Characterization of a Previously Unidentified Viral Protein in Porcine Circovirus Type 2-Infected Cells and Its Role in Virus-Induced Apoptosis

Abstract

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs. In this study, transcription and translation of a novel viral gene (termed ORF3 here) was detected during productive infection of PCV2 in PK15 cells. The results of infection with ORF3-deficient PCV2 by site-directed mutagenesis indicated that the protein is not essential for viral replication. To investigate the underlying mechanism of cell death caused by replication of PCV2, apoptosis characterized by chromosomal condensation and fragmentation, formation of apoptotic bodies, and significant increase in hypodiploids were detected in infected cells. We further demonstrated that PCV2-induced apoptosis required the activation of caspase-8 but not caspase-9. The activation of caspase-8 results in the activation of caspase-3 as shown by an increase in the cleavage of the caspase substrate in the infected cells. To determine whether ORF3 protein could trigger apoptosis, ORF3 as well as ORF1 and ORF2 genes were transiently expressed in PK15 and Cos-7 cells for apoptotic activity assay. Transfection of cells with the ORF3 alone induced apoptosis using a pathway similar to that described in the context of viral infection. This is further confirmed by a significant decrease in apoptotic activity of infected cells in the absence of the ORF3 expression, suggesting that the protein plays a major role in the induction of virus-induced apoptosis. Altogether, these results indicate that ORF3 is a novel PCV2 protein that is not essential for viral replication in cultured cells but is involved in PCV2-induced apoptosis by activating caspase-8 and caspase-3 pathways.

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).…”

“…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).…”

Development and evaluation of a recombinant DNA vaccine candidate expressing porcine circovirus 2 structural protein

“…The available vaccine formulations consist of inactivated virus (Castro et al 2007), Cap protein expressed in baculovirus system (Kolb 2007) and chimeric virus containing the portion of the immunogenic cap gene of PCV2 inserted into the PCV1 genome (Fenaux et al 2004). The PCV2 genome contains three major open reading frames; ORF1 encoding Rep and Rep' proteins involved in viral replication (Mankertz et al 1998); ORF2 encoding the capsid protein (Cap) (Nahagitgul et al 2000), major structural and immunogenic protein of PCV2; and ORF3 encoding a protein involved in virus induced apoptosis (Liu et al 2005). Cap protein has been reported to induce protective responses in pigs injected with baculovirusexpressed product or prepared ORF2 DNA (Blanchard et al 2003, Kamstrup et al 2004.…”