【作者】 王晓；

【导师】 赵蔚明； 赵孔南；

【作者基本信息】 山东大学 ， 病原生物学， 2008， 博士

【摘要】 人乳头瘤病毒(Human papillomavirus,HPV)是能引起人类皮肤粘膜多种良、恶性增殖性疾病的一类肿瘤相关病毒。目前,人们已经成功分离出了200多个型别的HPV。根据致病性的不同,又可将其中感染生殖道的HPV分为低危型和高危型。低危型HPV包括1,2,6,11等型,主要引起良性瘤或疣,如扁平疣和尖锐湿疣;高危型HPV包括16,18,45,58等型,主要引起皮肤粘膜的恶性肿瘤,如宫颈癌等。对HPV生物学特征的研究表明,HPV为无包膜的8kb左右的双链DNA病毒,基因组可分为3个区:非编码区,早期区和晚期区。其中,晚期区含有L1和L2两个ORFs,编码病毒的主要衣壳蛋白和次要衣壳蛋白,共同组成病毒的衣壳。然而,HPV的生活周期依赖于表皮宿主细胞的分化,晚期衣壳蛋白合成和病毒颗粒装配只能在上皮终末分化细胞中进行。HPV的这一特性限制了病毒的体外培养,延缓了对HPV各个基因的表达和功能的研究,尤其是阻碍了晚期蛋白表达的研究。迄今为止人们虽已成功地利用多种原核和真核的表达系统进行了HPV晚期基因的表达研究,并实现了HPV主要型别的病毒样颗粒的体外合成和组装,但是对于HPV L1基因表达的调控,病毒颗粒的形成和DNA包装的分子机制的研究仍不完全清楚。而进一步阐明这些分子机制对于疫苗设计,抗病毒药物筛选及病毒与宿主之间的相互作用的研究大有裨益。人乳头瘤病毒58型(HPV58)是一种高危型的HPV病毒,与宫颈癌的发生密切相关。流行病学研究表明,HPV58在亚洲地区宫颈癌的发生中扮演着重要角色;在我国HPV58型仅次于HPV 16型、18型与宫颈癌发生关系密切。目前,国内外对HPV58型的研究较少,基于HPV58型在我国妇女宫颈癌发生中的特殊位置,其相关研究对开发适合中国人群的HPV基因工程疫苗具有重大的现实意义。现已发现,大部分宫颈癌相关HPV型别的L1序列都可以产生两种L1蛋白,全长型L1(从第一个ATG开始翻译)和截短型L1蛋白(从第二个ATG开始翻译)。有研究推测,全长型L1蛋白主要在高危型HPV感染的上皮中合成,并可能参与宫颈癌的进展,而病毒颗粒主要由截短型L1蛋白组成。因此,全长和截短型L1蛋白的表达及病毒样颗粒的组装研究对于阐明HPV的致癌机制及疫苗的研究具有重要意义。经比较发现,HPV58L1的编码序列中也同样含有两个ATG,然而,目前对于HPV58全长和截短型L1蛋白表达的相关研究仍不清楚。HPV58能否形成这两种L1蛋白?哪一个L1蛋白可以组装成VLP颗粒?HPV58L1基因在不同翻译系统中的表达情况如何?这些问题的答案尚属未知。体外翻译系统是研究基因表达和调控的强大工具。酵母体外翻译系统已被用来进行多种哺乳动物和病毒的mRNA的翻译研究。与其他体外翻译系统相比,它存在众多优势,为研究病毒基因表达的调控机制和其对遗传和生理因素改变的反应等提供了独特的工具。然而,迄今为止,还没有关于HPV的mRNA在酵母体外翻译系统中的翻译的研究。在本研究中,我们首次建立了一种优化的适合于HPV58L1mRNA翻译的酵母体外翻译系统,并成功地对系统进行了几个因素的优化,进而利用该系统进行了HPV58长短L1 mRNA体外翻译和病毒样颗粒组装的研究。众所周知,PV晚期蛋白的翻译在转录后水平有复杂的调控机制,然而至今没有任何实验数据可以解释为何分化的角质细胞容许野生型PV L1序列的翻译以及宿主细胞的分化情况如何调节L1蛋白的表达。以往研究证实,基因序列中的密码子使用情况可以显著影响其在宿主细胞中的表达水平,通过优化密码可以增强PV早晚期蛋白的表达。这一现象提示,密码子的使用情况与tRNA种类之间的匹配可能是影响病毒基因表达机制的一个重要因素。有研究显示,PV L1蛋白不能有效表达与特定的tRNA的缺乏有关,特别是稀有密码子tRNAs。然而,tRNA丰度和其调控基因表达的功能只有非常有限的实验数据。至今我们也不清楚在细胞内tRNA的丰度如何调节细胞内靶基因的表达。优化密码提高蛋白表达的机制尚待进一步探讨。为进一步阐明L1基因表达的分子调控机制,我们利用不同分化阶段的小鼠和人的原代角质细胞培养系统和角质细胞体外翻译系统,研究野生型和优化型的HPV6bL1基因随细胞分化的不同转录和翻译特点,试图进一步解析影响L1蛋白在角质细胞中表达的分子调控机制。一、HPV58L1 mRNA在优化的酵母体外翻译系统中的表达研究研究中首先以含有HPV58全基因组的质粒pLink322-HPV58为模板,PCR法扩增HPV58全长型L1基因(LL1)和截短型L1基因(SL1)并连接到真核表达载体pcDNA3.0上,构建出真核重组质粒pcDNA3/LL1、pcDNA3/SL1,经酶切和测序鉴定序列无误。进而利用体外转录试剂盒将pcDNA3/LL1、pcDNA3/SL1中的L1基因体外转录为相应的长短L1 mRNA备用。为构建酵母体外翻译系统,我们首先制备了酵母的原生质体,然后根据以往报道方法制备啤酒酵母细胞的裂解物,并对制备过程进行了调整和改良。考虑到裂解缓冲液中的镁离子,钾离子浓度会影响外源性mRNA翻译活性,我们针对HPV58L1mRNA在酵母体外翻译系统中的翻译条件需求对镁离子和钾离子浓度进行了优化。为检测蔗糖对酵母裂解物稳定性的影响,我们在裂解物的制备过程中分别加入不同浓度的蔗糖,并检测其对系统的保护作用。为确定系统优化后翻译效率的提高程度,检测了优化前后酵母系统对HPV58SL1的翻译效率,并比较了与商品化的兔网织红细胞体外翻译系统(RRL)的翻译效率的差异,已知剂量的HPV6bL1蛋白作为信号强度对照。结果显示,我们构建的体外翻译系统对HPV58L1 mRNA翻译活性良好。镁离子和钾离子对L1蛋白翻译的影响显著,镁离子在裂解缓冲液中浓度为2.2mM时系统翻译活性最高,钾离子的最佳浓度为220mM。加入蔗糖后裂解物的抗冻融能力都有较大的提高,蔗糖的最佳浓度为100mM。对酵母体外翻译系统进行优化后,HPV58SL1 mRNA的翻译效率比RRL系统高出2倍。经计算,优化前1μg SL1mRNA在1毫升反应液中蛋白的产量为20—40μg,优化后为50-70μg,产量提高了1倍。为进一步研究HPV58长短L1 mRNA在该系统中的翻译特点和差异,我们在优化的酵母体外翻译系统中加入HPV58全长和截短型L1 mRNA,分别进行体外翻译反应。反应过程中改变反应时间、L1 mRNA模板量,加入外源性氨基酸或亮氨酸和酵母的氨基酰tRNA等,观察其对长短L1蛋白合成的影响。结果显示HPV58长短L1 mRNA均可以在酵母系统中获得良好的表达。对比两者的翻译模式,截短型L1翻译启动迅速,10分钟内即达最高表达量;全长型L1翻译启动较慢,反应30分钟后表达量与截短型持平。提示全长型L1蛋白的翻译依赖于反应时间,而截短型L1则不明显。随初始mRNA模板量的增高,两种L1蛋白信号均明显增强,但截短型L1 mRNA翻译水平仍明显高于全长型L1mRNA。外源性的氨基酸的加入仅可以显著提高全长型L1蛋白的翻译,而对截短型L1蛋白的翻译没有影响,提示内源性的氨基酸无法满足全长L1蛋白合成的需要。加入外源性酵母aa—tRNA对两种L1蛋白的翻译均无明显影响,提示系统中内源性的tRNA可充分满足HPV58两种L1 mRNA的表达。为进一步揭示长短L1蛋白合成效率的差异原因,我们在反应系统中加入不同浓度的外源性的亮氨酸,观察其对两种L1蛋白合成的影响。为检测体外合成的L1蛋白是否可以组成VLP颗粒,将体外翻译的反应液直接进行蔗糖超速离心后透射电镜检测VLP颗粒的形成。Western Blot结果显示,亮氨酸可以提高全长型的L1mRNA的翻译水平,且效应呈亮氨酸剂量依赖性;而截短型L1翻译不受影响。实验结果表明,系统中亮氨酸的缺乏是限制HPV58全长型L1表达的重要因素,这是由于长L1蛋白起始序列中亮氨酸的高频率使用所导致。电镜结果显示全长和截短型L1蛋白均可以组装成VLP颗粒。综上所述,本部分中我们成功地构建了一种新型的适合HPV58L1蛋白合成的酵母无细胞翻译系统。通过对HPV58L1蛋白合成所需的各成分浓度进行优化,显著提高了系统对L1 mRNA的翻译效率,经优化后首次观察到了系统中HPV58VLP的形成。因而该系统成为继RRL系统后的第二个可进行HPV L1病毒样颗粒组装的体外翻译系统。我们进一步利用该系统探索并揭示了导致长短L1 mRNA翻译差异的原因,并首次观察到了HPV58长短L1蛋白组装的VLP颗粒。HPW酵母体外翻译系统具有的开放性和高度可操作性等独特优势,因而该系统有望用于1)研究HPV 58 L1蛋白在酵母中表达的影响因素,为提高L1蛋白表达产量、完善VLP酵母基因工程疫苗的生产、开发提供重要的借鉴;2)探讨HPV VLP组装的分子机制,为提高酵母中VLP形成效率,减少其与天然病毒颗粒的免疫原性差异等研究提供一个方便快捷的工具;3)研究HPV DNA包装的分子机制,为今后探讨长短L1蛋白包装的病毒颗粒的感染力和感染机制的差异以及抗病毒新靶点的筛选等研究提供良好的平台。目前,研究成果已申请国家发明专利一项(公示期)。同类研究尚未见报道。二、HPV6b L1基因在原代角质细胞培养系统中的表达研究为了检测基因密码组成对L1基因在持续分化的角质细胞中表达的影响,我们用HPV6b野生型和优化型的L1真核表达质粒瞬时转染培养一天的鼠角质细胞。转染后的D3,D6,D9,D12分别收集鼠角质细胞提取RNA和蛋白。对细胞固定后进行免疫荧光染色后镜下观察。实时荧光定量PCR检测提取的RNA样品中L1基因的转录水平。Western Blot方法检测L1蛋白的合成情况。镜下显示:随着转染时间的增长,细胞显示出明显的逐步增强的细胞分化特征,免疫荧光染色结果也显示,角质细胞终末分化的标志性蛋白外皮蛋白(involucrin)在细胞内的表达也随转染时间的延长而明显增强。实时荧光定量PCR结果显示,转染后角质细胞可以持续转录野生和优化型的L1 mRNA至少12天,而且优化型的L1 mRNA的水平始终显著高于野生型L1,这表明对GC结尾的密码子进行优化可以促进L1基因在KC细胞内的转录。随着细胞的分化,野生型和优化型的L1基因的转录水平都显著下降。Western Blot分析显示,野生型L1蛋白的表达随着细胞转染时间的延长而增强;优化型L1蛋白水平随着细胞转染时间的延长而降低。这表明L1 mRNA的密码子成分组成是调节L1蛋白在KC细胞内持续表达的一个重要因素;HPV L1蛋白的表达是转录后水平调节,同时与细胞的分化水平显著相关。甲硫氨酸标记的L1蛋白免疫共沉淀实验显示,检测到的L1蛋白的持续表达是由于L1蛋白的持续合成,L1 mRNA的密码子组成是决定L1蛋白持续性合成的重要因素。考虑到人角质细胞是HPV感染的天然宿主细胞,我们检测了HPV6b L1真核表达质粒在原代人角质细胞内的表达情况。所得结果与鼠角质细胞中的结果相似。为了进一步证实tRNA对L1的翻译调控作用,我们检测了不同分化程度的人或鼠KC中分离的aa-tRNA对体外翻译系统中L1蛋白翻译的影响。结果表明,野生型L1 mRNA倾向于在分化的鼠KC细胞制备的裂解物中进行翻译,而优化型L1mRNA倾向于在低级分化的鼠KC细胞制备的裂解物中进行翻译。这一结果与L1转染的KC细胞中观察到的野生和优化L1蛋白的表达模式相同。这表明低级分化和完全分化的角质细胞中的aa-tRNA组成不同,因而可以不同程度的吻合HPV野生和优化型的L1 mRNA翻译的需要,从而可以调节它们在体外的翻译水平。综上所述,本研究中我们首次利用不同分化阶段的人和鼠原代角质细胞培养系统和体外翻译系统进行了L1蛋白翻译调控机制的研究。研究中首次探索了瞬时转染后的角质细胞中L1基因的转录和翻译时限,揭示了同义密码子的替换效应与细胞分化的密切关系。结果显示密码优化可以改变L1基因在细胞内的表达时效和表达模式,这种差异主要是由于分化阶段不同的角质细胞中的tRNA的组成不同,因而可以不同程度的调节野生型和优化型的L1基因的转录和翻译水平所致。所得结果为进一步阐明PV L1基因表达的分子调控机制提供了重要的实验数据和理论支持,同类研究尚未见报道。更多还原

【Abstract】 Papillomaviruses(PVs)are DNA viruses that infect keratinocytes in differentiating epithelia and induce a series of benign and malignant hyperproliferative lesions.To date, more than two hundreds of different HPV types have been identified.Infections by genital papillomaviruses are one of the most common sexually transmitted viral infections.The viruses that infect the genital tract are further classified as either high risk or low risk viruses.Low-risk HPV genotypes,such as HPV1,2,6,11,induce benign warts such as common warts and condyloma acuminata.In contrast,infections by high-risk HPV types including HPV 16,18,45,58 can lead to the development of cervical cancer which is one of the most common cancers worldwide.It is well known that HPVs are a family of approximately 8kb,non-enveloped and double-stranded DNA viruses.The genome of HPV can be divided into three regions:the non-coding region, the early and late regions.The late region includes two open reading frames(ORF),L1 and L2,which encode the major capsid protein and minor capsid protein assembling into the virus capsid together.However,the productive life cycle of HPVs is intimately linked to epithelial growth and cell differentiation.The expression of major capsid protein L1 only occurs in the terminally differentiated KCs.The link has posed a substantial barrier to the study of HPV in the laboratory because HPVs cannot be propagated in conventional cell lines.So far,different culture systems have been developed to mimick keratinocyte differentiation in vitro,however,it is unclear of the detailed mechanism responsible for the L1 gene expression,VLP assembly and DNA encapsidation.Further demonstration on these aspects will greatly help to the vaccine design,the anti-virus drug screen,and the research on the virus-cell interaction in the future.HPV 58 is an important HPV type tightly associated with cervical cancer.Several studies have suggested that HPV58 plays a prominent role in cervical cancer in Asia,and it is also the third most prevalent HPV type found in cervical cancer patients in China except HPV16 and 18.However,HPV58 has received little attention in the world.Considering the importance of HPV58 in China,studies on the HPV58 L1 protein expression and the development of its prophylactic vaccine is greatly desired.It has been demonstrated that the L1 sequences of the cervical carcinoma-associated HPV types always contain a second ATG codon downstream of the first translation initiation codon,which possess the ability to produce a variant L1 protein.The formation of virus-like particles(VLPs) occurs more efficiently when the second ATG codon is used for studies of the L1 gene expression in vitro.It has been postulated that the long L1 protein mainly occurs in the high-risk HPV-infected epithelium and plays a role in the carcinogenesis of cervical cancer.In contrast,the virus-like particles are mostly assembled by the short L1 protein. However,it is not clear whether the HPV 58 L1 gene is able to express two formats of the L1 proteins,with a long and a short L1 protein,which can self-assemble into virus-like particles.Also,little is known whether and how the HPV 58 L1 gene is expressed in different expression systems in which the L1 genes from the other HPV types are well expressed.In vitro translation system is a powerful tool to study gene expression and regulation. Yeast cell-free system(in vitro translation system)is a kind of in vitro translation system based on the extracts of the yeast cells.Yeast cell-free system has been widely used for the translation of various mammalian and viral mRNAs.Compared with other in vitro translation systems,yeast cell-free system shows a lot of advantages to study the regulatory mechanism of viral gene expression and its responses to the genetic and physiological changes.However,it is still unclear whether HPV mRNA could be translated in L1 protein in the yeast cell-free system.In this report,we first developed a new yeast cell-free lysate system that is suitable for efficient translation of HPV 58L1 mRNA.We successfully optimized the system in several aspects to achieve the optimal translation efficiency and demonstrated for the first time that HPV58 VLPs were assembled from the in vitro-translated L1 protein in yeast cell-free system.It is well known that complex mechanisms are involved in the regulation of PV late gene expression post-transcriptionally.However,no experimental data could clarify why the differentiated keratinocyte is permissive for the translation of the native L1 genes,and how the cell differentiation regulates the L1 gene expression freely.It has been proved that the codon usage of a target gene is tightly linked to its expression level in the host cells,and codon modification of PV early and late genes could greatly enhance their gene expression.It indicates that the match extent between codon usage and the tRNA species is an important determinant for viral gene expression,and the poor synthesis of PV L1 protein was due to the lack of specific tRNAs,especially some rare tRNAs. However,there are only very limited experimental data about the tRNA abundance and its regulatory function in gene expression.The detailed mechanism is still unclear.So efforts are still desirable to elucidate the mechanism of gene regulation in fine details.To explore the detailed molecular mechanism of L1 gene expression,we used the differentiating primary keratinocytes to establish the keratinocyte culture system and in vitro translation system,and investigated the transcription and translation pattern of both wild type and codon-modified HPV6b L1 genes during cell differentiation.Ⅰ.The in vitro translation of HPV58L1 mRNA in an optimized yeast cell-free systemWe first amplified the long and short L1 genes from the plasmid pLink322-HPV58 containing the complete genome of HPV58 using PCR method.The PCR products were then ligated into the eukaryotic expression vector pcDNA3.0 respectively to generate the constructs pcDNA3/LL1 and pcDNA3/SL1.The correct sequence and orientation of the L1 genes were further confirmed by the restriction analysis and DNA sequencing.Then the long and short L1 genes in the vector were in vitro transcribed into corresponding mRNAs using the in vitro transcription kit.To establish the yeast cell-free system,we used the protoplasts of the yeast cells to prepare the yeast cell-free lysate according to the previous procedure with some modification.Given that the concentration of the magnesium and potassium ions in the lysis buffer will greatly affect the translational activity of exogenous mRNAs,we optimized the concentration of them according to the requirements of the HPV58L1 mRNA translation.To test the effects of sucrose on the stability of the system,we applied different concentrations of sucrose during the lysate preparation.To determine the effects of optimization,we compared the translation efficacy between commercial rabbit reticulocyte system(RRL system)and the yeast cell-free system before and after optimization.The defined quantities of HPV6b L1 protein was used as a standard.Results showed that the in vitro translation system we established here is sufficient for the translation of HPV58L1 mRNA.The concentration of magnesium and potassium ions affects the L1 protein production greatly.The optimal concentration of magnesium was found to be 2.2mM,and that of potassium was 220mM.Intriguingly,sucrose seemed to provide a "cushion" that could assist the lysate against the environmentally thermometric changes and greatly increase the stability of the lysate.The optimal concentration of sucrose was 100mM.After optimization,the translation efficiency of HPV58L1 mRNA in the yeast cell-free system was twice higher than that of the RRL system.After calculation,50-70μg of the L1 protein expressed from 1μg of the short L1 mRNA in a volume of one ml translation reaction could be produced after optimization,which was significantly higher than that before optimization(20-40μg).To further explore the difference between HPV58 long and short L1 mRNA translation in the yeast in vitro translation system,we applied long and short L1 mRNAs into the system and performed the reaction by altering the reaction time,the amount of mRNA template,and by adding exogenous amino acids or leucine and yeast aminoacyl-tRNAs respectively.Results showed that both long and short L1 mRNAs could be translated in the yeast cell-free system.The translation could initiate within 10 min,but the level of the short L1 protein was significantly higher than that of the long L1 one.The short L1 protein level was slightly increased from 10 to 30 min in contrast with the long L1 protein level. The expression of both long and short L1 proteins was dramatically dependent on the amounts of the L1 mRNAs.Again,the level of the short L1 protein was significantly higher than that of the long L1 protein.Supplement of exogenous yeast aa-tRNAs slightly enhanced the translation of L1 mRNAs suggesting that endogenous aa-tRNAs were sufficient.Interestingly,supplement of the exogenous amino acids significantly enhanced translation of the long L1 mRNA,while hardly enhanced that of the short L1 indicating clearly that available endogenous amino acids are the major factor,which limits translation of the long L1 mRNA in the yeast cell-free lysates.To reveal the reason responsible for the difference between long and short L1 synthesis, exogenous leucine was supplied to the in vitro translation reaction.To clarify whether the in vitro translated L1 protein could self assemble into VLP particles,we applied the products to the sucrose ultracentrifugation and observed the formation of VLP directly. Western Blot analysis revealed that long L1 signal was greatly enhanced by the supplement of leucine in a dose-dependent manner.However,the short L1 signal had no response to it.So it seems that a single amino acid leucine can limit the rate of translation of the long L1 mRNA due to its high proportion in the L1 protein sequence. Electron microscopy result showed that both long and short L1 capsid proteins in the yeast lysate system could self-assemble into VLPs.Here we developed a yeast cell-free lysate system that was suitable for in vitro translation of HPV58L1 mRNA.The system was systematically optimized after which the translation efficiency was greatly enhanced and the VLP particles were observed.So the optimized yeast cell-free system was the second in vitro translation system sufficient for the HPV VLP formation except RRL system.Then we used this system to further explore the difference between long and short L1 mRNA translation and finally observed the VLPs assembled from both long and short L1 proteins.Considering that the HPV/yeast cell-free system is highly amendable and simple to be made,the system may be very useful in:1)exploring the factors affecting the expression of HPV58L1 protein in the yeast culture system,improving the protein production and modifying the vaccine design and development;2)clarifying the molecular mechanism of VLP assembly,increasing the VLP formation efficacy,and reducing the difference between the antigenicity of yeast-expressed VLP and native virions;3)revealing the molecular mechanism of DNA encapsidation,facilitating the research on the infectivity and infection mechanism of VLPs from both long and short L1 proteins,and providing possible new anti-virus targets.Currently,we have applied a national invention patent waiting for final approval.No similar reports have been found.Ⅱ.The HPV6b L1 gene expression in the primary keratinocyte systemTo explore the effects of codon composition on the L1 gene expression in the continuously differentiating keratinocytes(KCs),HPV6b wild type(Nat)and codon modified(Mod)L1 gene construct were transiently transfected into the human and mouse primary keratinocytes.Following transient transfection,cells were collected at D3,D6,D9,D12 post-transfection for RNA extraction,protein preparation and immunofluorescence staining.Quantitative RT-PCR was used to detect the L1 transcripts in the transfected KCs and Western Blot to determine the L1 protein synthesis.The morphological changes observed in the L1-transfected KCs over time reflected cell differentiation that was confirmed by the increased expression of involucrin,a marker of keratinocyte terminal differentiation,using immunofluorescence microscopy.QRT-PCR results showed that the transfected KCs could continuously transcribe the authentic and modified L1 mRNA for at least 12 days.The level of Mod L1 mRNA is higher than that of Nat L1 indicating that the codon modification promotes the transcription of L1 genes in KCs.The transcription of both L1 genes decreased as the cell differentiated.Western Blot analysis showed that the Nat L1 protein signal increased significantly over time.In contrast,Mod L1 signal decreased greatly over time.The results suggested that codon composition of the L1 mRNA plays a key role in regulation of the continuous expression of the L1 proteins.The expression of L1 protein was regulated in the post-transcription level and tightly associated with the cell differentiation.The result of the immunoprecipatation of[S35]methionine-labeled L1 protein showed that the detected continuous expression of the L1 protein was due to the continuous synthesis of the L1 protein and the codon composition of the L1 mRNA sequences was a major determinant of the continuous synthesis of the L1 protein.Considering that the natural host cell of HPV infection is human keratinocyte(HKC),we further explored the expression of L1 genes in HKC culture system.Similar results were obtained.To further confirm the regulatory role of tRNAs on the L1 gene translation,we extracted the aa-tRNAs from human and mouse KCs of different differentiation states and applied them to the KC in vitro translation system.Results showed that Nat L1 mRNA tended to be translated in the differentiated KCs,and the Mod L1 mRNA in the less-differentiated KCs.The result was consistent with those from the transfected KCs indicating that aa-tRNAs in less-differentiated and differentiated KCs differentially match the HPV Nat and Mod L1 mRNAs to regulate their translations in vitro.In this part,we first established the keratinocyte in vivo and in vitro systems using the continuously differentiating cells and investigated the regulatory mechanism of L1 gene expression.We showed for the first time the transcription and translation duration of L1 genes in the transiently transfected KCs,and revealed the tight link between isoencoding codons of viral gene and the host cell differentiation.It seems that the codon modification could change the duration and pattern of L1 gene expression in KCs which was mainly because the tRNA pools differ greatly during the cell differentiation,thus differentially regulate the transcription and translation of the Nat and Mod L1 mRNAs in vitro.Our results as important experimental data shed more light on the detailed regulatory mechanism of L1 gene expression.No similar reports were observed.更多还原