STRUCTURAL AND TRANSCRIPTIONAL ANALYSIS OF HUMAN PAPILLOMAVIRUS TYPE-16 SEQUENCES IN CERVICAL-CARCINOMA CELL-LINES
STRUCTURAL AND TRANSCRIPTIONAL ANALYSIS OF HUMAN PAPILLOMAVIRUS TYPE-16 SEQUENCES IN CERVICAL-CARCINOMA CELL-LINES
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DOI：
10.1128/jvi.61.4.962-971.1987
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发表时间：
1987-04-01
影响因子：
5.4
通讯作者：
HOWLEY, PM
中科院分区：
文献类型：
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作者：
BAKER, CC;PHELPS, WC;HOWLEY, PM
我们克隆和分析了整合的人乳头瘤病毒16型（HPV-16）基因组，存在于人宫颈癌细胞系SiHa和CaSki。将SiHa系中的单个HPV-16基因组克隆为10-kb HindIII片段。HPV-16基因组的整合发生在碱基3132和3384处，并破坏了E2和E4开放阅读框（ORF）。HPV-16序列的另外52个碱基对缺失融合了E2和E4 ORF。被破坏的E2 ORF的5“部分立即终止于连续的人右侧侧翼序列。异源双链分析，该克隆的整合病毒基因组与原型HPV-16 DNA显示没有其他的缺失，插入，或重排。然而，E1 ORF的DNA序列分析显示在核苷酸1138处存在额外的鸟嘌呤，导致E1 a和E1 b ORF融合成单个E1 ORF。人类侧翼序列的序列分析显示，一半的Alu序列在左侧交界处和一个序列高度同源的人类O重复的右侧区域。对来自CaSki系的三个最丰富的BamHI克隆的分析表明，这些克隆由（i）全长7.9-kb HPV-16 DNA;（ii）由长控制区的1.4-kb缺失产生的6.5-kb基因组;和（iii）由3“早期区的2.6-lb串联重复产生的10.5-kb克隆组成。这些HPV-16基因组在宿主染色体中排列为首尾相连的串联重复阵列。转录分析揭示了HPV-16基因组在这两种宫颈癌细胞系中的表达，尽管水平显著不同。用亚基因组链特异性探针对病毒RNA的初步作图表明，病毒转录似乎主要来自E6和E7 ORF。
We cloned and analyzed the integrated human papillomavirus type 16 (HPV-16) genomes that are present in the human cervical carcinoma cell lines SiHa and CaSki. The single HPV-16 genome in the SiHa line was cloned as a 10-kilobase (kb) HindIII fragment. Integration of the HPV-16 genome occurred at bases 3132 and 3384 with disruption of the E2 and E4 open reading frames (ORFs). An additional 52-base-pair deletion of HPV-16 sequences fused the E2 and E4 ORFs. The 5'' portion of the disrupted E2 ORF terminated immediately in the contiguous human right-flanking sequences. Heteroduplex analysis of this cloned integrated viral genome with the prototype HPV-16 DNA revealed no other deletions, insertions, or rearrangements. DNA sequence analysis of the E1 ORF, however, revealed the presence of an additional guanine at nucleotide 1138, resulting in the fusion of the E1a and E1b ORFs into a single E1 ORF. Sequence analysis of the human flanking sequences revealed one-half of an Alu sequence at the left junction and a sequence highly homologous to the human O repeat in the right-flanking region. Analysis of the three most abundant BamHI clones from the CaSki line showed that these consisted of (i) full-length, 7.9-kb HPV-16 DNA; (ii) a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and (iii) a 10.5-kb clone generated by a 2.6-lb tandem repeat of the 3'' early region. These HPV-16 genomes were arranged in the host chromosomes as head-to-tail, tandemly repeated arrays. Transcription analysis revealed expression of the HPV-16 genome in each of these two cervical carcinoma cell lines, albeit at significantly different levels. Preliminary mapping of the viral RNA with subgenomic strand-specific probes indicated that viral transcription appeared to be derived primarily from the E6 and E7 ORFs.