Polyomaviruses

1
Polyomaviruses

• Used to be considered part of same family as
  papillomaviruses (called Papovaviruses) but now
  recognized as distinct from papillomaviruses
• 13 members that infect several species of mammals
• Virion - nonenveloped icosahedral capsids about
  45 nm in diameter
• - circular double stranded DNA genome about
  5,000 bp (complexed with cellular histones)

2
Examples of Polyomaviruses

• Polyomavirus a mouse virus
• - widely studied in the lab
• SV40 a monkey virus
• - widely studied in the lab
• - originally identified in monkey kidney cells
  used to produce polio vaccine (around 1960)
• - causes tumours when injected into hamsters
  but not in natural host
• JC and BK human viruses closely related to SV40
• - found in brain (JC) and urinary tract
  (BK)
• - thought to be common but not normally
  associated with disease

3
Viral Genome
Viral DNA is double-stranded circular Viral DNA
is associated with nucleosomes composed of
cellular histones H2A, H2B, H3, H4
4
Genome Organization

• The genome of all polyomaviruses is
  organized into early and late regions separated
  by a regulatory region
• Regulatory region contains the origin of DNA
  replication
• promoters and enhancers for transcription
• Early region encodes T (tumour) antigens
• SV40 has small t and large T antigens
• Polyoma has tiny, small, middle and large T
  antigens
• produced from a common transcript by
  alternative splicing
• Late region encodes 3 capsid proteins
• generated from common transcript by
  alternative splicing

5
Polyoma
(regulatory region)
Divergent transcription
Fig 4 in text
6
SV40
7
T Antigens

• Tiny T
• In polyoma but not SV40
• 85 amino acids long has not been studied
• Small t
• N-terminal region is same as large T
• Not required for lytic cycle under most
  conditions
• C-terminus (SV40) binds and inhibits the cellular
  phosphatase pp2A
• Can contribute to cell transformation under some
  conditions (requires pp2A binding region)
• Middle T
• Contains small t and additional amino acids
• In polyoma but not SV40
• Plasma membrane protein
• Binds src kinase activating it resulting in
  phosphorylation of T and some cellular proteins
• Important for cell transformation
• Large T
• nuclear, sequence-specific DNA binding protein
• binds sequences in regulatory region
• Many functions

8
Functions of Large T Antigen

• Stimulates resting cells to enter cell cycle
• a) binds pRb and related proteins (like E1A)
• - causes release of pRb from E2F and
  activation of genes involved in cell
  proliferation by E2F
• b) SV40 T (but not polyoma T) binds p53 (like
  E1B)
• - inhibits p53 binding to DNA, resulting in
  decreased expression of cell cycle inhibitors
• - inhibits p53-mediated apoptosis
• c) binds p300 (histone acetyltransferase)
• - inhibits transcription activation by p300
• c) binds hsp70 (chaperone involved in protein
  folding)
• - stimulates the release of proteins from hsp70
• - N-terminal domain of T that binds hsp70 looks
  like a chaperone protein (this domain important
  for cell transformation)

9
Functions of Large T Antigen

• Regulates early mRNA production
• - represses early gene expression at end of
  early phase
• - by binding site I in regulatory region
• Required for viral DNA replication
• - binds origin (site II in regulatory region) to
  initiate DNA replication
• - melts origin DNA and serves as a DNA helicase
  during elongation
• - binds cellular DNA replication proteins

10
DNA Replication

• Occurs at ND10 sites
• Many rounds of replication per cell cycle
• Initiates from a single origin of replication
  that contains core and auxiliary sequences
• SV40 Core sequences 1) T binding site II
  (central)
• 2) palindrome on early side (early
  palindrome)
• 3) AT-rich region on late side
• SV40 auxiliary sequences 1) transcriptional
  elements
• 2) T binding site I

11
Initiation of DNA Replication (SV40)

• In presence of ATP, T forms 2 hexamers on site II
  in the core origin
• T unwinds DNA in the early palindrome
• As DNA unwinds, RPA binds to single stranded DNA
• T also recruits DNA polymerase a-primase to the
  origin to initiate DNA synthesis
  (species-specific interaction)
• Replication proceeds by theta structure mechanism
• T hexamer at each replication fork serves as DNA
  helicase

Large T hexamer
12
Elongation Phase of DNA Replication
Fig 11 in text
13
SV40 In Vitro Replication System

• Replication of SV40 and polyoma has been
  reconstituted in vitro by combining cell
  extracts, SV40 DNA and large T antigen
• Enabled the purification of all cellular proteins
  needed for SV40 replication
• Lead to the discovery of several cellular
  proteins that were subsequently shown to be
  important for cellular DNA replication

14
Cellular Proteins Required for SV40 Replication

• 1) RPA ssDNA binding protein
• - not previously known
• - essential for cellular DNA replication,
  repair and recombination
• 2) DNA polymerase a-primase required for
  initiating DNA replication on leading and
  lagging strands
• 3) DNA polymerase d shown to synthesize leading
  strand and extend DNA fragments on lagging
  strand
• 4) PCNA processivity factor for polymerase d
  (sliding clamp)
• - function not previously known
• 5) RFC loads PCNA on DNA
• - not previously known
• 6) Topoisomerase I and II relieves
  superhelicity during replication and resolves
  replicated molecules
• 7) RNaseI and FEN-1 (MFI) exonucleases that
  remove RNA primers
• 8) DNA ligase seals nicks in DNA

15
Permissive Infection

• also called productive Infection
• Viral DNA replicates and virions are produced
• Occurs in primate cells for SV40 and rodent cells
  for polyoma

16
Nonpermissive (Nonproductive) Infection

• Occurs in rodent cells for SV40 and primate cells
  for polyoma
• T antigens expressed but viral DNA cannot
  replicate (due to failure of large T to bind host
  polymerase a-primase)
• no virions produced
• Cells are transformed due to T antigen expression
• Transient Transformation
• Usual outcome
• Viral genomes are eventually lost from the cell
  and cell returns to normal due to lack of T
  antigen expression
• Permanent Transformation
• In absence of DNA replication, some viral DNA can
  integrate into cellular DNA
• Can result in continued expression of SV40 large
  T or polyoma middle T which causes permanent cell
  transformation