Baker C C, Phelps W C, Lindgren V, Braun M J, Gonda M A, Howley P M
J Virol. 1987 Apr;61(4):962-71. doi: 10.1128/JVI.61.4.962-971.1987.
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 full-length, 7.9-kb HPV-16 DNA; a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and a 10.5-kb clone generated by a 2.6-kb 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.
我们克隆并分析了人子宫颈癌细胞系SiHa和CaSki中存在的整合型人乳头瘤病毒16型（HPV-16）基因组。SiHa细胞系中的单个HPV-16基因组被克隆为一个10千碱基（kb）的HindIII片段。HPV-16基因组在碱基3132和3384处发生整合，E2和E4开放阅读框（ORF）被破坏。HPV-16序列额外缺失52个碱基对，使E2和E4 ORF融合。被破坏的E2 ORF的5'部分在相邻的人源右侧侧翼序列中立即终止。用原型HPV-16 DNA对该克隆的整合病毒基因组进行异源双链分析，未发现其他缺失、插入或重排。然而，对E1 ORF的DNA序列分析显示，在核苷酸1138处存在一个额外的鸟嘌呤，导致E1a和E1b ORF融合为一个单一的E1 ORF。对人源侧翼序列的序列分析显示，左侧连接处有一半的Alu序列，右侧侧翼区域有一个与人O重复序列高度同源的序列。对CaSki细胞系中三个最丰富的BamHI克隆的分析表明，这些克隆包括全长7.9 kb的HPV-16 DNA；由于长控制区缺失1.4 kb而产生的6.5 kb基因组；以及由3'早期区域的2.6 kb串联重复产生的10.5 kb克隆。这些HPV-16基因组在宿主染色体中以头对头、串联重复阵列的形式排列。转录分析显示，这两个人子宫颈癌细胞系中的HPV-16基因组均有表达，尽管表达水平差异显著。用亚基因组链特异性探针初步定位病毒RNA表明，病毒转录似乎主要来源于E6和E7 ORF。
