were down-regulated
in ORF5 compared to those in mock. Down-regulated genes are
indicated by in the schematic diagram of type I IFN signaling pathways
during viral infection. Viral RNA is recognized by IFIH1 (MDA5), DDX58
(RIG-1), and TLR3. DHX58 (LGP2) is a positive regulator of IFIH1
(c) (MDA5) and DDX58 (RIG-1). The resulting signal cascades utilize IRF3
and IRF7 to promote transcription of IFITM, IFIT, and type I IFN. IFITM
inhibits elements of virus life cycle such as entry, replication, fusion,
and release at cellular endosomal or lysosomal vesicles. IFIT
recognizes 5′-ppp of viral RNA and inhibits viral replication. IFN
receptor triggers activation of STAT proteins. Activated STATs are able
to form complexes as heterodimers. Heterodimeric STAT1/STAT2
binds to IRF9. This complex then translocates into the nucleus to
activate expression of ISG and IRF7. ISG causes viral RNA
degradation and inhibits assembly of progeny virus.

Fig 4. PCV2 ORF5 protein enhances the
replication of PCV2 by inhibiting type I IFN
production.
PK15 cells were infected with WT PCV2 (WT)
or ΔORF5. Real-time PCR was used to
quantitate viral DNA copy number at different
times after infection (upper panel). Viral
Fig 2.PCV2 ORF5 protein down-regulates replication was also assessed as TCID 50 via
mRNA transcripts of genes encoding the end-point dilution method using
proteins involved in type I IFN production in immunofluorescence assay (lower panel). All
porcine epithelial cells. results are shown as means ± standard errors.
PCV2 ORF5 expressing PK15 cells (ORF5) and * p < 0.05; ** p < 0.01; *** p < 0.001.
empty vector transfectant (mock) were treated
with Poly I:C. After treatment, cells were CONCLUSION
harvested and analyzed by Real-time PCR.
Relative quantitation of genes was normalized We found that PCV2 ORF5 protein participates in inhibition of
to pig gapdh. All results are shown as means type I IFN expression via down-regulating genes involved in
± standard errors. * p < 0.05; ** p < 0.01; *** p type I IFN production in porcine epithelial cells. As a result,
< 0.001. “No sti” means no stimulation and the replication of PCV2 is increasing..
“Poly I:C” means Poly I:C stimulation.
REFERENCES
Lv, Q., Guo, K., Xu, H., Wang, T., Zhang, Y. (2015).
Identification of putative ORF5 protein of porcine circovirus type 2
and functional analysis of GFP-fused ORF5 protein. PLoS One 10,
e0127859.
Ren, L., Chen, X., Ouyang, H. (2016). Interactions of porcine
circovirus 2 with its hosts. Virus Genes. 52, 437-444.
Fig 3. mRNA transcripts of genes encoding proteins involved in type I IFN production are increased in
ΔORF5 PCV2 infected PK15 cells compared to those in WT PCV2 infected cells. Contact information
PK15 cells were infected with WT PCV2 or ΔORF5 PCV2. After viral infection, cells were harvested and
analyzed by Real-time PCR. Relative quantitation of genes was normalized to pig gapdh. All results are Na Young Lee
shown as means ± standard errors. * p < 0.05; ** p < 0.01; *** p < 0.001. “WT” means WT PCV2 infected
cells and “ΔORF5″ means ΔORF5 PCV2 infected cells. Email : dmh04062@korea.ac.kr