1. Unit 6.2 Gene Expression and Replication in Small DNA Viruses
Small DNA Viruses of Animals (Papovaviruses)

2. What do the small DNA viruses have to tell us about viral approaches to gene expression and replication? What are their issues, problems and strategies? Are there common themes?

3. “Papovaviruses” Originally-
Papilloma, Polyoma, Vacuolating Virus (aka SV40)
Now-
Papillomaviridae (eg HPV)
Polyomaviridae (includes SV40)

4. Polyomaviridae Ubiquitous in mammals and birds
Mild disease in mammals, asymptomatic
Immunocompromised patients
Serious or fatal in birds
Small ds DNA viruses about 5000 bp
unenveloped

5. Polyomaviridae Such as SV40 Are Important in Biology
Understanding the structure of supercoiled DNA.
Identification of eukaryotic origins of DNA replication.
Insights into eukaryotic chromosomal DNA replication and cell cycle regulation.
Elucidation of promoter organization for mRNA Synthesis.
Discovery of RNA transcription enhancers.
Understanding of mechanisms of negative and positive
regulation of gene expression
Identification and characterization of viral oncogenes and host tumor suppressor genes.

6. Polyomaviruses and Particle Structure
Polyomaviridae Members:
Polyomavirus was discovered in the parotid salivary glands of mice and found to cause a variety of tumors in new born mice.
Simian Virus 40 was discovered in Rhesus monkey kidney cells. SV40 was
found to transform African Green Monkey Kidney cells and may be linked
to rare human cancers (Nonhodgkins Lymphoma & Mesothelomia).
JC and BK, two Human polyomaviruses, were discovered in the 1970’s and found to be related to SV40. Human JC can cause brain tumors in owl monkeys.
Virus particles are unenveloped icosahedra composed of a major protein, VP1 and two minor proteins, VP2 and VP3.
VP1 subunits are arranged in 72 capsomeres (12 pentavalent and 60 hexavalent capsomeres)
The viral genome is an 5.2 KB double stranded DNA that is supercoiled minichromosome associated with cellular histones accounting for about 20% of the total virion protein.

7. Polyomavirus (SV40) Genome Map and Proteins
Early Proteins
Large T antigen - Multifunctional protein
Small t antigen - Involved in the stimulation of cell proliferation
Late Proteins
Agnoprotein - Facilitates perinuclear-nuclear localization of VP1.
VP1 - Major capsid protein; capable of self-assembly into capsids.
VP2 - Minor capsid protein (myristoylated).
VP3 - Minor capsid protein (co-linear with the VP2 C-terminal region).
Ori - (Origin of Replication) - DNA sequence originating DNA synthesis – at control region.

8. SV40 mRNA Transcription and Processing
The genome is divided into early and late mRNA transcription units.
Early mRNAs are transcribed in a “counter clockwise” direction before DNA replication.
Early mRNAs are differentially spliced into two mRNAs for translation of the Large and Small tumor antigens.
Late mRNAs are transcribed in a “clockwise” direction after DNA replication.
Late mRNAs are also differentially spliced to produce the agnoprotein, and VPs.
All mRNAs are capped and polyadenylated-nucleus.
Late microRNAs for regulation of T

9. Features of Regulatory Region
Close resemblance to cellular regulators

10. SV40 Early Region Expression
poly A tail
2694
Large/Small T Antigens
Sm T
Lg T
Small T Inton
Large T Intron
Two splice sites occur on the mRNA to regulate the large and small T antigens.
Alternate splicing produces two mRNAs from the same gene
Early proteins are sufficient to elicit S phase in host

11. Early Protein Functions
Early gene expression produces both T and t T is the major regulatory protein Covalently modified Imported to nucleus Regulates viral gene expression, viral DNA replication, interacts with host proteins
t interacts with cytoplasmic proteins to help induce movement through cell cycle

12. Large T Antigen has Multiple Functions and Host Protein Interactions
Viral DNA Replication
DNA binding activity at origin of replication (ORI).
Initiation/elongation of viral DNA replication through pol alpha.
ATP-dependent helicase for DNA unwinding.
Host Cell Proliferation
Inactivates host p53 (to by pass G1 arrest and prevent apoptosis).
Inactivates the pRB family of proteins (to release E2F transactivator).
stimulates resting cells to move thru the cell cycle and replicate DNA.
Viral Gene Expression
Represses its own synthesis
Activates late viral gene expression via RpolI - functions as a transcription factor associated with TFII complexes.

13. T Antigen Binding to SV40 DNA
The T-antigen is composed of several functional parts, connected by flexible linkers. At one end, a helicase domain assembles with several other copies of the protein to form a six-fold ring.The hole is just big enough to encircle a DNA double helix. The central domain has a small patch, shown here in green, that binds specifically to the regulatory region in the SV40 genome, anchoring the T-antigen complex in the proper place. The third domain interacts with cellular proteins, directing the various stages in the viral life cycle. It is shown here on the right bound to the Rb protein, shown in red Twelve copies of the protein assemble around the DNA to form a long tube.

14. Bidirectional replication from an origin yields a theta form

15. T and host cell proliferation
T stimulates host cell proliferation
Relieves S phase block mediated by Rb (retinoblastoma) family proteins.
Blocks p53-mediated cell death
“tumor suppressor” genes
Interferes with regulator HSC70

16. SV40 and Cancer: a Timeline [Ferber, D., Science, 296:1012 (2002)]
1955 Salk introduces polio vaccine (injectable inactivated poliovirus).
1960 Monkey kidney extracts used to make vaccine shown to cause rare types of tumors in hamsters; SV40 discovered in extracts.
1961 SV40 is shown to cause four rare types of tumors in hamsters.
U.S. orders vaccine makers to eliminate SV40 from Salk vaccine.
1962 Sabin introduces oral polio vaccine.
1963 NCI study: No increased incidence of cancer in recipients (children).
1981 NCI study: No increased incidence of cancer in recipients (Now teenagers).
1992 SV40 detected in childhood brain tumors.
1994 SV40 detected in rare bone/lung cancers.
1995 NCI study: Tumors screened, found to be negative.
1997 NIH-sponsored SV40 consensus conference: no consensus
1998 Multi Lab study confirms SV40 in mesothelioma.
NCI study: Still no increase in cancers (now after 3 decades).
2001 Second NIH consensus conference: SV40 present in mesothelioma and perhaps other tumors; may cause cancer.
2002 SV40 detected in >40% of cases of non-Hodgkin’s lymphoma.

17. Small DNA Viruses-Issues and Problems
Small genome Extreme dependence on host

18. Small DNA Viruses-Strategies
Genetic Economy
Dependence on host machinery
Overlapping genes
Multifunctional proteins
Molecular mimicry
Manipulate intracellular environment
Functional Clustering of Genes
Temporal Expression

19. Readings
POV textbook: Ch. 8 pp (leave out sections on retroviruses, TAR and tat), also Ch. 9 pp
Also see: