Unit 6.2 Gene Expression Unit 6.2 Gene Expression and Replication in Small and Replication in Small

DNA VirusesDNA Viruses

Small DNA Viruses of Animals Small DNA Viruses of Animals (Papovaviruses)(Papovaviruses)

What do the small DNA viruses have to What do the small DNA viruses have to tell us about viral approaches to gene tell us about viral approaches to gene

expression and replication?expression and replication?What are their issues, problems and What are their issues, problems and

strategies?strategies?Are there common themes?Are there common themes?

““Papovaviruses”Papovaviruses”

Originally-Originally-

PaPapilloma, pilloma, PoPolyoma, lyoma, VaVacuolating Virus (aka cuolating Virus (aka SV40)SV40)

Now-Now-

Papillomaviridae (eg HPV)Papillomaviridae (eg HPV)

Polyomaviridae (includes SV40)Polyomaviridae (includes SV40)

Polyomaviridae 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

Polyomaviridae Such as SV40 Polyomaviridae Such as SV40 Are Important in BiologyAre 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.

Polyomaviruses and Particle StructurePolyomaviridae 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.

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.

VP1 subunits are arranged in 72 capsomeres (12 pentavalent and 60 hexavalent capsomeres)

Polyomavirus (SV40) Genome Map and ProteinsEarly Proteins

Large T antigen - Multifunctional protein

Small t antigen - Involved in thestimulation 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.

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

Features of Regulatory RegionFeatures of Regulatory Region

Close resemblance to cellular regulators

SV40 Early Region Expression

4571 4638 5163poly A tail

2694

2694 4571 4918 5163

Large/Small T Antigens

Sm T

Lg T Lg T

Small T Inton

Large T Intron

poly A tail2694

Two splice sites occur on the mRNA to regulate the large and small T antigens. Alternate splicing produces two mRNAs from the same geneEarly proteins are sufficient to elicit S phase in host

Early Protein FunctionsEarly Protein FunctionsEarly gene expression produces both T and tEarly gene expression produces both T and t

T is the major regulatory proteinT is the major regulatory protein

Covalently modifiedCovalently modified

Imported to nucleusImported to nucleus

Regulates viral gene expression, viral DNA replication, Regulates viral gene expression, viral DNA replication, interacts with host proteinsinteracts with host proteins

t interacts with cytoplasmic proteins to help t interacts with cytoplasmic proteins to help induce movement through cell cycleinduce movement through cell cycle

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.

Large T Antigen has Multiple Functions and Host Protein Interactions

T Antigen Binding to SV40 DNAT 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.

Bidirectional replication from an origin yields Bidirectional replication from an origin yields a theta forma theta form

T and host cell proliferationT and host cell proliferation

T stimulates host cell proliferationT stimulates host cell proliferation Relieves S phase block mediated by Rb Relieves S phase block mediated by Rb

(retinoblastoma) family proteins.(retinoblastoma) family proteins. Blocks p53-mediated cell deathBlocks p53-mediated cell death ““tumor suppressor” genestumor suppressor” genes Interferes with regulator HSC70Interferes with regulator HSC70

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 consensus1998 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.

Small DNA Viruses-Issues and Small DNA Viruses-Issues and ProblemsProblems

Small genomeSmall genome

Extreme dependence on hostExtreme dependence on host

Small DNA Viruses-StrategiesSmall DNA Viruses-StrategiesGenetic EconomyGenetic Economy

Dependence on host machineryDependence on host machinery

Overlapping genesOverlapping genes

Multifunctional proteinsMultifunctional proteins

Molecular mimicryMolecular mimicry

Manipulate intracellular environmentManipulate intracellular environment

Functional Clustering of GenesFunctional Clustering of Genes

Temporal ExpressionTemporal Expression

ReadingsReadings

POV textbook: Ch. 8 pp 240-265 (leave POV textbook: Ch. 8 pp 240-265 (leave out sections on retroviruses, TAR and tat), out sections on retroviruses, TAR and tat), also Ch. 9 pp 288-297.also Ch. 9 pp 288-297.

Also see: Also see: http://www.microbiologybook.org/mhunt/http://www.microbiologybook.org/mhunt/

dna1.htmdna1.htm