Novel Human Polyomavirus non-coding control regions differ in bi-directional gene expression according to host cell, large T-antigen expression, and clinically occurring rearrangements

Human polyomavirus (HPyV) DNA genomes contain three regions denoted early viral gene region (EVGR) encoding the regulatory T-antigens and one microRNA, late viral gene region (LVGR) encoding the structural Vp capsid proteins, and non-coding control region (NCCR). The NCCR harbours the origin of viral genome replication and bi-directional promoter/enhancer functions governing EVGR- and LVGR-expression on opposite DNA strands. Despite principle similarities, HPyV-NCCRs differ in length, sequence, and architecture. To functionally compare HPyV-NCCRs, sequences from human isolates were inserted into a bi-directional reporter vector using dsRed2 for EVGR- and GFP for LVGR-expression, respectively. Transfecting HPyV-NCCR reporter vectors into human embryonic kidney (HEK)-293 cells and flow cytometry normalized to archetype BKPyV-NCCR revealed a hierarchy of EVGR-expression levels with MCPyV-, HPyV12, and STLPyV-NCCRs conferring stronger, and HPyV6-, HPyV9-, and HPyV10-NCCRs weaker levels, while LVGR-expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing SV40-large T-antigen (LTag) increased EVGR-expression for most HPyV-NCCRs, which correlated with the number of LTag-binding sites (Spearman r 0.625; p