Interferon-mediated enhancement of in vitro replication of porcine circovirus type 2 is influenced by an interferon-stimulated response element in the PCV2 genome.
Porcine circovirus type 2 (PCV2) is an economically important swine pathogen. It has been shown that treatment of PCV2-infected cells with interferon gamma (INF-gamma) or alpha (IFN-alpha) increases PCV2 replication in vitro, and that an interferon-stimulated response element (ISRE)-like sequence was identified in the PCV2 genome. To determine if the ISRE is involved in viral response to IFNs, several ISRE mutants of PCV2 were created by serial mutations of the ISRE sequence. Treatment of ISRE mutants-infected cells with IFN-gamma or IFN-alpha showed a progressive diminishment in the enhanced viral replication in correlation with increasing alterations to the ISRE sequence. To determine if the ISRE was necessary and sufficient for IFN-mediated enhancement of PCV2 replication, DNA fragments spanning the ISRE-containing promoter region of the rep gene from wildtype and ISRE-mutant PCV2 were tested in a luciferase reporter gene system. 3D4/31 cells transfected with reporter gene constructs were treated with IFN-gamma or IFN-alpha, respectively. The results showed that the untreated controls for both ISRE-mutant and wildtype PCV2 had higher levels of luciferase reporter activity than IFN-treated samples, indicating that, when removed from the context of whole viral genome, the ISRE-like activity of the sequence was lost. Furthermore, treatment with IFNs diminished the promoter activity regardless of the mutation, suggesting that other cis elements in the viral genome may be required for regulating the ISRE-mediated gene transcription. In conclusion, the PCV2 ISRE, when present in the context of intact virus but not in isolation, influences the interferon-mediated enhancement of PCV2 replication in vitro.
Ramamoorthy
S
S
Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0913, USA.
Huang
F F
FF
Huang
Y W
YW
Meng
X J
XJ
D000818:Animals; D019677:Artificial Gene Fusion; D017925:Circovirus / Q000235:genetics / Q000276:immunology* / Q000502:physiology*; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D016898:Interferon-alpha / Q000378:metabolism*; D007371:Interferon-gamma / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020218:Response Elements; D013552:Swine; D015533:Transcriptional Activation; D014779:Virus Replication
D004279:DNA, Viral; D016898:Interferon-alpha; D007371:Interferon-gamma; D008156:Luciferases