Inimese papilloomiviiruse genoomi replikatsiooni ja oligomeeride tekke analüüs

Väitekirja elektrooniline versioon ei sisalda publikatsioonePapilloomiviirused on kaheahelalise DNA genoomiga viirused, mis on ülimalt liigi- ja koespetsiifilised ning nakatavad imetajate, lindude ja roomajate epiteelkoes paiknevaid keratinotsüüte. Inimese papilloomiviirused (HPV-d) on populatsioonis laialt levinud patogeenid, millega nakatumisel kaasneb enamjaolt epiteeli healoomuliste vohandite tekkimine (tüükad, papilloomid). Mõningad HPV tüübid võivad põhjustada ka halvaloomuliste kasvajate teket. Inimese papilloomiviiruse poolt põhjustatud emakakaelavähk on kolmas enimlevinud vähk naistel, mistõttu selle valdkonna uuringud on väga olulised. Käesolevas doktoriväitekirjas uurin HPV18 DNA lühiajalise paljunemise käigus tekkivaid multimeerseid viiruse molekule, mis sisaldavad tandemina mitut viiruse genoomi koopiat (oligomeerid). Seda, et replikatsiooni käigus moodustab HPV oligomeere, märgati juba üle 30 aasta tagasi, aga nende tekkepõhjused on seni teadmata. Üks võimalik oligomeeride tekkepõhjus oleks see, et HPV-de puhul kasutatakse ühe multimeeri paljundamiseks vaid ühte replikatsioonikompleksi (oligomeeris on aktiivne ainult üks replikatsiooni alguspunkt), mis tähendab, et väheste rakuliste ja viiruslike valkude abil saab paljundada suurt arvu HPV genoome. See võimaldab ära hoida või edasi lükata HPV tuvastamist peremeesraku immuunsüsteemi poolt. Mitmedimensionaalseid geelelektroforeese on läbi viia keerukas ja veelgi keerukam on analüüsida tulemusi. Selletõttu annan käesolevas töös ülevaate, kuidas analüüsida ja tõlgendada kahe- ja kolmedimensionaalsest agaroos-geelelektroforeesist (2D ja 3D AGE) saadud tulemusi. Näitan rakendades 2D ja 3D AGE, et HPV kasutab vähemalt kahte eri replikatsioonimehhanismi genoomi paljundamiseks ja seega ka oligomeeride moodustamiseks.Papillomaviruses are double-stranded DNA viruses that are extremely species and tissue specific; they only infect the epithelial keratinocytes of amniotes (mammals, birds, and reptiles). Human papillomaviruses (HPVs) are prevalent pathogens, and infection leads to hyperproliferative lesions, such as warts and condyloma. However, some HPV types can cause malignant tumors. Cervical cancer caused by HPV is the third most common cancer worldwide; thus, great effort needs to be put into patient screening, vaccine development, and therapeutic strategies. The current study focuses on analyzing HPV DNA multimeric molecules that consist of several viral genome copies (oligomers) that arise during initial amplification. The fact that HPV forms oligomers was demonstrated in biopsies from cervical carcinomas over 30 years ago, but the reason is still unknown. The reason why the oligomeric state has been used by the virus can be explained by the necessity to keep a low profile in the host organism. HPV can replicate an entire oligomer consisting of many monomers by using as many host and viral replication proteins as a monomer would. Therefore, much more viral DNA can be replicated while eluding the host adaptive immune system. Multidimensional gel electrophoresis experiments are technically challenging to carry out; however, analyzing the results is even harder. Therefore, I included an overview of how to analyze the results obtained from two- and three-dimensional agarose gel electrophoresis (2D and 3D AGE, respectively). Utilizing 2D and 3D AGE, we showed that HPV uses two distinct mechanisms to replicate its genome and therefore form oligomers

Inimese papilloomiviiruse tüüp 18 DNA replikatsiooni ja oligomerisatsiooni jälgimine U2OS rakkudes lühiajalise replikatsiooni tingimustes

Ligipääs piiratud kuni 27.05.20162016-05-2

The Cell Cycle Timing of Human Papillomavirus DNA Replication.

Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research

Difference in cell cycle timing of HPV18 DNA replication during transient and stable replication phases.

<p>Difference in cell cycle timing of HPV18 DNA replication during transient and stable replication phases.</p

Stable replication of the HPV18 genome in the U2OS cell line.

<p>Stable replication of the HPV18 genome in the U2OS cell line.</p