Krϋppel-like factor 12a and 12b inhibit Cyvirus cyprinidallo3 replication by repressing viral ORF8 promoter activity

Cyvirus cyprinidallo3, also known as Cyprinid herpesvirus 3 (CyHV-3), is a highly infectious and lethal DNA virus causing considerable economic losses in the common carp and koi farming sectors. The pathogenesis caused by CyHV-3 is still poorly understood and effective control measures remain unavailable. Previously, we found that the immediate early (IE) gene ORF8 promoter of CyHV-3 exhibits the highest activity in the host cells. However, the transcriptional regulatory mechanism of the viral ORF8 promoter remains unknown. Herein, the potential transcription factors Krϋppel-like factors (KLFs) 12a and 12b from common carp (Cyprinus carpio) targeting the ORF8 promoter were characterized. KLF12a and KLF12b were widely expressed in various tissues of common carp and the corresponding proteins are primarily localized to the nucleus of common carp brain cell line (CCB). Particularly, KLF12a and KLF12b overexpression significantly suppressed the ORF8 promoter activity and deletion of the ORF8 promoter region containing KLF12 binding sites resulted in a marked decrease in activity. Continuous deletion of the viral ORF8 promoter at 75 bp intervals further revealed that −203∼-53 region is strongly required for the viral ORF8 promoter activity. More importantly, both KLF12a and KLF12b overexpression markedly reduced viral genome copies number, and virions in CCB cells. Taken together, Cyprinus carpio KLF12a and KLF12b inhibit CyHV-3 replication by repressing viral ORF8 promoter activity, which provides a foundation for further investigation into the transcriptional regulatory mechanisms of IE genes in CyHV-3.