Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements
Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements
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DOI：
10.1128/jvi.02231-17
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发表时间：
2018-04-01
影响因子：
5.4
通讯作者：
Hirsch, Hans H.
中科院分区：
文献类型：
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作者：
Ajuh, Elvis T.;Wu, Zongsong;Hirsch, Hans H.
人类多瘤病毒（HPyV）DNA基因组包含三个区域，即早期病毒基因区（EVGR），编码调节性T抗原和一个microRNA，晚期病毒基因区（LVGR），编码结构性Vp衣壳蛋白，以及非编码控制区（NCCR）。NCCR具有病毒基因组复制的起点和双向启动子/增强子功能，其控制相对DNA链上的EVGR和LVGR表达。尽管主要相似，HPyV NCCR在长度，序列和结构上不同。为了在功能上比较HPyV NCCR，将来自人分离株的序列插入双向报告载体中，使用dsRed 2用于EVGR表达，使用绿色荧光蛋白（GFP）用于LVGR表达。将HPyV NCCR报告载体转染到人胚肾293（HEK 293）细胞中，并将流式细胞术标准化为原型BKPyV NCCR，揭示了EVGR表达水平的层次结构，其中MCPyV、HPyV 12和STLPyV NCCR赋予较强的水平，HPyV 6、HPyV 9和HPyV 10 NCCR赋予较弱的水平，而LVGR表达变化较小，并显示出相当的活性水平。转染表达猿猴病毒40（SV 40）大T抗原（LTag）的HEK 293 T细胞增加了大多数HPyV NCCR的EVGR表达，其与LTag结合位点的数量相关（斯皮尔曼r，0.625; P < 0.05），并在SV 40 LTag小干扰RNA（siRNA）敲低后降低。在表达同源MCPyV LTag的293 MCT细胞中，对两种不同的MCPyV NCCR特异性确认了LTag依赖性激活。来源于皮肤（A375）、宫颈（HeLaNT）、肺（A549）、脑（Hs 683）和结肠（SW 480）的不同细胞系中的HPyV NCCR表达证明宿主细胞特性显著调节基线HPyV NCCR活性，其与SV 40 LTag表达部分协同。发现临床上发生的HPyV 7 PITT-1和-2和HPyV 9 UF 1的NCCR序列重排与各自的HPyV原型相比增加EVGR表达，但这部分是宿主细胞类型特异性的。在这里，我们表明，HPyV NCCR不仅在序列长度，数量和LTag和共同的转录因子结合位点的位置不同，但也赋予双向病毒基因表达的差异。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著调节。含有缺失和插入的HPyV 7和HPyV 9 NCCR的临床序列变体与EVGR表达增加相关，类似于BKPyV和JCPyV重排，强调HPyV NCCR序列不仅是宿主细胞嗜性而且是致病性的主要决定因素。这些结果将有助于定义HPyV表面受体以外的次级HPyV细胞嗜性，以确定塑造病毒生命周期的关键病毒和宿主因素，并开发HPyV持久性和复制的临床前模型以及合适的抗病毒靶点。
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. LTagdependent 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.