Brown D R, McClowry T L, Sidner R A, Fife K H, Bryan J T
Department of Medicine, Indiana University School of Medicine, Indianapolis, Ind., USA.
Intervirology. 1998;41(1):47-54. doi: 10.1159/000024914.
The abundant human papillomavirus type 11 (HPV 11) E1E4,E5 transcript potentially encodes the E1E4,E5a and E5b proteins. It is not known if either of the E5 proteins are expressed from this transcript. For HPV 16, E5 is a single open reading frame (ORF), and the E5 protein is expressed from an unspliced E2,E5 transcript but not from the spliced E1E4,E5 transcript. This study was undertaken to determine if the HPV 11 E5a protein is expressed from the E1E4,E5 transcript. To detect E5a expression in eukaryotic cells, the green fluorescent protein (GFP) gene was fused to the 3' end of the E5a gene in the pEGFP-N1 vector. Several recombinant plasmid constructs were made to determine if E5a translation is influenced by upstream sequences present in the E1E4,E5 transcript. COS-7 cells were transfected with each construct, and flow cytometry was performed after 24 h of growth. The amount of E5a-GFP expressed from each construct was determined by the mean fluorescence of 2,000 transfected cells. Although the E5a-GFP fusion was expressed by all but one construct, the quantity of expressed E5a-GFP varied considerably. The most abundant expression was detected in cells transfected with the E1E4,E5a construct that lacked the 5' noncoding sequence between nucleotides (nts) 714 and 831 that is present in the authentic transcript. Other constructs expressed E5a-GFP in variable amounts, suggesting that sequences between nt 714 and the start of the E5a ORF affect expression of the E5a protein. An E2,E5a construct was made to compare the HPV 11 E5a expression to that of HPV 16. In contrast to HPV 16, no E5a-GFP was expressed from the HPV 11 E2,E5a construct. E1wedgeE4 protein was detected by immunofluorescence in COS-7 cells transfected with a construct that expressed E1E4 as a T7-epitope-tagged protein, and E5a as a GFP fusion. We conclude that the abundant HPV 11 E1E4,E5 transcript is a functional message that can support both E1E4 and E5a expression in eukaryotic cells.
人乳头瘤病毒11型（HPV 11）丰富的E1E4、E5转录本可能编码E1E4、E5a和E5b蛋白。目前尚不清楚这两种E5蛋白是否由该转录本表达。对于HPV 16，E5是一个单一的开放阅读框（ORF），E5蛋白由未剪接的E2、E5转录本表达，而不是由剪接后的E1E4、E5转录本表达。本研究旨在确定HPV 11 E5a蛋白是否由E1E4、E5转录本表达。为了检测真核细胞中E5a的表达，将绿色荧光蛋白（GFP）基因与pEGFP-N1载体中E5a基因的3'末端融合。构建了几个重组质粒，以确定E5a的翻译是否受E1E4、E5转录本中上游序列的影响。用每个构建体转染COS-7细胞，生长24小时后进行流式细胞术检测。通过2000个转染细胞的平均荧光强度来确定每个构建体表达的E5a-GFP的量。尽管除了一个构建体之外，所有构建体都表达了E5a-GFP融合蛋白，但表达的E5a-GFP的量差异很大。在用缺少真实转录本中存在的核苷酸（nts）714和831之间的5'非编码序列的E1E4、E5a构建体转染的细胞中检测到最丰富的表达。其他构建体表达的E5a-GFP量各不相同，这表明nt 714与E5a ORF起始之间的序列影响E5a蛋白的表达。构建了一个E2、E5a构建体，以比较HPV 11 E5a与HPV 16 E5a的表达情况。与HPV 16不同，HPV 11 E2、E5a构建体未表达E5a-GFP。在用一个将E1E4表达为T7表位标签蛋白、将E5a表达为GFP融合蛋白的构建体转染的COS-7细胞中，通过免疫荧光检测到E1ΔE4蛋白。我们得出结论，丰富的HPV 11 E1E4、E5转录本是一个功能性信息，能够在真核细胞中支持E1E4和E5a的表达。
