Unit 6.2 Gene Expression and Replication in Small DNA Viruses

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, lifelong (usually) • 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 (Pvy) 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 capsomers (12 pentavalent and 60 hexavalent capsomers) 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 Small T Inton poly A tail 2694 4571 4638 5163 Sm T Lg T Lg T Large T Intron 2694 4571 4918 5163 poly A tail 2694 Large/Small T Antigens 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. 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.

12. 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.

13. Bidirectional replication from an origin yields a theta form

14. Summary/Chronology of T Functions Early gene expression produces both T and t T is the major regulatory protein It also determines host range. T controls the switch from early (pre-replication) to late (post-replication) gene expression

15. Summary/Chronology of T Functions • T stimulates host cell proliferation • Relieves S phase block mediated by Rb (retinoblastoma) family proteins. • Blocks p53-mediated cell death • Stimulates replication of DNA by host cell machinery • Directs machinery to viral DNA

16. Summary/Chronology of T Functions • T binds at Sites I,II,III of control region to block its own expression: autoregulation, down-regulation • 21 bp GC repeats and 72 bp enhancer binding. • Promotes assembly of transcription complex • Binds to TBP, TAFs and also specific TS factors • Stimulates late gene expression

17. 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.

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

19. 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

20. Readings • POV textbook: Ch. 8 pp 240-265 (leave out sections on retroviruses, TAR and tat), also Ch. 9 pp 288-297. • Also see: http://microbiology.columbia.edu/G6021/007DNAreplication08.pdf