Nonstructural proteins of Torque teno sus virus 2 from O2AUG: Prediction to experimental validation

The expression profiles of nonstructural proteins (NSPs) in Torque teno sus virus 2 (TTSuV2) have not yet been characterized. Here, we determined the coding sequences of the TTSuV2 NSPs ORF2, ORF2/2, and ORF2/2/3 by overlapping polymerase chain reaction (PCR) and subsequent expression in bacterial and mammalian cells. We generated two monoclonal antibodies (mAbs), 2E5 and 6F8, from mice immunized with mixed Escherichia coli expressing His-tagged ORF2 and ORF2/2. Enzyme-linked immunosorbent assay (ELISA) and western blot analysis revealed that, 2E5 mAbs bound to the consensus sequences of ORF2, ORF2/2, and ORF2/2/3, while 6F8 recognized the common sequences of ORF2/2 and ORF2/2/3. Immunofluorescence assay (IFA) revealed that ORF2 was localized in the cytoplasm, ORF2/2, in the nucleus but not the nucleolus, and ORF2/2/3, in the peri-nuclear region. To identify the expression profiles of TTSuV NSPs, a circular TTSuV2_ZJ (GenBank: KF660540) genomic DNA clone was constructed and transfected into HEK293T and HeLa cells. Splicing mRNAs and the expression and localization of ORF2/2 and ORF2/2/3 were identified by RT-PCR, western blot analysis, and IFA, respectively. However, ORF2 was not detected either at the RNA or protein level. Our study is the first to provide experimental evidence of the existence of ORF2/2 and ORF2/2/3 at the protein level. Moreover, the mAbs have potential applications in future research on TTSuV2 viral protein function and diagnosis of related diseases.