Molecular evolution and genomic diversity in carcinogenicity of primate papillomaviruses

Cervical cancer is the 4th most common cancer in women, with more than 0.5 million new cases and 0.2 million deaths occur worldwide annually. As one of the most carcinogenic infectious pathogens, the nature of human papillomavirus (HPV) has been well studied in past four decades, and persistent infection of high-risk (HR) types of HPV has been confirmed as the root causative factor for cervical cancer and its premalignant lesions. Papillomaviridae is a diverse family of small and non-encapsulated viruses with double stranded circular DNA genome that include early genes (e.g., E1, E2, E4, E5, E6 and E7), late genes (L1 and L2) and upstream regulatory region (URR). Thereinto, E6 and E7 oncoproteins target and inactivate tumour suppressors p53 and pRb, respectively, by proteasomal degradation to drive malignant progression.Given high genetic diversity of PVs and the complex scenario of virus-host interaction, the underlying mechanism of HPV carcinogenicity in cervical cancer, however, is still not clearly understood. Genetic comparison between human and non-human primate (NHP) PVs suggests key similarities in evolution implying ancient origin of viral carcinogenicity. Thus, we hypothesize that the emergence of cancer-causing PVs was a product of biological niche adaptation, resulted by the evolution of success amongst viral infections under natural selection and DNA mutation.In an effort to understand the origin and evolution of PV carcinogenicity, we firstly collected paired oral, perianal and genital swab samples from 117 noncaptive wild rhesus macaques in Kam Shan Country Park, Hong Kong. Using a combination of phylogenetic and bioinformatic algorithms, the genetic diversity and evolutionary skeleton of primate PVs were elucidated. To further evaluate the evolutionary relationship of primate PV carcinogenicity at functional level, we characterized the oncogenic property of E6 proteins encoded by phylogenetically related human and macaque papillomaviruses and detected their interactions with host p53 protein using in vitro cell model.All putative macaque PV types clustered into α-, β- and γ-PV genera, with strong tissue tropism. Ancient intra-host divergence of primate PVs was estimated to occur 41 million years ago, in which multiple ancestors had split and adapted to specific host ecosystems prior to the speciation events of primate host species. Following subsequent divergence and niche adaptation, distinct but phylogenetically related PVs might be transmitted to similar host ecosystems by closely related host animals when host speciation occurred, which may explain in part the origin of carcinogenicity of human and non-human primate PVs that evolved from a most common ancestor (MRCA) containing the determinants for cervicovaginal colonization and cervical cancer.The in vitro model suggested strong species barrier of HPV and macaque PV E6s in degrading host p53. While an amino acid change of p53 at site 129 was found to be critical for crossspecies degradation and has a potential for genetic therapy. Systematic investigation of E6 proteins encoded by distinct macaque PV types in degrading host p53 revealed evolutionary relatedness of PV carcinogenicity: the majority of macaque PVs within the oncogenic species α12 were able to degrade macaque p53 while other α, β and γ macaqye PVs did not. Alignment of macaque PV E6 ORFs found four amino acid sites (MfPV3 E6 aa site 2, 13, 46 and 47) potentially correlated with the biochemical ability to degrade p53. Furthermore, MmPV1 and MfPV10 E6s can degrade both human and macaque p53, suggesting an ancient origin of oncogenic HPVs splitting from their most recent mucosal viral ancestors before host speciation.In conclusion, this thesis used analytic tools of evolution and systematic biology via expanding the repertoire of human and non-human primate PV genomes and in vitro experiments to elucidate the evolutionary dynamics, genetic basis and biological function of papillomavirus carcinogenicity. Our data found a similar evolutionary history of NHP-PVs as observed in their human counterparts on both genetic and functional levels. The findings reveal an important role of niche adaptation sharing similar tissue tropism and pathogenic properties between human and macaque PVs, which lays a genetic foundation for research on parasite-host interactions and carcinogenic outcomes.子宮頸癌是第四大常見婦科癌症，全世界每年有超過50 萬的子宮頸癌新發病例，以及20 萬的死亡病例。作為致癌性傳染病原體之一的人類乳頭瘤病毒（HPV），其病毒學特性在過去的三至四十年中已經被充分地研究。子宮頸被高危型HPV 持續感染已被廣泛認為是子宮頸癌以及癌前損傷產生的最根本因素。乳頭瘤病毒科是雙鏈環狀DNA 病毒的一科，包含了多種小型無包膜病毒。其基因組編碼早期基因（如：E1，E2，E4，E5，E6及E7）、晚期基因（L1和L2）和上游調控區域（URR）。E6和E7作為乳頭瘤病毒的致癌基因之一，能分別介導蛋白酶體的降解作用使宿主的抑癌基因p53和pRb失活，從而誘發癌變。即使HPV誘導子宮頸癌的致癌機理已經被廣泛揭示，但由於PV高度的基因多樣性，以及複雜的病毒-宿主相互作用，HPV 在基因水準的致癌機理依然不得而知。通過比對人類乳頭瘤病毒和非人類靈長動物乳頭瘤病毒（NHP-PV），我們發現了其在進化中的相似性。在本研究中，我們假設致癌的PV 的產生是生態適應的產物，是在自然選擇和基因突變的作用下帶來的進化結果。為探究HPV 致癌性的起源以及進化方式，我們首先從分散在香港金山郊野公園中的117 只野生恆河猴身上採集了配對的口腔、肛門以及生殖道的拭子樣本。通過系統發育學和生物資訊學，我們闡述了靈長類動物PV的基因多樣性和進化框架。為了進一步驗證靈長動物PV 在致癌功能性方面的進化關係，我們研究歸納了親緣性較近的人類與恆河猴PV的E6蛋白在降解宿主p53蛋白方面的特性。所有的恆河猴PV都劃分在α、β和 γ乳頭瘤病毒屬，並且具有和HPV一樣很強的組織親嗜性。我們估算到靈長類動物PV 在宿主體內的分化發生在大約4100 萬年前，當時最古老的病毒開始分化並且適應宿主體內特定的生態環境，然後與宿主共同進化。隨著一系列的分化和生態環境適應，親緣性相近的PV 在相似的宿主生態環境中交叉傳播，這就是人和非人靈長類動物PV 從共同祖先進化出來，並且都擁有定植在子宮頸並且誘發子宮頸癌的能力的原因。我們的體外模型闡釋了HPV 和恆河猴PV 的E6 蛋白在降解宿主p53 方面有物種間屏障。並且我們找到位於p53 上的129 位點對突破該種間屏障起重要作用，該位點的發現也為基因治療和藥物研發奠定了基礎。 另外我們發現，大部分位於α12中的恆河猴PV都能降解宿主p53，而其他α或者β/γ-PV不能，這也清楚地揭示了PV致癌性的進化關係。 通過比對NHP-PVE6 開放閱讀框，我們篩選出四個（位點2，13，46 和47）蛋白上的可能與降解p53 蛋白相關的關鍵氨基酸。另外，我們發現MmPV1 和MfPV10的E6可以同時降解人類和恆河猴的p53，說明致癌性的靈長類動物PV先從粘膜型病毒祖先分化，然後與宿主共同進化。總而言之，本研究論文運用進化分析和系統生物學的方法，通過廣泛調查NHP-PV 的基因組多樣性以及建立細胞實驗，來闡述進化方式以及在基因層面和功能層面揭示乳頭瘤病毒的致癌機理。數據顯示NHP-PV 擁有和HPV 相似的進化史。我們的發現有效的說明了人類和恆河猴PV 具有相似的組織親嗜性以及致病特性，這些發現為今後病原-宿主相互作用以及致癌性研究奠定了基因學基礎。Long, Teng.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …)