Human papillomaviruses (HPVs) are prevalent pathogens that cause epithelial hyperproliferation. More than 170 HPV genotypes have been cloned. HPV types trophic for cutaneous skin cause planter and palmar warts and common warts. The low risk mucoso-tropic HPV types cause 90% of ano-genital warts and all laryngeal papillomas. Persistent infections by the high-risk HPVs in the mucosal epithelia can lead to cancers, including cervical, penile, anal, and a subset of head & neck carcinomas. Worldwide, well over 500,000 cases of cervical cancer arise each year, leading to at least 275,000 deaths, primarily in developing countries for lack of early diagnosis and treatment. Indeed, HPV is the second most frequent cause of mortality due to cancer among women globally. This presentation and discussion will cover the systematic, career-long efforts in our lab to understand the natural history and biology of the viruses, regulation of viral gene expression, mechanisms of DNA replication, and the viral protein functions and interactions with the host cells that form the basis for pathogenesis. We learned that the productive phase of these viruses takes place in epithelial tissues undergoing squamous differentiation. Our laboratory has developed a three-dimensional tissue culture system with which primary human keratinocytes can be developed into squamous epithelia. Individual papillomaviral genes can be studied in such organotypic raft cultures growing at the liquid medium-air interface following retroviral-mediated gene transfer into the keratinocytes. Moreover, we also generate the complete high-risk HPV-18 genome in the cells by in vivo recombination and are able to recapitulate the entire virus reproductive cycle and produce abundant infectious virions. Using this system, we have elucidated the viral productive cycle during epithelial differentiation. We are examining various site-directed mutations in the HPV DNA to confirm the roles of the gene products in the life cycle. I will also discuss progress made in vaccine development by other laboratories as well our own recent efforts toward antiviral drug discovery and validation.

人類乳突病毒(簡稱HPV)，為造成上皮細胞過度增生的流行病原。HPV有超過170種的基因亞型，而其中有多種的亞型會造成手足部的蹠疣、掌疣與一般疣。低風險的黏膜型(mucoso-tropic) HPV會感染肛門及生殖區以及喉乳頭狀瘤(laryngeal papilloma)。而黏膜上皮細胞若持續受到高風險型的HPV感染則會引發多種癌症，如：子宮頸癌，生殖器、肛門與頭頸部的癌症。全世界每年有超過50萬的子宮頸癌新增病例，並有超過27萬5千人死亡，且主要是發生在缺乏早期診斷和治療的開發中國家。事實上，HPV是造成女性因罹癌而死亡的第二大原因。這次的演講將探討本實驗室對於病毒自然史以及生物學、病毒基因表達的調控、DNA複製的機轉、發病機制裡病毒蛋白與宿主細胞的交互作用和功能之研究。從中我們了解到，在病毒產生時期，上皮組織會發生鱗狀分化(squamous differentiation)。我們利用本實驗室所建立的三維組織培養系統，可使人類初級角質細胞(keratinocyte)分化成鱗狀上皮細胞(squamous epithelia)。我們藉由反轉錄病毒(retrovirus)為載體將基因導入角質細胞後，再以氣液介面 (liquid medium-air interface)活體組織培養來研究乳突病毒之基因。此外，我們還在活體內重組出高風險HPV 18型的基因組來表現整個病毒的繁殖週期並產生大量感染性的病毒粒子。利用這個系統，我們能說明在分化的角質細胞中，病毒的生產週期。我們還透過研究HPV DNA 中不同的點突變來確認其在生命週期中所扮演的角色。之後，我還會討論其他實驗室所做的疫苗開發之進展以及我們對抗病毒藥物的發現與驗證。 （感謝陽明大學腦科學研究所蔡金吾教授與周婉儒研究生協助翻譯）

The presentation will discuss scientific pursuits from the late 1960's and early 1970's when I was a beginning scientist to the present days of molecular medicine. I will describe the early state of the art in investigating the genomic organization of bacteriophages and animal viruses. Phages and viruses rely on host cells to perform all the essential functions for their propagation, including RNA transcription, protein translation, and DNA replication. Since their genomes are a mere tiny fraction of the sizes of host cell chromosomes, phages and viruses are model organisms to probe macromolecular metabolism in their respective hosts. Prior to the advent of molecular cloning of nucleic acids, electron microscopic (EM) examination of heteroduplexes of nucleic acids was the only convenient tool to study sequence relationships between related DNA and RNA strands. I will discuss the various biochemical experiments carried out by colleagues in the Cold Spring Harbor Laboratory (New York, USA) that eventually led to our EM discovery of split genes, RNA splicing and alternative RNA processing. In addition, EM also revealed the usage of alternative transcription promoters and alternative poly-adenylation sites for overlapping transcripts. These discoveries proved to be the beginning of completely new fundamental principles in the regulation of gene expression in higher eukaryotic cells. In these systems, mRNAs are composed of multiple but usually relatively short exons after much longer intervening intron sequences are removed. Generally over 90% of the primary RNA transcripts consist of introns. For decades, it was thought over 90% of the human genomes were noncoding "junk" DNA and their existence was a puzzle and a term "selfish" DNA was coined because no function could be ascribed to it. With the invention of diverse new technologies and investigations of many additional model organisms, small regulatory RNAs called miRNAs have been discovered and they regulate mRNA translation or stability. Thus, they can regulate the expression of oncoproteins or tumor suppressors. In fact much of the human genome is indeed transcribed, possibly to regulate gene expression. In addition to the regulations based on DNA and RNA sequences, epigenetic regulations through the modification of histone proteins and chromatin structures add layers of complexity to gene regulation. These progressive discoveries all contribute to advance in molecular and personal medicine, limited only by one's imagination, dedication, commitment, persistence, and the availability of funds for research and development.

本演講將探討從1960年代末期和1970年代初期我開始成為科學家到現今對分子醫學之研究，並描述早期對噬菌體(bacteriophage)和動物病毒基因體(genome)結構的最先進技術。噬菌體和病毒利用宿主細胞來進行其複製繁殖所需要之功能，包括RNA的轉錄、蛋白質的轉譯和DNA的複製。由於噬菌體和病毒的基因組來自於宿主細胞染色體的一小部份，所以他們成為很好的模式物種以用來研究其宿主的大分子代謝過程。而在核酸的分子純化繁殖技術發明之前，利用電子顯微鏡(electron microscopy; EM)觀察異源雙股(heteroduplexes)核酸分子是唯一方便的工具來檢測相關的DNA與RNA之間序列的關係。我將討論各種由冷泉港實驗室(Cold Spring Harbor Laboratory)的同事所開發的生化實驗，而這些實驗也幫助了我們增進對中斷基因(split gene)、RNA剪接(RNA splicing)與選擇性加工(alternative RNA processing)之研究，此外，利用EM也發現了選擇性轉錄啟動子(alternative transcription promoter)和選擇性多腺苷酸化位點(alternative poly-adenylation sites)在重疊轉錄物(overlapping transcripts)中的功用。這些發現證明了在高等真核細胞如何調控基因表現全新的基本原則。在轉錄所產生的RNA之中，有超過90%都含有內含子(intron)，RNA上的內含子會在RNA進行轉譯前被剔除，而在成熟mRNA中保留下來為較短的外顯子(exon)。數十年以來，人們認為人類基因組裡有超過90%為沒有編碼的"垃圾"DNA，而他們的存在一直是一個謎。因為這些DNA在當時無法被賦予任何功能，因此被稱為「自私」的DNA。然而隨著許多新技術的發明和對模式物種的研究，科學家發現許多稱為「微小RNA (miRNAs)」的短序列RNA可調控RNA的轉譯與穩定性，因此他們能調控癌蛋白(oncoprotein)和腫瘤抑制基因(tumor suppressor)的表現，實際上大部分的人類基因組確實會被轉錄，可能就是為了調控基因的表現。除了直接控制DNA與RNA序列之外，基因表現還可以透過修飾組蛋白(histone)和重塑染色質(chromotin)的結構來調控，此機制稱為表觀遺傳調控(epigenetic regulation)，使基因的調節過程更加上一層複雜度。透過人們的想像力、奉獻、決心、毅力和研究開發的經費，這一點一滴累積的發現對分子與個人化醫療的進展都產生重要貢獻。 （感謝陽明大學腦科學研究所蔡金吾教授與周婉儒研究生協助翻譯）