Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2

Abstract

bPorcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. To date, viral proteins Cap, Rep, Rep=, and ORF3, encoded by the PCV2 genome, have been described. Here, transcription and translation of a novel viral gene within the PCV2 genome (designated ORF4) was determined and functionally analyzed in vitro and in vivo. Northern blot analysis indicated that the RNA transcribed from the ORF4 gene is about 180 bp in length and overlaps ORF3 in the same direction. Site-directed mutagenesis confirmed that the viral ORF4 protein is not essential for virus replication in PK-15 cells and in mice infected with an ORF4-deficient PCV2 (PCV2⌬). PCV2⌬ triggered higher activity levels of caspase-3 and -8 than wild-type PCV2 (wPCV2) in PK-15 cells. The antigenic epitopes of two mouse monoclonal antibodies (MAbs) raised against the viral ORF4 protein were mapped to the same 19KSSASPR25 peptide. Expression of ORF4 was confirmed using the specific MAbs in wPCV2-infected PK-15 cells and mice. Mice infected with PCV2⌬ had a higher serum viral load (genomic copies) and more severe lymphoid tissue damage in the spleen than those infected with wPCV2. Meanwhile, flow-cytometric analysis indicated that the PCV2⌬ infection caused a significant decrease of CD4 ؉ and CD8 ؉ T lymphocytes. Our results demonstrate that ORF4 is a newly discovered viral protein that is not essential for PCV2 replication but plays a role in suppressing caspase activity and regulating CD4؉ and CD8 ؉ T lymphocytes during PCV2 infection.

The ORF3 protein of porcine circovirus type 2 promotes secretion of IL-6 and IL-8 in porcine epithelial cells by facilitating proteasomal degradation of regulator of G protein signalling 16 through physical interaction

“…Among them, PCV2 ORF1 encodes a gene for virus replication, and ORF2 encodes a gene for capsid formation (Mankertz et al, 1998;Nawagitgul et al, 2000). ORF3 and ORF4 may regulate host immune responses by interacting with host proteins (Liu et al, 2005(Liu et al, , 2006(Liu et al, , 2007He et al, 2013). The function of PCV2 ORF3 may be to activate host cell signalling, whereas the function of PCV2 ORF4 may be to downregulate host cell signalling (Liu et al, 2007;He et al, 2013).…”

“…ORF3 and ORF4 may regulate host immune responses by interacting with host proteins (Liu et al, 2005(Liu et al, , 2006(Liu et al, , 2007He et al, 2013). The function of PCV2 ORF3 may be to activate host cell signalling, whereas the function of PCV2 ORF4 may be to downregulate host cell signalling (Liu et al, 2007;He et al, 2013). In particular, a previous experiment demonstrated that PCV2 infection in host cells activates NFkB by IkBa degradation (Wei et al, 2008).…”

“…Among PCV2 ORFs, ORF3 and ORF4 are not essential for replication of PCV2, but are associated with PCV2 pathogenicity (Liu et al, 2005(Liu et al, , 2006He et al, 2013). Previously, the interaction of PCV2 ORF3 with regulator of G protein signalling 16 (RGS16) has been reported (Timmusk et al, 2006).…”

A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

“…ORF1 encodes the Rep and Rep' proteins involved in viral replication (Cheung 2003). ORF3 (Liu et al 2005(Liu et al , 2006 and ORF4 (He et al 2013) encodes non-structural (NS) proteins related to viral pathogenesis. ORF2 encodes the major structural capsid protein (Cap, 30 kDa), which is also the main antigenic determinant of the virus (Nawagitgul et al 2000).…”

ORF2 protein of porcine circovirus type 2 promotes phagocytic activity of porcine macrophages by inhibiting proteasomal degradation of complement component 1, q subcomponent binding protein (C1QBP) through physical interaction

“…Among the four major ORFs of PCV2, ORF1, which is also termed the 'rep' gene, encodes the viral replicase and ORF2 (the 'cap' gene) encodes the capsid protein (Mankertz et al, 1998;Nawagitgul et al, 2000). ORF3 and ORF4 encode nonstructural genes that may modulate the phenotype of host cells (Liu et al, 2005(Liu et al, , 2006(Liu et al, , 2007He et al, 2013).…”