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      Correction: Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein

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          Abstract

          There is an error in the legend for Fig 1, "Genomic location of ORF5 gene and Detection of ORF5 transcripts by RT-PCR and Northern blot from PCV2-infected cells," panel D. Please see the complete, correct Fig 1 legend here. 10.1371/journal.pone.0134203.g001 Fig 1 Genomic location of ORF5 gene and Detection of ORF5 transcripts by RT-PCR and Northern blot from PCV2-infected cells. A) Genomic schematic of PCV2 Yangling strain (wPCV2). Coding sequences of the five ORFs are annotated with nucleotide coordinates that indicate the nucleotide site of each gene. The ORF2, ORF3 and ORF4 genes are transcribed leftward, while the ORF1 and ORF5 genes are transcribed rightward. The EcoRI restriction enzyme site is also indicated. (B) Nucleotide and amino acid alignments of the putative ORF5 reported by Hamel et al in 1998 (pmws, GenBank accession no. AF027217 [19]) and our team (wPCV2, this study) using the ClustalV Method. Homologous nucleotides and amino acids are indicated by asterisks. (C) Analysis of the ORF5 gene in PCV2-infected cells by RT-PCR. RNA was isolated from PCV2- or mock-infected cells and copied into cDNA. The cDNA was amplified with a pair of ORF5 primers. Positive fragment was amplified from the PCV2 genome using PCR. (D) Northern blot identification of the ORF5 transcript in PAMs infected with wPCV2. Total RNA samples were isolated from mock- and wPCV2-infected PAMs. Lane a, no RNA signal was detected in mock-infected PAMs after hybridization with a DIG-labeled ORF5 DNA probe (probe D). Lane b, mixtures of pMD-19T-ORF1 (3625 bp), PCR products of ORF1 (945 bp) and ORF5 (180 bp) were hybridized with probe D. Lane c, Rep, Repˊ, ORF3 and ORF5 transcripts were hybridized with probe D in wPCV2-infected PAMs.

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          Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein

          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.
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            Author and article information

            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, CA USA )
            1932-6203
            28 July 2015
            2015
            : 10
            : 7
            : e0134203
            Article
            PONE-D-15-30395
            10.1371/journal.pone.0134203
            4517912
            26218681
            778c02a8-6b59-46bc-b521-c046012dae45
            Copyright @ 2015

            This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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