Lecture 14 Flashcards
(20 cards)
Socio-historical importance of polyomavirus?
SV40 discovery: It was during polio pandemic that affected mostly children => then polio vaccine came out with tissue cultures of harvested kidneys from monkeys => observed CPE => took the media => did some purification, formalin treat the virus (cross-link) to not make infectious anymore but keeping all antigens => this was the vaccine woohoo => but some people had adverse effects CPE but not from polio, from monkey vaccine => SV40 (polyomavirus) purified in cultures like polio => infected rodents and saw tumours like in adenovirus => PANIC => immediate research, lots of firsts
parallel to adenovirus but 30K orginal research, half of which was studying SV40 (1st genome sequences, 1st TF discovered, 1st transcription enhancer, 1st NLS (organelle targeting sequence) discovered) PolyA first shown in SV40
What research implications did SV40 have after the initial PANIC?
- was there more cancer? what types of cancer? Were there SV40 sequences?
- Unforch a few unlucky people may have actually developed cancer from the vaccine => mesotheliomas (some SV40 sequences) but tkt not millions
- helped research in tumour development, cancer research but also basic biology as seen by all the firsts
- helped study how DNA replication happens / similar to mammalian cells, biochemically almost identical, all knowledge put together (for humans) from studying virus
Family of polyomavirus
Polyomaviridae
Polyomavirus morphology
- Small (45 nm in diameter)
- non-enveloped
- icosahedral
- Genome: dsDNA 4.5-5.5 kb circular, similar to papillomaviruses, lots of //s there too, once upon a time even grouped into the same family but then molecules and sequencing and they were like nawr
What are some representative polyomaviruses? (well studied ones)
- Human (BK virus: Kidney epithelium, hemorrhagic cystitis, ureteral stenosis, allograft failure / JC virus: kidney epithelium, and brain oligodendrocytes, progressive multifocal leukoencephalopathy)
- Humans (SV40 Virus of Asian macaques: kidney epithelium, PML-like illness in some colonies)
- Mouse (Mouse polyomavirus 1st discovered also caused cancer in mice multiple cancer, kidney epithelium, tumours in athymic mice)
Human Pathology associated with polyomavirus infection
- Everywhere but IS good control => pathology when immunocompromised (HIV, organ transplant, etc.)
- JC virus (JCV): neutrophils => oligodendrocytes (make myelin) => dementia, change of personality, loss of balance => progressive multifocal leukoencephopathy (PML) (observed more during HIV pandemic in 1980s) 5% of AIDS patients have PML / is fatal in about 50% of those cases (6 months to a year)
- BK Virus (BKV): common early childhood infection, can cause mild respiratory or urinary tracks disease, viral DNA is found associated with some types of cancer
JC + BK named initials of 1st people infected, no other meaning - Merkel Cell Polyomavirus (MCV): Recently discovered in 2008, causes most cases of Merkel Cell (specialized skin cell, meant to support sensory neurone and their function) Carcinoma, mostly seen in AIDS patients, very rare before HIV
- SV40: A contaminant in Salk Polio vaccine, may have contributed to a variety of rare tumour types in vaccine recipients, potential oncogene
Pathologic characteristics of JCV infection in the brain
- Light patches demyelination, little particles, macrophage presence inflammation, presence of viral DNA
Merkel Cell Carcinoma pathology
- dangerous tumour goes deep in skin, becomes metastatic, 40% of people die within 5 years, 60% make it to five years (when metastatic, obvi way more death)
Structural details about the capsid of polyomaviruses
- 5 copies of VP1 come together for bulges => tough disulphide bonds, self-assembly
- 72 pentamers of VP1, 1 copy of VP2 or VP3 per pentamer (inside accessory proteins that glue)
- Similar to HPV in structure
- Histones like P7 high + charge protein that coats adenovirus genome (condense DNA to fit in capsid) / stolen from host cells
- Genomic DNA => circular viral genome
- Similar structure to eukaryotic genome => “mini chromosomes” we refer to / has similar replication cycle to eukaryote hence why has been useful to study our own DNA replication but don’t have H1 like in eukaryotes
- In eukaryotes histone October: H2A, H2B, H3, H4 (2 of each) + h1 in human cells to act as a latch
About genomes of JCV, BKV, MPyV, SV40?
- Some conserved, some not conserved
- They all have RR, regulatory region (2 functions) also acts as a promoters
- bidirectional promoter (late structural proteins / early prepare host cell for viral replication) which is temporally regulated
Functions of the virus-coded polyomavirus proteins
- Mice get injected with virus -> tumours -> take lysates to make cell extracts -> then take sera and run on western blot to get antigen
- Large T: most well studied protein period, T antigen, named T and size since tumour antigen of Dif sizes collected on western blot, initiation of viral DNA replication, stimulation of host DNA synthesis, regulation of transcription (binds to RR and enhances transcription of late genes)
- Middle T: cell transformation
- Small T: efficient viral DNA replication (PP2A binding)
- VP1: major capsid protein, attaches to cellular receptors
- VP2: minor capsid protein
- VP3: minor capsid protein
- Agnoprotein: release of viral particles
How does polyomavirus enter the cell?
- Clathrin coated pit => goes in endosome that then acidifies
- Caveolae => no clathrin, stay pH neutral
Macropinocytocis - Binds to glycolipids instead of protein receptor (not specific, allows for wide tropism)
- Presents on most cells
- Low affinity but many many interactions => kinda like velcro, also how it gets enveloped and just sucked in
- JCV: neuron receptor serpentine receptor, 5-hydroxy tryptamine serotonin receptor then clathrin
- SV40: glycolipids
- BKV, mPyV: caveolae, pH neutral vacuoles
- all use actin cytoskeletons to move and deliver mini chromosome in nucleus, then almost immediately starts making early genes little t, medium T, large T
Structure of the SV40 LgT protein
- Swiss Army knife of functionality, lots of functionality in small sequence space
- Pol alpha binding and J domain (Hsc70 binding chaperone, also required to support DNA replication by SV40 Large T), Rb binding releasing E2F for S phase and cell division,
Helicase domain: DNA binding, specific DNA binding, Pol alpha and p53 binding, Zn finger domain, ATPase activity (used energy to unwind DNA for replication), p300/CBPbinding, unwinds and can recruit DNA replication machinery
host range - Helicase, Hexamers, DNA binds right in the middle
What is one obstacle polyomaviruses must overcome to replicate?
Target cells, not dividing, quiescent, terminally differentiated cells like epithelial or oligodendrocyte cells (same issue as adenovirus)
- SV40 forces cells into S phase, also has TF, 1st thing it does
- Dna viruses that force cell division are more likely to cause cancer, because yes caused by progression of cell cycle, but also lots of inflammation (like Hep C), also alcohol use, excessive liver use can increase chance of liver cancer
Where is the origin of replication and how does it work?
- in RR region, so SV40 binds there and does helices, replication bubble that then moves over time
Late promoter region of polyomaviruses
AT rich (TATA) and central perfect palindrome (core origin Tag binding site II) which is the sequence where hexametric Large T organizes and binds to genome and initiation of replication
How was SV40 DNA replication fork discovered?
- process discovered through adding Large T and nuclear extracts and determining all the other necessary proteins, bound at perfect palindrome, 2 bind head-to-taol unwind and recruit the necessary machinery (bidirectional, leading and lagging strand synthesis)
Many viruses and other organisms have the “end replication problem” where due to lagging strand synthesis, the ends may be lost to nucleases, how do polyomaviruses deal with it?
- Has a circular genome, easiest way around the end replication problem, bacteria also have circular genome, papilloma and polyoma do this but also other viruses / also adenovirus circularization of genome, no lagging strand protein bound right at the end, no missing nucleotides
Rb binding motif
L-X-C-X-E
Mechanism of the Large T protein
pRB bound to E2F is G1/S boundary gatekeeper, point of no return
T binds Rb which lets go of E2F
E2F transcriptional activator => DNA synthesis, Thymidine kinase, DHFR (increase nucleotide pool in the cell), DNA pol alpha
p14 ARF tumor supresor gene, turns on p53 activator
Convergently evolved mechanism by DNA tumour viruses to do their replication, 2 conserved regions, adenovirus, polyoma, papilloma
LXCXE => molecular crowbar to displace E2F, fits perfectly into pocket of Rb to inhibit it
Different packages/proteins to do the same conserved activity with same conserved motifs but like SV40 Tag (1 protein), adenoviruses (2 proteins E1A, E1B) HPV (2 proteins E7 and E6)
