Replication of Small DNA Virus

1. Replication of Small DNA Virus

2. Animal Virus DNA Genomes • Larger size range (5 – 200 kbp) than RNA viruses • Similar to host genome, may use cell machinery for DNA replication & transcription • Problems of dependence on host cell for DNA replication: • Cell must be in S phase for DNA synthesis • DNA synthesis can’t occur at ends of linear DNA molecules (“end problem” due to use of RNA primer)

3. Virus Solutions For Cell Cycle S Phase Requirement • Small DNA viruses only infect cells that have entered the S phase of the cell cycle • Other viruses induce its host cell to enter S phase • Large viruses encode their own enzymes and thus not dependent on cell enzymes

4. Virus Solutions To DNA Synthesis “End Problem” • Use protein primers instead of host cell RNA primer • Circular genome form concatemers (rolling circle model) with no ends • May resort to reverse transcription (host cell telomerase), RNA to DNA

5. Family Papovaviridae • Two groups of similar viruses of animals and humans • Papillomavirus • Polyoma virus, vacuolating virus • Circular dsDNA • Icosahedral capsid, 45-55 nm • Replication occurs in nucleus

6. Genus: Papilloma Virus • “nipple-shaped” projection, tumor • Human papillomaviruses (HPV) 1-48 • Benign warts and tumors (oral/pharyngeal, skin) • Malignant tumors (oral/pharyngeal, genital carcinoma) • Difficult to culture in vitro

7. Genus: Polyoma Virus • “many tumors” in experimental animals • SV40 (simian vacuolating virus) - isolated in 1960 from monkey • JC virus – isolated in 1970 from patient with neurologic disease • BK virus – isolated in 1971 from kidney transplant patient

8. Virus Infections • Asymptomatic – no disease symptoms • Acute – disease symptoms • Persistent – long term • Chronic: infectious virus • Latent: no virus replication, virus reactivation • Transformation – alter cell regulation, tumor production • No infectious virus • Viral DNA, complete or partial

9. SV40: Host / Infections • Primary monkey kidney cell culture = Latent infection • Many experimental animals (host cell lacks some requirement for virus replication) = Nonpermissive (“abortive”) infection • Newborn hamsters = no virus replication, instead Transform cells to many different tumors

10. SV40: Type of Infection Due to Host • Monkey: Acute productive – infectious virus • Monkey: Persistent chronic – low level of virus replication over long term • Monkey: Persistent latent – no virus replication, possible later reactivation of virus • Hamster: Nonproductive – no virus replication; may lead to transformation by disrupting cell regulation; complete or part of viral DNA present

11. JC Virus Infection • Patient with rare fatal neurologic disease – PML (progressive multifocal leukoencephalopathy) • Common infection in young children via respiratory route • Persistent latent infection (no infectious virus) of lungs & kidneys • Host immunosuppression leads to activation of virus that spreads to brain

12. BK Virus Infection • Isolated from urine of renal transplant patient • Common mild respiratory tract infection • Persistent latent infection of lungs and kidney • Host immunosuppression leads to activation of virus • Overt disease is rare

13. Papovavirus Genome: ds DNA • Circular, supercoiled, condensed by cell histones • Papillomavirus, 8 kb; may exist as episome in host cell • Polyoma virus, 5 kb; may integrate into host cell DNA • Promoter, enhancer regions • “early” and “late” genes

14. Virus Infection: Cell Transformation

15. SV40 Genome: Control Region • ori – origin of replication for DNA • PE, PL– promoter region for “early” and “late” mRNA • “21”bp & “72”bp – “early” promoter enhancer regions • T – three different binding sites for large T (tumor) antigen

16. SV40: Entry / Uncoating • Receptor mediated endocytosis • Transport of vesicle to nucleus • Fusion of vesicle with nuclear membrane and virus enters nucleus • Uncoat and release of viral DNA in nucleus

17. SV40: “Early” mRNA Transcription • Uses #1 DNA strand (counter-clockwise) • “early” promoter (PE) directs mRNA transcription • “TATA” box – conscensus sequence for cell RNA pol II • Viral enhancer region for cell DNA binding proteins • One “early” mRNA • Alternative splicing for two mRNA

18. SV40: “Early” Proteins • Large T antigen (multifunctional): • Activate host cell (bind and inactivate cell growth-suppressor proteins: p105 Rb, p53) • Block cell apoptosis (programmed death) • Viral DNA replication • Down-regulate “early” mRNA • Activate “late” mRNA • Role in virus assembly • Small t antigen (viral DNA replication)

19. Tumor (Cellular Growth) Suppressor Genes: Rb, p53 • Cellular genes whose function is to block uncontrolled cell replication • Rb (retinoblastoma susceptibility gene): • Gene product (p105 RB) repress transcription • Mutation results in tumor (uncontolled cell growth) of retina • p53 gene product (p53) leads to: • G1 arrest; contact-inhibition • Apoptosis (programmed cell death) • Inactivation of p53 results in loss of cell division repression

20. SV40: Semi-conservative DNA Replication • Formation of initiation complex: • T antigen (numerous enzymes) • Cell DNA primase-DNA pol • Cell DNA binding proteins • dsDNA opens up, RNA primer • Bidirectional DNA (continous, discontinous strand) synthesis • Forms two dsDNA “loops”

21. SV40: DNA Replication • dsDNA opens up, RNA primer • Bidirectional DNA (continous, discontinous strand) synthesis • Forms two dsDNA “loops”

22. SV40: “Late” mRNA Transcription • Follows viral DNA replication • Uses #2 DNA strand (clockwise) • T antigen binds near Ori and blocks “early” mRNA transcription • T antigen binds to Enhancer region and turns-on “late” promoter (PL) • One “late” mRNA • Alternative splicing for two mRNAs

23. SV40: “Late” Proteins • Two “late” mRNAs (both are bicistronic) • Each mRNA translates for two proteins using different start codons: • #1 mRNA: VP 1, agnoprotein • #2 mRNA: VP2, VP3

24. SV40: Maturation • Assembly of virus in nucleus • Release by cell lysis • In cell culture, produce ~ 104 - 105 progeny virus

25. Oncogenes • Genes encoding the proteins originally identified as the transforming agents of oncogenic viruses (SV40; T antigen) • Some oncogenes were shown to be normal components of cells • For retrovirus: • v-onc is viral oncogene • c-onc is cellular version and termed proto-oncogene and are cellular growth control genes • Most likely v-onc “stolen” from host cell

26. Nonpermissive SV40 Infection • “abortive” infection as viral DNA can not replicate in host cell • Viral T antigen stimulates cell DNA replication and cell division; continued stimulation may lead to cell transformation • Viral DNA may integrate into cell DNA by random recombination event, results in stable cell transformation

27. pSV40T7 Recombinant Expression Vector • SV40 signals for mRNA transcription of cDNA cloned into polycloning site • SV40 “early” promoter (PE) • SV40 polyadenylation site

28. Reading & Questions • Chapter 16: Replication Strategies of Small and Medium-Sized DNA Viruses

29. Class Discussion – Lecture 9 • 1. Why does SV40 require its host cell to be active metabolically (in S phase of cell cycle)? • 2. How does SV40 insure that its host cell is active metabolically? • 3. Is SV40 mRNA transcription and DNA replication similar to its host cell?