In vitro evaluation of bidirectional transcription levels of five types of non-coding control regions of Merkel cell polyomavirus

Abstract

Merkel cell polyomavirus (MCPyV) has been identified as the causative agent of Merkel cell carcinoma, and its non-coding control region (NCCR) has been demonstrated to play a critical role in regulating viral transcription. While NCCR variants exist, their comparative impact on bidirectional promoter activity remains poorly characterized. The present study conducted an in vitro evaluation of bidirectional transcription levels of five major MCPyV NCCR types (I, IIa-1, IIa-2, IIb, IIc). The NCCRs were subsequently cloned into a bidirectional reporter vector, which expresses green (EGFP, early) and red (RFP, late) fluorescent proteins. Subsequent to transfection into HEK293 cells, promoter activity was quantitatively analyzed via fluorescence imaging and flow cytometry. Bioinformatic analysis revealed high sequence similarity (> 94%) among the five NCCRs and predicted conserved transcription factor binding sites. The results indicated that all the variants exhibited stronger late promoter activity compared with the early promoter activity (p < 0.01). These observations are in alignment with the established biology of MCPyV. However, no statistically significant differences in the early/late transcription ratio or overall fluorescence intensity were observed between the different NCCR types under these conditions. These findings suggest that the core promoter function is conserved among these major NCCR variants in this model system. This study provides a foundational comparison of MCPyV NCCR activity, highlighting the need for further investigation in more physiologically relevant models to understand how NCCR diversity may influence viral pathogenesis in vivo. Moreover, incorporating models of viral genome integration is essential to understand mechanism of MCPyV carcinogenesis and viral-host interaction.

Graphical abstract

Introduction

Merkel cell polyomavirus (MCPyV) is an established oncogenic pathogen implicated in the pathogenesis of Merkel cell carcinoma (MCC), a rare but aggressive neuroendocrine skin cancer with rising global incidence [1, 2]. Since its serendipitous discovery in 2008 through digital transcriptome subtraction, MCPyV has been detected in approximately 80% of MCC tumors, with clonal viral integration into the host genome recognized as a critical oncogenic driver [3]. In contrast to other polyomaviruses, MCPyV has been observed to exhibit a distinct tumorigenic mechanism, whereby persistent expression of viral early genes—specifically the truncated large T-antigen (LT) and small T-antigen (sT)—dysregulates cell cycle control and apoptotic pathways [4–6]. It is imperative to note that the non-coding control region (NCCR) of MCPyV orchestrates this transcription programme, which is subject to stringent regulation. This region functions as the master switch for viral replication and persistence [7–9].

The NCCR, located between the early and late gene regions, contains the origin of replication (ori) and bidirectional promoter-enhancer elements that govern temporally distinct phases of transcription [8]. Early transcription, directed towards T-antigen expression, facilitates viral genome replication and host cell manipulation, while late transcription produces structural capsid proteins (VP1/VP2) that are essential for virion assembly [4, 10]. In MCPyV-positive MCC tumors, the NCCR frequently undergoes structural rearrangements during viral integration, resulting in sequence diversity across isolates. To date, five major NCCR types (Ⅰ, Ⅱa-1, Ⅱa-2, Ⅱb, and Ⅱc) have been characterized based on their variations in core promoter elements, enhancer motifs, and tandem duplication [7, 11]. These structural polymorphisms have the potential capacity to exert a critical influence on promoter strength, cell tropism, and oncogenic potential by means of altering transcription factor binding affinities or chromatin accessibility [12, 13].

Notwithstanding the pivotal role of the NCCR, comparative analyses of its impact on early versus late transcriptional efficiency across naturally occurring variants remain fragmented. Previous studies have primarily focused on single NCCR types or correlative genomic surveys in clinical samples, thereby neglecting systematic in vitro quantification of bidirectional promoter activity [7, 14]. This discrepancy is of significance as it suggests that transcriptional dysregulation, whether through NCCR mutations, epigenetic silencing, or integration-induced disruptions, may underpin viral latency, reactivation, or oncogenic progression [13, 15, 16]. For instance, enhanced early-phase transcription could potentiate T-antigen-driven transformation, while suppressed late-phase expression might facilitate immune evasion by limiting antigen presentation [2, 17, 18]. Furthermore, the interplay between NCCR architecture and cellular transcription factors in different host environments remains poorly resolved, hindering mechanistic understanding of MCPyV tropism and pathogenesis.

This study addresses these knowledge gaps through a comprehensive in vitro evaluation of the transcriptional kinetics driven by five predominant MCPyV NCCR variants (Ⅰ, Ⅱa-1, Ⅱa-2, Ⅱb, and Ⅱc). Utilizing luciferase reporter assays in human cell lines modelling cutaneous and neuronal lineages, we quantify real-time activity of both early and late promoters across NCCR types under identical experimental conditions. It is hypothesized that structural heterogeneity within NCCRs is responsible for significant differences in promoter strength and temporal dynamics associated with transcription factor binding sites (TFBSs), thereby influencing viral fitness and pathogenic outcomes [4]. The present study aims to elucidate the regulatory mechanisms critical for MCPyV persistence and oncogenesis by delineating how specific NCCR motifs modulate transcription. Such insights could identify novel targets for disrupting viral replication or inform prognostic biomarkers based on NCCR signatures in MCC.

In summary, the MCPyV NCCR represents a genomic nexus where minor sequence variations may exert major biological consequences. This study provides the first comparative experiment for understanding how natural NCCR diversity sculpts the transcriptional landscape of an oncogenic polyomavirus. Our findings indicate that while all five NCCR variants consistently promoted significantly stronger late viral gene (LVG) expression relative to early viral gene (EVG) expression, no statistically significant differences in bidirectional transcription levels were observed among the different NCCR types under the tested conditions. This suggests that these NCCR types may confer similar basal promoter activities.

Methods and materials

1. TFBS prediction

Two different online prediction programs were used with moderate stringency to locate major TFBSs in silico. Software for searching transcription factor binding sites (including TATA boxes, GC boxes, CCAAT boxes, transcription initiation sites (TIS)). This tool uses weight matrix in transcription factor database TRANSFAC R.3.4 developed by Dr. Wingender et al., and the cut-offs originally estimated by Tsunoda and Takagi (https://tfbind.hgc.jp/). The potential TFBSs identified were further predicted using ConTra v3 (http://bioit2.irc.ugent.be/contra/v3/), a tool can analyze promoter regions, 5΄-UTRs, 3΄-UTRs and introns or any other genomic region of interest. Then the predicted TFBSs were visualized in NCCR genes (positive and negative strand).

• 2. In silico sequence analysis and gene synthesis of MCPyV NCCRs

In this study, the NCCR classification criteria was based on the previous study by Hashida et al. [11]. The structural validation of five representative Merkel cell polyomavirus (MCPyV) non-coding control region (NCCR) subtypes I, IIa-1, IIa-2, IIb and IIc was performed through in silico sequence analysis. Synthetic gene fragments were designed to incorporate Kozak sequences (to enhance translational efficiency) flanking bidirectional fluorescent reporters: upstream red fluorescent protein (RFP) and downstream enhanced green fluorescent protein (EGFP). Each NCCR sequence was verified using NCBI BLAST to confirm fidelity to target viral regulatory regions, with specific attention to structural variations defining the five subtypes. Sequence annotations explicitly demarcated functional elements: RFP (red), NCCR (black), EGFP (green), and Kozak sequences (yellow), ensuring precise downstream cloning.

• 3. Plasmid construction and purification

All reporter plasmids were derived from the phRG (R2.2) backbone (Novopro V005502) (Fig. 1). First, five RFP-Kozak-NCCR-Kozak-EGFP fragments (I-NCCR, IIa-1-NCCR, IIa-2-NCCR, IIb-NCCR, IIc-NCCR) were synthesized in vitro and cloned into a T-vector for intermediate storage. The plasmid was linearized by PCR amplification targeting the region between its two poly(A) signals. The initial construct (phRG-I-NCCR) was generated via homologous recombination, integrating the RFP-Kozak-I-NCCR-Kozak-EGFP fragment into linearized phRG using overlapping homologous arms. Subsequent constructs (IIa-1, IIa-2, IIb, IIc) were created by replacing the I-NCCR insert in linearized phRG-I-NCCR with corresponding synthetic fragments through homologous recombination. All experimental plasmids were purified and validated by Sanger sequencing.

Fig. 1.

Plasmid construction of 5 types of NCCRs

• 4. Transfection and fluorescence imaging

HEK293 cells were cultured in DMEM-H supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under 5% CO₂. For transfection, cells at 60–80% confluency in 12-well plates were transfected with 1 µg of phRG-NCCR plasmids or mock-treated (control) using Lipofectamine 3000, following the manufacturer’s protocol. At 48 h post-transfection, live-cell imaging was performed using an Mshot MZX81 stereo fluorescence microscope. RFP (ex: 555 nm) and EGFP (ex: 488 nm) signals were captured from five random fields per well. Fluorescence intensities were quantified using ImageJ software, with the RFP and EGFP ratio calculated to assess NCCR-mediated bidirectional transcriptional activity. Each ratio was obtained by the following formula:

• 5. FACS-based bidirectional reporter assay

For the bidirectional reporter assay, HEK293 cells were seeded in 12-well plates and transfected as described above. After 48 h post-transfection, cells were rinsed once with PBS-2.5 mM EDTA and then detached, and resuspended in 1 mL PBS-2.5 mM EDTA. Directly before each measurement, samples were stained with DAPI (final concentration, 1 ng/mL) to exclude dead cells and analyzed on a BD Fortessa flow cytometer (BD, Franklin Lakes, NJ, USA). Instrument parameters were optimized as follows: forward/side scatter voltages at 220 V; EGFP, excitation at 488 nm (blue laser) and emission at 530/30 nm at a detector voltage of 373 V; RFP, excitation at 561 nm (yellow-green laser) and emission at 586/15 nm at a detector voltage of 500 V; DAPI, excitation at 405 nm (violet laser) and emission at 450/50 nm at a detector voltage of 302 V. In order to calculate the weighted mean fluorescence intensity (MFI) for red (early) and green (late) expression, the cell number (N) and mean fluorescence (I) of quadrant 1 (Q1) (red cells), Q2 (red and green cells), and Q4 (green cells) were inserted into the following formulas:

• 6. Statistics

All experiments were performed in triplicate (n = 3). Fluorescence intensity data were expressed as mean ± standard deviation (SD). Statistical comparisons across NCCR subtypes were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. A threshold of p < 0.05 was considered statistically significant. Analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA), with graphical representations generated to illustrate subtype-specific regulatory differences.

Results

Phylogenetic analysis and TFBS prediction of NCCR

To explore the diversity of NCCR genes, representative MCPyV strains were selected and NCCR-based phylogenetic tree were built. According to NCCR diversity, MCPyV were classified into 5 major types, I, IIa-1, IIa-2, IIb and IIc (Fig. 2A). Type I (I-NCCR) contains a tandem repeat, and there is “GTTGA” insertion and “CCTTTTGTT” deletion in IIa-1-NCCR and IIb-NCCR, respectively (Fig. 2B). In spite of this, the five types of NCCR share 94.2%−98.8% similarity. To investigate the role of TFBS in different types of NCCRs, two different computer prediction programs were used with moderate stringency to locate major TFBS in silico. Due to high similarity of gene sequences of NCCR, approximately 18 TFBSs were consistently predicted by both programs and included multiple sites for some factors like Sp1 or NF1 (Fig. 3).

Fig. 2.

Phylogenetic analysis of 5 types of NCCR of representative MCPyV strains. A phylogenetic tree; B. partial sequences of 5 types of NCCR. Core variable regions (insertion or deletion) were highlighted. LVGR: late viral gene region; EVGR: early viral gene region

Fig. 3.

In silico prediction of common transcription factor- and LTag-binding sites in the NCCRs of 5 genotypes of MCPyV. The direction of EVGR and LVGR transcription and the start codon (ATG) are depicted in green and red arrows, respectively. Binding sites are indicated as follows: green triangles, LTag; red triangles, Sp1; yellow triangles, NF-kB; black square, TATA box; light purple triangles, TBP1; blue triangles, NF1; purple triangles, Ets1; dark blue triangles, Spi-B; pink rectangle, origins of replication (ori); gray rectangle, tandem repeat; TI-E1, transcriptional initiation sites (early stage); TI-L1, transcriptional initiation sites (late stage) [19]; ins, insertion; del, deletion

In vitro bidirectional transcription analysis

To experimentally quantify the effect on viral gene expression, the 5 types of NCCRs were cloned into the reporter gene vector pHRG-2.2, which, as reported earlier [8], recapitulates the bidirectional organization of the polyomavirus DNA genome and allows monitoring of NCCR-directed EVGR and LVGR expression by the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), respectively. The corresponding NCCR reporter constructs were transfected into the embryonic kidney cell line HEK293. As shown in Fig. 4A, all the five types of NCCRs conferred strong LVGR and weak EVGR expression, as indicated by the mean fluorescence intensity (MFI) determined by fluorescence imaging (Fig. 4B). Despite the higher LVGR expression in all types of NCCRs compared with the EVGR expression, there was no significant difference of bidirectional transcription levels among these groups (Fig. 4B and C). Results of flow cytometry quantification also indicated higher LVGR expression (Fig. 5A and B), while IIc-NCCR group had the highest double-positive cells (22.45%) compared with the other four groups (16.82%−20.63%) (Fig. 5A).

Fig. 4.

Phenotypic characterization of NCCR bidirectional reporter constructs transfected into HEK293 cells. (A) The indicated constructs were transfected into HEK293 cells, and fluorescence images (20×objective) were taken 2 days post transfection (dpt); (B) Mean fluorescence intensity (MFI); (C) Fluorescence intensity ratio (FIR) of EGFP (early) and RPF (late). *, p < 0.05; **, p < 0.01; ns, non-significant; Bar, mean ± SD. Quantification results are from 3 independent replicates

Fig. 5.

Flow cytometry and quantification of indicated NCCRs cloned into the bidirectional reporter vector pHRG 2.2. (A) Flow cytometry analysis. x axis, EGFP fluorescence; y axis, RFP fluorescence. For each measurement, 5,000 transfected (fluorescent) cells were gated except for mock transfection, where 10,000 untransfected cells were measured. Numbers in the quadrants (Q) are absolute numbers of detected cells and percentages with respect to the gated cells. FITC-A, 488 nm excitation wavelength; PE-CF594-A, 561 nm emission wavelength. (B) Weighted mean fluorescence intensity (MFI). Red bars, sum of red cells (Q1 + Q2); green bars, sum of green cells (Q2 + Q4). (C). RFP/EFGP ratio. For each measurement, the weighted MFI was calculated (see Materials and Methods)

Discussion

This study presents an in vitro comparison of the bidirectional transcriptional activity driven by five major MCPyV NCCR variants (I, IIa-1, IIa-2, IIb, and IIc). Using a dual-fluorescence reporter system in HEK293 cells, we quantified the relative strengths of the early and late promoters across these naturally occurring NCCR types. Our findings indicate that while all five NCCR variants consistently promoted stronger LVGR expression relative to EVGR, no statistically significant differences in bidirectional transcription levels were observed among the different NCCR types under the tested conditions. This suggests that, despite their structural variations, these NCCR subtypes may confer similar basal promoter activities in this embryonic kidney cell model.

The conserved high LVGR/EVGR ratio across all NCCRs basically consists with the previous study, in which natural NCCRs consistently directed higher LVGR expression in vitro [20]. However, NCCR rearrangement might alter the landscape of bidirectional transcription levels. For instance, NCCR rearrangements of BKV significantly increased early gene expression and decreased late gene expression, irrespective of insertion or deletion architectures [21]. In this study, all the five natural NCCR types of MCPyV directed higher late gene expression, suggesting different mechanisms of viral latency, (re)activation and immune escape among human PyVs. The slightly higher proportion of double-positive cells observed for the IIc-NCCR variant in flow cytometry may hint at subtle regulatory differences, possibly due to its unique sequence architecture, though this did not translate into significant differences in overall fluorescence intensity. The in silico prediction of TFBSs revealed a high degree of conservation among the five NCCRs, which may explain the lack of pronounced functional divergence in the assay. Key factors such as Sp1, NF-1, and NF-κB binding sites were consistently present, suggesting redundant or compensatory mechanisms that maintain promoter output despite structural variations. As demonstrated in a preceding study by Bethge et al., Sp1 was found to be instrumental in the regulation of bidirectional BKPyV gene expression [13]. These findings illuminate the mechanisms by which TFBSs regulate bidirectional gene expression and their roles in viral pathology, offering novel perspectives for the development of targeted anti-viral therapies in the future. Despite the bidirectional regulatory role of TFBSs, circMCV-T has been identified from MCPyV genome to act as a competitive inhibitor of miR-M1, partially alleviating miR-M1–mediated repression of T antigen transcripts and thereby modulating viral replication [22]. A similarly located circRNA was also identified in rat polyomavirus 2 (RatPyV2) [23], suggesting a conserved regulatory mechanism among polyomaviruses. These findings reveal a dynamic circRNA/miRNA/mRNA network that fine-tunes viral gene expression and replication.

However, this study has several limitations. First, the use of only HEK293 cells, an embryonic kidney line, may not fully recapitulate the transcriptional environment of MCPyV’s natural target cells, such as cutaneous or neuroendocrine lineages. Cell-type-specific transcription factors and epigenetic landscapes likely influence NCCR activity, and their absence in this model may mask variant-specific behaviors. Second, the experimental setup focused on transient transfection under standard conditions, which does not account for potential effects of viral integration, chromatinization, or long-term regulation that occur in natural infections or MCC tumors. incorporating models of viral genome integration will certainly help to understand these effects. Third, while fluorescence-based reporters provide robust quantitative data, they do not capture post-transcriptional or translational regulation that may affect functional T-antigen or capsid protein levels. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties [20].

Future studies should employ more physiologically relevant cell models, including Merkel cell lines or primary keratinocytes, and consider using integrated reporter systems or viral context models to better mimic the native state of MCPyV infection. Additionally, exploring the role of specific TFBS mutations or chromatin immunoprecipitation assays could help dissect the mechanistic basis of NCCR-mediated transcription.

In conclusion, our work provides a foundational in vitro profile of MCPyV NCCR’s bidirectional regulating activity and underscores the need for more complex models to fully understand how NCCR diversity influences viral gene expression and pathogenesis. For instance, incorporating models of viral genome integration or using reporter constructs that can replicate or episomally maintain (to mimic viral DNA) might reveal additional differences in promoter regulation. However, exploration on both NCCR/TFBS and circRNA/miRNA/mRNA networks is also essential to comprehensively understand the early and late transcription regulation mechanisms of virus-host interactions and viral pathogenicity.

Author contributions

Conceptualization: X.Z. and X.T; Resources and experiment: M.Z., X.Q., X.W., H.M., L.L. and Y.A.; Funding Acquisition: X.Z.; Writing-Original draft: X.Z.; Writing-Review and Editing: all authors. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Jiangxi Provincial Natural Science Foundation (20242BAB25355).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.