Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements

Abstract

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's , 0.625; < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific. HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

Characterization of six Merkel cell polyomavirus‐positive Merkel cell carcinoma cell lines: Integration pattern suggest that large T antigen truncating events occur before or during integration

“…In one case, such a deletion which was present both in an episomal and integrated viral genome was shown to confer decreased viral replication capacity but increased T‐antigen expression . Similarly, increased T‐antigen expression has also been described for clinically occurring NCCR rearrangements in HPyV7 and HPyV9 . Only in one cell line (MKL‐2) a deletion in the NCCR region was present without having an obvious effect on TA expression, while the NCCRs of all others matched described wild‐type sequences (Fig.…”

“…43 Similarly, increased T-antigen expression has also been described for clinically occurring NCCR rearrangements in HPyV7 and HPyV9. 45 Only in one cell line (MKL-2) a deletion in the NCCR region was present without having an obvious effect on TA expression, while the NCCRs of all others matched described wild-type sequences (Fig. S3).…”

Promoter activity of Merkel cell Polyomavirus variants in human dermal fibroblasts and a Merkel cell carcinoma cell line

“…Yet, in another study by Ajuh and co-workers, the authors showed that LT trans-activated the MCPyV R17b (= consensus) early and late promoter in HEK293T cells [29]. The discrepancy between their results and the findings by Kwun et al and us can be explained by the use of different LT. HEK293T cells express both SV40 LT and sT, but not MCPyV LT [29]. Moreover, the possible contribution of sT in the trans-activation of the early and late MCPyV promoters in these cells was not investigated.…”

“…Kwun et al found that MCPyV LT repressed early and late promoter activity of MCPyV isolate MCC339 in HEK293 cells, but they did not examine the effect of LT in MCC13 or HDF cells [42]. Yet, in another study by Ajuh and co-workers, the authors showed that LT trans-activated the MCPyV R17b (= consensus) early and late promoter in HEK293T cells [29]. The discrepancy between their results and the findings by Kwun et al and us can be explained by the use of different LT. HEK293T cells express both SV40 LT and sT, but not MCPyV LT [29].…”

“…Mutations in the non-coding control region (NCCR) of human polyomaviruses like BKPyV, JCPyV, KIPyV, HPyV7, HPyV9 and HPyV12 have an impact on the transcriptional activity of the promoter, and may affect the virulence of the virus [24][25][26][27][28][29][30][31][32][33]. Whether changes in the NCCR of MCPyV have an effect on the promoter activity, and have pathogenic consequences, has not been investigated.…”