CHAPTER 21: POLYOMAVIRUS Simian virus 40 was found as a contaminant of Salk poliovirus

vaccine
Polyomaviridae- Greek poly (many) and -oma (tumor)
VIRION Simian virus 40 (SV40): discovered 10 years ago as a
• Naked icosahedral capsid (T = 7). contaminant of rhesus monkey kidney cell cultures that were
• Diameter 45 nm. being used for production of the Salk inactivated poliovirus
• Formed from 72 capsomers, pentamers of VP1. vaccines.
GENOME - Usually cause tumors only under very specific conditions:
• Circular dsDNA, 5.3 kb. o Injection of high titers of virus into particularly
• DNA: packaged as “minichromosome” with nucleosomes formed
susceptible animals shortly after birth.
from cellular histones.
- Not “professional” tumor viruses.
GENES AND PROTEINS
• Two transcription units: early and late. - Can occasionally transform a normal cell into a tumor cell
• Divergent transcription from a central control region containing due to their complex interactions with cellular signaling
origin of DNA replication. pathways that facilitate high levels of viral DNA replication
• Differential splicing produces 3 to 4 mRNAs from each and progeny virus production
transcription unit. - Mouse polyomavirus and simian virus 40 are closely
• Early proteins (T antigens) regulate cell cycle and direct DNA related and initially grouped with the papillomaviruses
replication. o Polyomaviridae and Papillomaviridae was later on
• Late proteins (VP1, 2, 3) make virus capsid. reclassified due to differences in genome size and
VIRUSES AND HOSTS organization and in virus replication strategies
• Mouse polyomavirus
• Simian virus 40
Human polyomaviruses are widespread but cause disease
• Polyomaviruses infecting birds, rodents, cattle
only rarely
• Five human viruses: BK, JC, MC, WI, and MC viruses.
DISEASES
• Usually persistent, non-symptomatic infections. Five distinct human polyomaviruses are presently known: BK,
• Progressive multifocal leukoencephalopathy (PML): rare JC, MC, WI, and MC viruses.
demyelinating disease caused by human polyomavirus JC. - Appear to be widespread in the human population and are
DISTINCTIVE CHARACTERISTICS acquired during early childhood, but usually cause no
• Dependent on cellular enzymes for RNA synthesis and apparent disease.
processing, and DNA replication. BK and JC viruses: first isolated in the early 1970s.
• T antigens interact with multiple signaling pathways and activate - Progressive multifocal leukoencephalopathy (PML): rare
the cell cycle to facilitate viral DNA replication. but fatal demyelinating disease caused by JC virus
• T antigens are oncogenes that can transform nonpermissive cells o Particularly in immunosuppressed individuals,
in vitro.
including patients suffering from AIDS.
• Tumorigenic in animals when administered at high conc, but
WI and KU viruses: recently discovered in the respiratory
rarely cause tumors in nature.
tracts of children
Mouse polyomavirus was discovered as a tumor-producing - Association with human disease is not clear.
infectious agent MC virus: recently isolated from Merkel cell carcinomas.
- MC DNA: found in a large proportion of these rare but
Mouse polyomavirus: commonly found in wild mice and their aggressive human skin tumors
inbred laboratory cousins but causes little or no overt disease. - Viral DNA: integrated in the cellular genome and contains
- Can cause multiple tumors in parotid glands, skin, kidneys, mutations that inactivate its ability to replicate.
bone, lungs, liver, and many other tissues when injected - Appears mainly in immunosuppressed individuals or in
into susceptible rodents and other mammals at high conc people over 65.
o Can grow rapidly and can eventually kill the host
animal. Polyomaviruses are models for studying DNA virus
- Distinguished from retroviruses that cause mouse replication and tumorigenesis
leukemia by its smaller size and greater stability.
- Can cause tumors at numerous sites in experimental Mouse polyomavirus and simian virus 40 have: have been
animals. studied intensively as models for understanding
Ludwig Gross: discovered that extracts of mouse tissues tumorigenesis due to their ability to transform cells in vitro
caused salivary gland tumors when injected into baby mice and cause tumors in experimental animals
while studying the transmission of leukemia in mice (early - Also used for studying mammalian RNA synthesis and
1950s) processing, DNA replication, and signal transduction due
- Later on showed that these tumors were caused by a to their small number of genes and their strong
filterable agent (Gross, S. Stewart and Bernice Eddy) dependence on cellular macromolecular synthesis

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Polyomavirus capsids are constructed from pentamers of the - Supercoiled DNA: migrates more rapidly during
major capsid protein electrophoresis in agarose gels than relaxed circle or linear
DNA since the supercoiled form is more compact.
Surface capsid: made from 72 capsomers visible by EM, each - Nicked circular or linear DNA: bind more of the
made of five molecules of the major capsid protein VP1 fluorescent dye ethidium bromide than covalently closed
- Capsomers: linked by the flexible carboxy-terminal arms circular DNA since the strands can freely rotate about each
of VP1, which extend across the gap and embrace VP1 other and therefore unwinding is less constrained.
molecules in neighboring capsomers, stabilizing the capsid
structure Polyomavirus genes are organized in two divergent
- VP1: major capsid protein; forms stable pentamers, not transcription units
hexamers, and these pentameric capsomers fit into
“sixfold holes” on the surface of the capsid. - Most polyomaviruses code for only 6 to 8 proteins as a
- VP2 or VP3: minor capsid proteins located below the result of their small genome size.
capsid surface. - Viral genes: organized as two families: early and late gene
- Myristate (C14 saturated fatty acid): common protein Divergent transcription: transcription pattern
modification enables interaction of proteins with lipid - Early genes: transcribed on one DNA strand from near the
membranes “12 o’clock” position in a counterclockwise direction
o Linked to N-terminal amino acid of VP2 - Late genes: transcribe on the complementary DNA strand
in a clockwise direction
The circular DNA genome is packaged with cellular histones Intergenic region or control region: noncoding region of
approx 400 nucleotides that contains several important
Viral genome: 5.3-kb circular, double-stranded DNA molecule sequence elements involved in both transcription and genome
- Packaged in nucleosomes formed from two molecules replication
each of the four cellular histones H2A, H2B, H3, and H4. Polyadenylation signal (5'-AAUAAA-3'): specifies the 3' ends of
- Toroidal coil: condenses the viral DNA into a each transcript
“minichromosome” containing about 25 such.3
nucleosomes giving a total of 200 histone molecules per Virions enter cells in caveolae and are transported to the
viral DNA molecule. nucleus

Circular DNA becomes supercoiled upon removal of histones Mouse polyomavirus and simian virus 40: BINDS to sialic acid-
containing glycoproteins and gangliosides on the cell surface
Supercoiled form of DNA: results from the toroidal coilinga of and subsequently internalized in caveolae (specialized vesicles
the double helical DNA around the nucleosomes in the intact formed at the plasma membrane.)
minichromosome. - After internalization, virions are transported through
- Toroidal coils: distributed around the DNA molecule after cytosol to the lumen of ER near nuclear membrane
removal of histones - Then viral DNA will be transported to the nucleus
- Becomes coiled when histones are removed using sodium VP2 molecules: mediate the passage of virions across the ER
dodecyl sulfate and proteins are extracted w/ organic membrane
solvent such as phenol - Lie below the surface of each capsomere.
Relaxed circular DNA: results from the cleavage of
phosphodiester bond bet two nucleotides of one of the DNA Extrusion of N-terminus of VP2 through capsid surface (due to
strands conditions w/in ER) → N-terminal myristate will interact with
Topoisomerase I: enzyme which can alternatively unwind lipid membranes → release of the virions into the cytosol.
supercoils by breaking a phosphodiester bond, allowing one of
the broken ends to rotate around the opposite DNA strand, Transport and release of viral DNA into the nucleus appears to
and then resealing the phosphodiester bond. be a two-step process.
DNA gyrase: specialized enzymes found in bacteria w/c can - Capsids can dissociate in vitro by treatment with reducing
catalyze the supercoiling of relaxed DNA molecules agents (breaks disulfide bonds) and chelating agents
- Carry out the reverse reaction of topoisomerase I, using (remove divalent cations bound to capsid proteins).
ATP as an energy source. - Upon entry into the cytosol, the capsid dissociates but VP1
remains associated with the viral minichromosome
Supercoiled DNA can be separated from relaxed or linear - The nuclear localization signal on VP1 directs this complex
DNA molecules to the nucleus via the nuclear pores.
o Once in the nucleus, VP1 dissociates from the viral
Supercoiled DNA has diff physical properties than relaxed minichromosome, which remains intact during
circular DNA and linear DNA of the same nucleotide sequence transcription and DNA replication.
and molecular weight.
E.g.

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The viral minichromosome is transcribed by cellular RNA 1. Small T antigens (STAg)
polymerase II - 195-amino acid protein
- Ribosomes translate first 191 codons and continue
- A complex process that involves numerous cellular translation in the same reading frame downstream of the
proteins. splice junction.
Transcriptional enhancer: contains a number of consensus - They encounter a UAG stop codon only 12 nt after the
DNA sequences that are recognized by a variety of cellular splice junction
transcription factors. Found within the intergenic region Excision of the smallest intron (48 nt) → removal of 16 triplet
- Bound transcription factors: facilitate the association codons (5’ splice site at nucleotide 748 and 3’ splice at
with viral DNA of the multiple cellular proteins required nucleotide 797)
for transcription.
o TATA-binding protein (TBP): present in a complex 2. Middle T antigen (MTAg)
called TFIID, which binds to a consensus TATA box - 421 amino acid protein
sequence element on the viral DNA - Ribosomes translating this mRNA make a protein that
▪ TFIID recruit RNA polymerase II and a number of contains the same first 191 amino acids as STAg but
general transcription factors (TFII A to H) to a continue in a different RF (–1) downstream of the splice
site approximately 30 nt beyond the TATA box. junction.
- Initiators: alternative transcription start sequences o In RF –1, the protein is extended to a size of 421
present at the beginning of many viral and cellular genes amino acids before a termination codon is
that lack TATA boxes encountered.

Four early mRNAs are made by differential splicing of a 62-nt intron excision → removal of 20 triplet codons plus 2 nt
common transcript (5’ splice site at nucleotide 748 and 3’ splice at nucleotide 811)

2.7-kb EARLY TRANSCRIPTS of mouse polyomavirus 3. Large T antigen (LTAg)

- Capped at their 5' ends, and cleaved and polyadenylated - protein 785 amino acids in length (708 amino acids for
at their 3' ends, by protein machinery used by the cell for simian virus 40).
generating its own mRNAs - Ribosomes translating this mRNA make a protein that
- Undergo alternative splicing w/in the nucleus to generate shares the same first 79 amino acids with STAg & MTAg
four different mRNA species. since the 5' splice site lies exactly 79 triplet codons beyond
o Tumor antigens, or T antigens- mRNAs w/c code for the initiator AUG.
early proteins that were first detected in virus- o RF downstream of the splice junction is now +1;
induced tumors codes for a totally different amino acid sequence
Alternative splicing: because different triplet codons are recognized by
- Occurs between two possible 5' splice sites (nt 411 & 748) the ribosome.
and two possible 3' splice sites (nt 797 & 811) o RF does not terminate until very near the 3' end of the
- Approxequal amounts of the four mRNAs are made. mRNA

Spliceosome: ribonucleoproteins which bind to both ends of 385-nt intron excision → removes 128 codons plus 1 nt (5’
the intron as well as to an internal branch site near the 3' splice splice site at nucleotide 411 and 3’ splice at nucleotide )
site.
Unusually small polyomavirus introns evolved to reduce 4. tiny T antigen (TTAg).
splicing efficiency between the 5' splice site (nt 748) and either - 85-amino acid protein
of the two 3' splice sites. - Has the same 79 N-terminal amino acids as the other T
- If these small introns are artificially lengthened by antigens.
insertion of as little as 40 nt into the viral genome, almost - A 399-nt intron (133 triplet codons) keeps the ribosome
all splicing events target the nearest 5' splice site at nt 748, in reading frame 0, as with STAg mRNA.
and no large T antigen mRNA is made. o 3' splice site at nt 811 lies just downstream of the
- These small introns therefore ensure a balanced UAG that terminates STAg translation.
production of the different early mRNAs. o Another termination codon, UGA, is present just 7
codons beyond the splice junction, making an 85-
T antigens share common N-terminal sequences but have amino acid protein
different C-terminal sequences
Between nt 797 and 811: 2 diff RF used to make parts of STAg
Ribosomes initiate translation on all four early mRNAs at the and LTAg,
first AUG codon they encounter (nt 175) → reading frame 0 Between nt 811 and 831: all three RF used to make parts of
MTAg, LTAg, and TTAg.
Four mouse polyomavirus early mRNAs:

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Simian virus 40: has two 5' splice sites analogous to those of - Mutated and highly active form is encoded by the acutely
mouse polyomavirus, but only one 3' splice site. transforming retrovirus Rous sarcoma virus, which
- Makes a small and large T antigen, but no middle T produces cancer in chickens
antigen. - Loses a phosphate group at tyrosine 527 and
- Rare doubly spliced simian virus 40 mRNA codes for a phosphorylates itself at tyrosine 416 when associated
protein similar to mouse polyomavirus tiny T antigen. with middle T antigen
o greatly stimulate its protein kinase activity.
T antigens bring resting cells into the DNA synthesis (S) phase - Phosphorylates several tyrosine residues on MTAg itself.
of the cell cycle - The following set of cellular proteins will then bind to
phosphotyrosines:
Early proteins: principal function is to enable viral DNA o Srchomology 2 domain protein (Shc)
replication. o Phosphatidylinositol 3-kinase (PI3K)
- Induce entry of the cell into the DNA synthesis (S) phase o and phospholipase C (PLC)
of the cell cycle. - These proteins will then be activated by phosphorylation
Most cells are differentiated in a resting (G0) state (not of activated c-Src upon binding to middle T antigen
dividing) - Leads to a cascade of signaling events
- Under these conditions, enzymes and precursors needed Phosphorylated Shc: activates MAP kinase pathway through
to support cellular or viral DNA synthesis are not normally an activated Ras protein
present in the cell nucleus. PI 3-kinase: synthesizes phosphatidylinositol 3-phosphate,
a phospholipid that controls proliferation, vesicle trafficking,
The T antigens of mouse polyomavirus and simian virus and organization of the cytoskeleton.
40: have been shown to interact with numerous cellular Phospholipase C: cleaves phosphatidylinositol, generating
signaling pathways, leading to stimulation of the cell cycle. inositol triphosphate, which releases calcium ions from the
endoplasmic reticulum.
Small T antigen inhibits protein phosphatase 2A and induces
cell cycling Large T antigen activates or suppresses transcription of
cellular genes by binding to a number of important cellular
Small T antigen: binds/forms a complex with protein regulatory proteins
phosphatase 2A (PP2A) to inhibit the phosphatase activity of
this protein Large T antigen: a complex protein with a surprisingly large
PP2A: important in several cellular signaling pathways by number of different functions.
removing phosphate groups that stimulate the activities of - Most are localized in the nucleus of infected cells, some
proteins in those pathways. are found in the cytoplasm and at the plasma membrane.
Mitogen-activated protein kinases (MAP kinases): cellular - Interacts with a number of cellular proteins that control
targets of PP2A cell metabolism and the cell cycle.

Phosphorylation of MAP kinase at specific serine & threonine The nuclear localization signal of simian virus 40 large T antigen
residues → activates the nuclear transcription factor AP-1 → was one of the first such signals identified.
activates transcription of the cyclin D1 (important activator of
the cell cycle) 1. Retinoblastoma protein (pRb) controls cell cycle and S-
phase gene expression.
Inhibition of PP2A activity by STAg allows MAP kinases to
remain phosphorylated and therefore enzymatically active The retinoblastoma protein (pRb): named due to mutations in
- STAg indirectly stimulates the MAP kinase pathway and the Rb gene
helps bring the cell into the S phase. - one of the major cell-cycle control proteins
- can lead to retinal tumors in humans.
Middle T antigen stimulates protein tyrosine kinases that pRb, p107 and p130 (related proteins): repress transcription
signal cell proliferation and division from a large number of cellular genes when bound to members
of the E2F family of transcriptional activator proteins.
Middle T antigen: localized at the PM of infected cells, where
it is inserted via a hydrophobic region near its C-terminus Phosphorylated pRb → dissociation of pRb from E2F partner
- Can also bind to and inactivate PP2A since it contains most → cellular genes transcription* → cell cyle entry → protein
of the amino acid sequences of small T antigen expression required for cellular and viral DNA replication
- Major functions: association with several cellular protein
tyrosine kinases (c-Src) LTAg has a conserved binding site for the Rb protein
C-Src:
- Modifies number of other cellular proteins by Binding of LTAg to pRb → mimics pRb phosphorylation →
phosphorylating specific tyrosine residues.

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releasing E2F → transcription of cellular genes targeted by E2F
(DNA polymerase alpha, thymidine kinase, DNA ligase, Binding of LTAg to these proteins can lead to both stimulation
histones) and repression of gene expression.
E.g.:
A number of DNA viruses make early proteins that interact p300: associated with a number of cellular transcription
with pRb and stimulate the cell cycle. factors, including p53 and AP-1.
- Believed to cooperate with these factors in stimulating
2. The DNA J domain acts as a cochaperone to dissociate pRb transcription by acetylating nucleosomal histones,
from E2F. therefore reducing their affinity for DNA and making DNA
DNA J domain: N-terminal region of large T antigen required more accessible to components of the transcription
for gene activation via Rb pathway machinery.
- Cochaperone protein (first described in E. coli as a - Binding of LTAg to p300 can inhibit its transcriptional
bacterial protein required for replication of phage lambda coactivation function and therefore reduce transcription
DNA) of genes targeted by these factors.
- Can complement DNA J-minus mutants of E. coli and However, LTAg is known to increase transcription of many
enable phage lambda DNA replication other genes, probably via its interactions with the machinery
- Believed to act by helping to separate the Rb protein from that assembles RNA polymerase II at promoters.
a complex containing E2F and other proteins, thus
stimulating transcription of cell-cycle and S-phase genes. Large T antigen hexamers bind to the origin of DNA
Hsp70 (DNA K in E. coli): cellular chaperone where DNA J and replication and locally unwind the two DNA strands
eukaryotic counterparts interact and take part in ATP
dependent protein folding and unfolding reactions vital in LTAg binds to viral DNA and directs the assembly of a number
assembling or disassembling multiprotein complexes. of cellular proteins that carry out DNA replication once the cell
has entered the S phase and viral DNA replication has begun.
3. p53 blocks the cell cycle and induces apoptosis in response
to virus infection. DNA-binding domain of LTAg: recognizes G(A/G)GGC
pentanucleotide sequences on dsDNA.
p53: cellular control protein first discovered as a 53-KDa Simian virus 40 DNA and mouse polyomavirus DNA: have 12
protein present in immunoprecipitates of simian virus 40 large such sequences that lie within a 160-nt span in the intergenic
T antigen. region.
- Binds and activates transcription of genes that can lead In mouse polyomavirus: these 12 binding sequences are
either to blockage of the cell cycle or apoptosis grouped in four sites denoted 1/2, A, B, and C
- Controls the cell cycle by the fact that majority of human - LTAg molecules bind cooperatively to these sequences
tumors have undergone mutations in the p53 gene. and assemble at a specific site denoted the DNA
o For a tumor cell to thrive, it must rid itself of the replication origin (Ori) to form two circular hexamers that
control exercised by the p53 protein. enclose the DNA like a wheel on an axle.
- Rapidly degraded and present in very low conc in the cell EM and x-ray crystallographic studies: revealed the detailed
and is inactive as a transcription factor. structure of the hexamer form of large T antigen
o Infections by polyomavirus can lead to increases in - Each LTAg monomer in a hexamer binds a molecule of
the conc and the activity of p53. ATP.
▪ Partly mediated by LTAg interacting with pRb, - LTAg acts as a DNA helicase.
which raise levels of p53.
- However, binding of LTAg to p53 suppresses its ATP hydrolysis to ADP → release of ADP molecule →
transcriptional activation function, allowing the cell to conformational change →rotates adjacent protein domains
enter S phase and avoiding p53- mediated apoptosis. with respect to each other → changes the size of the central
o This allows the infected cell to survive long enough to opening of the hexamer in contact with the DNA → 5 to 6 bp
produce progeny virus particles. of dsDNA pulled through the hexamer by the motion of a β-
- Both papillomaviruses and adenoviruses make analogous hairpin in contact w/ DNA → locally separated two strands
proteins that interact with p53.

4. p300 and general transcription factors control levels of Large T antigen assembles the cellular DNA synthesis
transcription of viral and cellular genes. machinery to initiate viral DNA replication
LTAG interacts directly with a number of cellular proteins
involved in transcription including: Generates a localized region of ssDNA that can now serve as a
- TATA-binding protein (TBP) template for DNA replication.
- TFIIB (general transcription factor)
- p300 (transcriptional co-activator which has histone DNA-bound LTAg hexamers bind to three cellular proteins
acetylase activity) required for initiation of DNA replication:

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Replication protein A: binds to the unwound DNA strands and - LTAg concentration increases, early transcription
keeps them from renaturing into a double-helical form. decreases.
DNA polymerase α/primase: binds to ssDNA and lays down a o controls its own levels of synthesis.
short (6–9 nt) RNA primer that remains H-bonded to the DNA
template. 2. Activation or induction of new transcription factors.
- The same enzyme then extends the primer by adding a - The combined effects of STAg, MTAg & LTAg on cellular
short (20–30 nt) DNA molecule. signaling pathways and the ability of LTAg to bind to a
- Primers are formed and elongated on both DNA strands, variety of transcriptional repressors and activators,
and as a result, bidirectional DNA synthesis is initiated. changes the mix of transcription factors available to bind
Topoisomerase I: required to help release the tension created to the transcriptional enhancer.
in the circular, dsDNA molecule as the DNA polymerase and o Changed factors specifically activate the late
helicase proceed along the DNA molecule, twisting the double promoters.
helix in front as it is unwound behind. o the exact mix of transcription factors that activate
late transcription is not fully understood
Three other cellular proteins (replication protein C (RPC),
proliferating cell nuclear antigen (PCNA), and DNA polymerase 3. Derepression of late transcription by dilution of a
δ (delta), are recruited to the complex repressor.
Replication protein C: helps to dissociate DNA polymerase - Increase number of viral DNA molecules = smaller
α/primase and load DNA polymerase δ onto the 3' ends of the proportion of viral DNA contain bound repressor
growing DNA chains. o Allows activation of the late promoter and increased
PCNA: binds to DNA polymerase δ and ensures that it does not levels of late transcription.
dissociate from the DNA template. - Simian virus 40 late transcription: initially blocked by
DNA polymerase δ: then extends the DNA chains. binding of a cellular protein to viral DNA near the late
promoter.
LTAg continues to act as a helicase by unwinding the template o Concentration of this repressor in the cell is limited
DNA as the leading strands are extended toward each
replication fork 4. Increased efficiency of processing and export of late
RNAs.
As DNA synthesis progresses, cellular histones present in the - Activation of late transcription leads to initiation at
S-phase nucleus assemble on the growing DNA chains to form multiple sites upstream of this promoter sequence, and
nucleosomes. RNAs with these 5' ends are more efficiently processed
- Final result: synthesis of two circular, double-stranded into mRNAs and exported to the cytoplasm
viral minichromosomes. The progeny DNAs can be used as - Most mouse polyomavirus: late transcripts initiate at a
a template for further transcription or can be replicated. single promoter sequence.
o RNA transcripts with this 5' end are not efficiently
High levels of late transcripts are made after DNA replication exported to the cytoplasm, perhaps because they are
begins missing a critical RNA export sequence.

Initiation of late transcription on both mouse polyomavirus 5. Readthrough RNAs hybridize with early transcripts
and simian virus 40 DNA occurs at multiple promoter sites and lead to their degradation.
within a 100-nt region that overlaps with the transcriptional - The presence of complementary early and late RNAs in the
enhancer. nucleus leads to the formation of dsRNAs, which are
- Most of late promoter sequences are not associated with degraded by cellular enzymes.
a consensus TATA box. o Most of the early transcripts are degraded compared
to late transcripts which are in excess
Before DNA replication: low but detectable levels of late - Most RNA polymerases that transcribe the late genes of
transcripts are made mouse polyomavirus do not terminate transcription after
After DNA replication: much higher levels of late transcripts passing through the late polyadenylation signal but
are made, and synthesis of early transcripts is reduced. continue transcription along the DNA template.
o Leads to the production of very long late RNAs that
Five different reasons for the increase in the ratio of late to can contain multiple, tandem repeats of the DNA
early transcription: sequence, similar to the production of long DNAs by
rolling circle DNA replication due to circular DNA.
1. Repression of early transcription by large T antigen. - Giant viral RNAs contain sequences that are
- Binding of LTAg to G(A/G)GGC binding sites can interfere complementary to early transcripts, and they are made in
with the binding of RNA polymerase and general large amounts for reasons explained above.
transcription factors such as TFIID, reducing the amount
of early RNA synthesized. Three late mRNAs are made by alternative splicing

6 | CAÑETE, DNP
 important cellular regulatory pathways that are disrupted
Three different late messenger RNA species are formed from or modified during transformation and tumor formation.
mouse polyomavirus late transcripts by alternative splicing - Their discovery has helped provide a detailed
- Unspliced late mRNA: translated to produce viral capsid understanding of the process of tumorigenesis.
protein VP2.
- Splicing: removes either a small or a large intron, starting Only non-permissive cells can be transformed
at the same 5' splice site but cutting at two possible 3'
splice sites. Transformation of cells can take place only if the cells are
o Removal of small intron: generates VP3 mRNA nonpermissive for viral replication.
o Removal of large intron: generates VP1 mRNA - Nonpermissive: means that the virus can enter the cell
VP3 protein (translation): and express its early genes, but can neither replicate its
- Starts in the same reading frame as VP2 but at an AUG that DNA nor express late proteins.
codes for an internal methionine in VP2 - Infectious cycle is abortive and does not result in the
o VP3 is identical to the C-terminal two-thirds of VP2. production of progeny virus or kill the infected cell.
VP1 mRNA The inability of rat and hamster cells to replicate mouse
- Coded in a different reading frame that overlaps with the polyomavirus or simian virus 40 DNA has been traced to a lack
end of the VP2/VP3 reading frame. of interaction between some elements of the cellular DNA
Alternative splicing of early transcripts: removes introns replication machinery and the viral LTAg.
downstream of the AUG start codon, w/in ORF, and generates
distinct proteins from a single transcription unit by changing Transformed cells integrate viral DNA into the cell
RF or removing parts of the same RF. chromosome
Alternative splicing of late transcripts: generates distinct
proteins by removing AUG start codons, leading to initiation of - 1 to 10 out of every 100,000 infected nonpermissive cells
translation at different start sites, in the same or different end up being transformed w/c can be detected as dense,
reading frame. rapidly growing colonies on a monolayer of normal
fibroblasts growing on the surface of a Petri dish.
Capsid proteins: imported into the nucleus, where they form o Invariably contain most or all of the early region of the
capsids that package viral minichromosomes. viral genome, integrated into the cellular genome at
- Under certain conditions, fragmented cellular DNA, apparently random positions.
resulting from cleavage during cell death, can also be - Low frequency of cell transformation in vitro (and tumor
packaged into capsids to form “pseudovirions”. formation in vivo): probably due to the low probability of
- Pseudovirions may enable passage of certain cellular DNA integration of the early region of viral DNA into the cellular
sequences from one host to another. genome.
o Polyomaviruses do not normally integrate their DNA
How do polyomaviruses transform cells in vitro and cause into the host cell chromosome during replication;
tumors in vivo? o Integration is carried out by cellular enzymes that pick
up degraded fragments of viral DNA and insert them
Polyomaviruses were first discovered as agents able to induce into the host genome.
formation of tumors in mice and other rodents. - Cells w/ integrated functional early region: can continue
- Can transform normal rat or hamster fibroblasts grown in to express T antigens, and all daughter cells inherit this
vitro into cell lines that are immortal, form dense and ability.
rapidly growing colonies, have reduced requirements for o Various signaling pathways that turn on cell cycling,
GF, can cause tumors when injected into animals. reduce dependence on growth factors, and increase
Productive infection of permissive cells by polyomaviruses cell growth rate are permanently altered.
leads to cell death and release of progeny virions. o Such cells can eventually evolve to form tumors when
this occurs in vivo
Paradoxically, polyomaviruses cause few if any tumors in their - Transgenic animals that express one or more of the early
natural hosts. genes of polyomaviruses in specific tissues are being
studied as model systems for tumor formation.
- Allowed scientists to study the biochem and signaling
pathways that are altered upon infection by
polyomaviruses due to the ability to study cell
transformation in vitro by
- Polyomavirus early genes: act as oncogenes both in cells
cultured in vitro and in intact animals.
- Study of the structure and activity of the early gene
products (T antigens) led to the discovery of a number of

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