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 r, 0.625; P < 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.

IMPORTANCE 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.

人多瘤病毒（HPyV）DNA基因组包含三个区域，分别表示编码调节性T抗原和一个microRNA的早期病毒基因区域（EVGR）和编码结构性Vp衣壳蛋白的非病毒性对照基因（晚期病毒基因区域（LVGR））区域（NCCR）。NCCR具有病毒基因组复制和双向启动子/增强子功能的起源，这些功能控制着相反DNA链上的EVGR和LVGR表达。尽管主要相似之处，但HPyV NCCR的长度，序列和结构均不同。为了在功能上比较HPyV NCCR，使用dsRed2进行EVGR表达，并使用绿色荧光蛋白（GFP）进行LVGR表达，将来自人类分离株的序列插入双向报告载体。将HPyV NCCR报告载体转染到人类胚胎肾293（HEK293）细胞中，并将流式细胞仪标准化为原型BKPyV NCCR，揭示了EVGR表达水平的层次结构，MCPyV，HPyV12和STLPyV NCCR的水平较高，而HPyV6，HPyV9和HPyV10的NCCR水平较弱，而LVGR的表达变化较小，并显示出可比的活动水平。表达猿猴病毒40（SV40）大T抗原（LTag）的HEK293T细胞的转染提高了大多数HPyV NCCR的EVGR表达，这与LTag结合位点的数量有关（Spearman'sr为0.625；P <0.05），并在SV40 LTag小干扰RNA（siRNA）敲低后降低。在表达同源MCPyV LTag的293MCT细胞中，针对两种不同的MCPyV NCCRs明确证实了LTag依赖性激活。HPyV NCCR在源自皮肤（A375），子宫颈（HeLaNT），肺（A549），脑（Hs683）和结肠（SW480）的不同细胞系中的表达表明，宿主细胞特性显着调节了基线HPyV NCCR活性，这部分协同作用带有SV40 LTag表达式。与相应的HPyV原型相比，发现HPyV7 PITT-1和-2以及HPyV9 UF1的临床发生的NCCR序列重排增加了EVGR表达，但这部分是宿主细胞类型特异性的。

重要性HPyV NCCR整合了有关宿主细胞特异性，持久性，病毒复制和疾病的基本病毒功能。在这里，我们表明，HPyV NCCRs不仅在序列长度，数量以及LTag和常见转录因子结合位点的位置上不同，而且在双向病毒基因表达方面也带来了差异。重要的是，EVGR报告基因的表达受到LTag表达和宿主细胞特性的显着调节。与BKPyV和JCPyV重排相似，含有缺失和插入的HPyV7和HPyV9 NCCR的临床序列变异与增加的EVGR表达相关，强调HPyV NCCR序列不仅是宿主细胞嗜性的主要决定因素，而且是致病性的主要决定因素。这些结果将有助于确定HPyV表面受体以外的继发性HPyV细胞向性，