Brown D R, Pratt L, Bryan J T, Fife K H, Jansen K
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
Virology. 1996 Aug 1;222(1):43-50.
Detection of E1-E4 protein in human papillomavirus (HPV 11)-infected tissue is tightly linked to detection of L1 major capsid protein. The only L1-containing transcript identified in HPV 11-infected tissue is the bicistronic E1-E4-L1 mRNA, potentially encoding both the E1-E4 and the L1 proteins. It has not been established that these proteins can be expressed from the E1-E4-L1 transcript. The HPV 11 E1-E4-L1 sequence was cloned by reverse transcriptase polymerase chain reaction into the p1393 vector to produce recombinant baculoviruses. Immunoblots of recombinant baculovirus-infected Sf9 cell lysates demonstrated both the E1-E4 and the L1 proteins. An ELISA was performed on infected Sf9 cells using a monoclonal antibody specific for nondenatured L1, demonstrating that 10 ng of native L1 protein was present per microgram of total nuclear protein. Electron microscopic analysis revealed 50- to 60-nm icosahedral virus-like particles. In vitro transcription/translation was performed using pSPORT constructs containing the E1-E4-L1 sequence or, as controls, monocistronic pSPORT-E1-E4 or L1 constructs. The pSPORT-E1-E4-L1 construct produced the E1-E4 and L1 proteins at a ratio of 17:1. For E1-E4 protein, expression was greater from the pSPORT-E1-E4-L1 construct than from the monocistronic pSPORT-E1-E4 construct. In contrast, more L1 protein was expressed from pSPORT-L1 than from pSPORT-E1-E4-L1. A mutant E1-E4-L1 construct containing no E1-E4 start codon expressed L1 protein in amounts nearly equal to that expressed from the pSPORT-L1 construct. Addition of an antisense oligonucleotide directed at the E1-E4 start codon region to in vitro reactions using pSPORT-E1-E4-L1 was associated with inhibition of E1-E4 protein synthesis and increased translation of L1 protein.
在人乳头瘤病毒（HPV 11）感染组织中检测E1-E4蛋白与检测L1主要衣壳蛋白紧密相关。在HPV 11感染组织中鉴定出的唯一含L1的转录本是双顺反子E1-E4-L1 mRNA，可能编码E1-E4和L1蛋白。尚未证实这些蛋白可从E1-E4-L1转录本中表达。通过逆转录聚合酶链反应将HPV 11 E1-E4-L1序列克隆到p1393载体中以产生重组杆状病毒。重组杆状病毒感染的Sf9细胞裂解物的免疫印迹显示了E1-E4和L1蛋白。使用对未变性L1特异的单克隆抗体对感染的Sf9细胞进行酶联免疫吸附测定（ELISA），结果表明每微克总核蛋白中存在10 ng天然L1蛋白。电子显微镜分析揭示了50至60纳米的二十面体病毒样颗粒。使用含有E1-E4-L1序列的pSPORT构建体或作为对照的单顺反子pSPORT-E1-E4或L1构建体进行体外转录/翻译。pSPORT-E1-E4-L1构建体以17:1的比例产生E1-E4和L1蛋白。对于E1-E4蛋白，pSPORT-E1-E4-L1构建体的表达高于单顺反子pSPORT-E1-E4构建体。相反，pSPORT-L1表达的L1蛋白比pSPORT-E1-E4-L1更多。不含E1-E4起始密码子的突变E1-E4-L1构建体表达的L1蛋白量几乎与pSPORT-L1构建体表达的量相等。在使用pSPORT-E1-E4-L1的体外反应中加入针对E1-E4起始密码子区域的反义寡核苷酸与E1-E4蛋白合成的抑制和L1蛋白翻译的增加相关。
