Paragraph: Porcine circovirus type 2 (PCV2) is the essential infectious agent responsible for causing porcine circovirus-associated diseases in pigs. To date, eleven RNAs and five viral proteins of PCV2 have been detected. Here, we identified a novel viral gene within the PCV2 genome, termed ORF5, that exists at both the transcriptional and translational level during productive infection of PCV2 in porcine alveolar macrophages 3D4/2 (PAMs). Northern blot analysis was used to demonstrate that the ORF5 gene measures 180 bp in length and overlaps completely with ORF1 when read in the same direction. Site-directed mutagenesis was used to show that the ORF5 protein is not essential for PCV2 replication. To investigate the biological functions of the novel protein, we constructed a recombinant eukaryotic expression plasmid capable of expressing PCV2 ORF5. The results show that the GFP-tagged PCV2 ORF5 protein localizes to the endoplasmic reticulum (ER), is degraded via the proteasome, inhibits PAM growth and prolongs the S-phase of the cell cycle. Further studies show that the GFP-tagged PCV2 ORF5 protein induces ER stress and activates NF-κB, which was further confirmed by a significant upregulation in IL-6, IL-8 and COX-2 expression. In addition, five cellular proteins (GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3) were found to interact with ORF5 via yeast two-hybrid assay. These findings provide novel information on the identification and functional analysis of the PCV2 ORF5 protein and are likely to be of benefit in elucidating the molecular mechanisms of PCV2 pathogenicity. However, additional experiments are needed to validate the expression and function of the ORF5 protein during PCV2 infection in vitro before any definitive conclusion can be drawn. piglets and is clinically characterized by fever, unthriftiness, respiratory distress, enlarged lymph nodes, and occasional jaundice and diarrhea [7] [8] . Many other conditions, including congenital tremors (CT), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), reproduction disorders and fetal myocarditis, are also closely related to PCV2 infection [9] [10] [11] [12] [13] [14] . PCV2 infection leads to the development of microscopic lesions mainly dominated by lymphoid depletion and histiocytic infiltration in lymphoid tissues [9, [15] [16] [17] . Mortality rates may vary from 15 to 20% and can be significantly higher in the presence of co-infection with either porcine reproductive and respiratory syndrome virus (PRRSV) or mycoplasma hyopneumoniae [7] [8] 18] . PCV2 infection has thus far caused huge economic losses to the swine industry worldwide. The PCV2 genome is a single-stranded, ambisense, closed circular DNA containing between 1,766 and 1,768 nucleotides [19] [20] . Although the nucleotide homology within individual isolates of either PCV1 or PCV2 is over 90%, the homology between PCV1 and PCV2 as serotypes is only 68% to 76% [19, 21] . This diversity may be responsible for the differences in pathogenicity or virulence observed for these serotypes. The virus was predicted to possess 11 overlapping open reading frames (ORFs) [19], of which four have been characterized in detail in replicating PCV2. On the viral strand, there is the ORF1 (nucleotides 51-995) gene, which encodes the Rep and Rep' proteins essential for PCV2 replication [22]. On the complementary strand, there are the ORF2 (nucleotides 1735-1034), ORF3 (nucleotides 671-357) and ORF4 (nucleotides 565-386) genes, which encode the capsid protein [23], apoptotic protein [21] and controversial anti-apoptotic protein, respectively [24-25]. Previous transcriptional analysis conducted during productive PCV2 infection of porcine kidney cells measured nine unique PCV2-specific RNA transcripts (designated-CR, Rep, Rep 0 , Rep3a, Rep3b, Rep3c, NS515, NS672, and NS0) [26]; however, only CR, Rep and Rep 0 transcripts were proven capable of translation into functional proteins [22, [26] [27] . In contrast, ORF3 and ORF4 transcripts, which are not included in the above nine RNAs, were subsequently confirmed to play important roles in the pathogenesis of PCV2; ORF3-deficient PCV2 is attenuated in its natural host [28] , and ORF4-deficient PCV2 induces a higher viral load and more severe microscopic lesions in the spleen during early stages of infection in mice [24] . Collectively, these research findings indicate that PCV2 pathogenesis is both complicated and multigenic and that the individual viral proteins encoded by ORF2, ORF3 or ORF4 are insufficient in determining pathogenicity. Previous computational analysis has revealed that the PCV2 genome contains at least seven ORFs with the potential to encode proteins > 5 kDa [29] ; to date, four have been identified. However, it has not yet been determined whether the three additional predicted proteins exist or whether there are still other viral proteins encoded by PCV2 that control its pathogenicity. Sequence alignment analysis of PCV2 isolates from varying geographic regions suggested that the putative ORF5 gene (proposed to exist by Hamel et al) might not encode a functional protein, as translation of the protein was found to terminate after only seven amino acids in some Chinese PCV2 strains [19, 30] . However, several groups have aligned the genomic sequences of various PCV2 isolates and found a region of extremely high identity (over 95%) at the amino acid level, suggesting that PCV2 encodes a novel protein, although different names for this possible protein have been used in the literature (Gen Bank accession no. O92288, Q77GS3, Q77S04, etc). This new putative viral gene (designated ORF5 in this study), which is entirely different from that predicted by Hamel et al in 1998 [19], completely overlaps the ORF1 gene and is read in the same direction. The ORF5 gene is approximately 180 bp (spanning nucleotides 553-732) and encodes a polypeptide that is predicted to be of 6.5 kDa in molecular weight, whereas the corresponding region of PCV1 does not appear to produce a peptide. As PCV1 isolates are naturally nonpathogenic [31] [32] and PCV2 isolates are pathogenic, this putative polypeptide may be important for the pathogenesis of PCV2. Presently, no Functional Analysis of GFP-Fused Putative PCV2 ORF5 Protein PLOS ONE | 
 Title: Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein
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