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Principles of Virology
Welcome to the lecture series Principles of Virology Thomas Kietzmann Principles in Virology TK Thomas Kietzmann Viral DNA replication Parvovirus Hepatitis B virus Retrovirus VII Adenovirus Herpes simplex virus Polyoma-, and Papilloma Poliovirus Reovirus Rotavirus Influenzavirus Ebolavirus 1 Principles in Virology Thomas Kietzmann Universal rules of DNA replication TK • DNA is synthesized by template-directed incorporation of dNMPs into 3’-OH of DNA chain • DNA is always synthesized 5’-3’ via semiconservative replication (two daughter strands) • Replication initiates at specific sites on template called origins • Catalyzed by DdDp + accessory proteins • Primer-dependent Principles in Virology Thomas Kietzmann Tasks of the host TK • Viral DNA replication always requires synthesis of at least one viral protein, sometimes many (hence always delayed after infection) • Simple viruses require more host proteins - genetic economy • Complex viruses encode many, but not all proteins required for replication 2 Principles in Virology Thomas Kietzmann TK Where does the polymerase come from? • Small DNA viruses do not encode an entire replication system - Encode proteins that orchestrate the host -Papillomaviridae, Polyomaviridae, Parvoviridae • Large DNA viruses encode most of their own replication systems -Herpesviridae, Adenoviridae, Poxviridae Principles in Virology Thomas Kietzmann Viral proteins TK DNA polymerase and accessory proteins Origin binding protein, helicases, Exonucleases Enzymes of nucleic acid metabolism (thymidine kinase, ribonucleotide reductase, dUTPase) 3 Principles in Virology Thomas Kietzmann TK Thomas Kietzmann TK Diverse viral DNA genomes Principles in Virology Principal mechanisms of viral DNA synthesis RNA primed Never RNA primed 4 Principles in Virology Principles in Virology Thomas Kietzmann TK Thomas Kietzmann TK The 5´end problem Lessons from SV40 Circular dsDNA, associated with cellular histones in a chromatin-like complex 5 Principles in Virology TK Thomas Kietzmann ds DNA-Virus: Simian Virus 40 (SV 40) replication Precondition: Cells in S phase of the cell cycle ORI 5' 3' 3' 5' After completion of the genomes initiation of a second replication round at the new ORIs Binding of large T-antigen-P(Thr124) to ORI, Synthesis of primers by primase and Pol continuous discontinuous Topoisomerase; Separation of the circular genomes Principles in Virology TK Thomas Kietzmann Recognition and unwinding of the SV40 origin Large T has 3’-5’ helicase activity 6 Principles in Virology TK Thomas Kietzmann Function of SV40 large T • Activation of cellular DNA and RNA synthesis by binding to p53 and Rb leading to loss of contact inhibition • Block of apoptosis • Binding to SV40 ori to initiate viral replication (helicase activity) • Shutting off early viral transcription due to binding near the early promoter • Activating late transcription • Contributing to viral assembly Principles in Virology TK Thomas Kietzmann Synthesis of leading and lagging strands Synthesis of RNA primers Synthesis of short DNA fragments Rf-C binds 3’OH along with PCNA and pol δ -Rf-C a clamp loading protein -Allows entry of PCNA on DNA -Causes release of Pol α Form sliding clamps along DNA Synthesis of long DNA 7 Principles in Virology Thomas Kietzmann TK Synthesis of leading and lagging strands Principles in Virology Thomas Kietzmann An SV40 replication machine TK 8 Principles in Virology Thomas Kietzmann TK Cell proteins required for polyomavirus DNA replication Principles in Virology Thomas Kietzmann TK Function of topoisomerases 9 Principles in Virology TK Thomas Kietzmann ds DNA-Virus: Simian Virus 40 (SV 40) gene expression different frame Capsid proteins Splicing VP1 40 kD, 364 aa VP3 27 kD, 234 aa VP2 39 kD, 351 aa Promoter Ori (A)n 5,2 Kb (A) n Small t-antigen Regulator proteins 20 kD, 174 aa Accumulation Viral proteins Large T-antigen Splicing 81 kD, 708 AS Viral replication Viral transcription Cellular DNA synthesis Cell immortalisation different frame All genes are transcribed by host RNA pol II Principles in Virology Thomas Kietzmann SV40 replication TK • Attachement to host receptors and endocytosis • Import of genomic DNA into host nucleus Transcription of early genes (LT and sT genes) • • Association of LT with p53 and Rb • Replication of the DNA genome in the nucleus • Transcription of late genes encoding for structural proteins (VP1, VP2 and VP3 • Assembly of new virions in nuclear viral factories • Virions are released by lysis of the cell 10 Principles in Virology Thomas Kietzmann Replication of papillomaviruses TK • Non-enveloped • Small, icosahedral 55 nm • Genome is a single circular dsDNA about 8 kb, associated with cellular histones in a chromatin-like complex • • • Only one strand of the genome is transcribed and yield two classes of proteins expressed by alternative splicing : a) Early Proteins: non-structural regulatory proteins (E1-E7). b) Late Proteins: the structural proteins L1 and L2. On rare non-specific recombination, the viral genome can be integrated in host chromosome. This inactivates the integrated virus but can gives the host cell a replicative advantage sometimes leading to malignant tumors Principles in Virology Thomas Kietzmann Replication of papillomaviruses TK Replication is divided in two distinct steps linked to the differentiation state of the host epithelial cell: a) Basal squamous epithelial cells: Plasmid/Episome replication. It is in synchrony with the host cell chromosome in order to ensures an average of one viral genome per basal cell. b) Differentiated keratinocytes: Vegetative replication. In these cells, which no longer undergo cellular DNA synthesis, is a burst of viral DNA synthesis with active production of virions. Basal squamos cells Differentiated keratinocytes 11 Principles in Tumor Biology Thomas Kietzmann TK Latent infection of a cell by DNA viruses Cancerogenesis Principles in Tumor Biology Thomas Kietzmann TK Human Papilloma Viruses and Diseases HPV Types Diseases HPV Type 1, 2, 3 und 4 („low risk“) HPV Type 3 und 10 („low risk“) HPV Type 6 und 11 („low risk“) Verruca vulgaris and plantaris Verruca plana juvenilis Condylomata acuminata and BuschkeLöwenstein tumor HPV Type 16 („high risk“) Bowenoid papulosis, mainly in males as intraepithelial neoplasia of the penis HPV Type 18, 45 and 31(„high risk“) Cervical intraepithelial neoplasia (CIN): which can lead to cervical carcinoma Vulvar intraepithelial neoplasia (VIN) Vaginal intraepithelial neoplasia (VAIN) Anal intraepithelial neoplasia (AIN) HPV Type 13 and 32 („low risk“) Morbus Heck (Verrucae in the mouth) HPV Type 5 und 8 („intermediate risk“) Epidermodysplasia 12 Principles in Tumor Biology Thomas Kietzmann TK Prevalence of various HPV types in cervical cancer Principles in Tumor Biology Thomas Kietzmann TK Human papilloma viruses can cause cancer Low risk HPV -no cancer- High risk HPV -cancer- 13 Principles in Tumor Biology Thomas Kietzmann TK Control of the cell cycle by cyclins cdk1 Cyclin A cdk1 Cyclin B M phase G2 phase Mitogen Cyclin D S phase cdk4/6 G1 phase cdk2 Cyclin A E2F cdk2 Cyclin E Principles in Tumor Biology Thomas Kietzmann TK Function of the retinoblastoma (Rb) protein CDK4/6 CDK2 Cyclin D Cyclin E S phase genes: OH OH Rb OH E2F X OH E2F OH P HPV E7 P Rb OH HPV E7 OH OH Rb P P TK TS DHFR Myc DNA-Pol Cyclin E E2F CDK1 Cyclin A P OH OH 14 Principles in Tumor Biology Thomas Kietzmann TK Cell cycle stop and activation of apoptosis by p53 UV , Damaged DNA Various noxes HDM2 + ATM ATR HPV- E6 OH P p53 p53 labile stabile HDM2 p21 CDK2 Principles in Virology BAX GADD45 Cyclin E CDK1 Cyclin B Apoptosis Thomas Kietzmann TK Key terms and questions What is an ori and what are it´s functions? What are the functions of T antigen during the SV40 infectious cycle? How are circular replicating viral genomes separated? What is a permissive and non-permissive cell/infection? A papilloma virus enters a cell and does not produce progeny virus; however, episomal viral DNA is maintained in the cell and some gene expression occurs. Of which kind of infection is this a sign for? Pappillomavirus can affect the cell cycle; at which point and how doe papilloma viral proteins interfere with the cell cycle? Some papilloma viruses have a high potential to cause cancer; how can this be prevented? 15
