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, 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 ~ 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?