Lecture 16- SV40 Flashcards
How was SV40 discovered?
As a contaminant in some earlier poliovaccines. We now know a large number of people were injected with SV40 as a part of the polio vaccine. It is likely that the SV40 virus remained active in those vaccines.
What does transformation mean?
A normal cell transforms into a cancerous cell. This is a concern with SV40. IT is a tumor causing agent. One indication is that it can transform cells in the laboratory.
What are permissive vs non permissive cells?
A permissive cell or host is one that allows a virus to circumvent its defenses and replicate. Usually this occurs when the virus has modulated one or several of the host cellular intrinsic defenses and the host immune system.
Permissive/non permissive cells and SV40?
Monkey cells are permissive; will become infected but not transformed. Rodent cells are not permissive; replication of viral DNA is undetectable and progeny virions are not produced. Instead, rodent cells will transform.
SV40 does not cause tumors in monkeys.
Here, we are only interested in permissive cells
SV40 codes for Large T antigen- why is this significant?
Holds the record for the number of functions located in one protein?
The Virion- Capsid
Naked capsid virus
T = 7, but capsid construction is unconventional
72 pentamers of VP1 rather than 12 pentamers and 60 hexamers
One molecule of either VP2 or VP3 associated with each pentamer
(5 VP1s surrounding either VP2 or VP3)
The Virion- Genome
Genome is ds circular DNA, 5243 base pairs
Within virion, DNA associated with histones
H2A, H2B, H3, H4
approximately 25 nucleosomes on DNA.
Agno protein (62 aa): facilitates assembly. binds to VP1, facilitates nuclear localization of VP1
t-Ag dispensable for lytic cycle
Origin region has lots of info. Has an early promoter and a late promoter control region. Will go two separate directions (early vs late). For early genes: amino terminus indicates that you’ll be synthesizing counter clockwise.
Late coding region- VP2 and Vp3 overlap. VP2 is in the same reading frame as VP3. aa seq of VP3 are same as overlapping region of VP2
Note function of agno protein: not required, without agno just get smaller burst size
Will only really talk about how it’s made
t-antigen is important for transformation
can identify large T antigens using serum
keep an eye out for the many functions of large T antigen
Genetic Map
maybe diagram
General features of infection
Infection of permissive monkey cell:
1) enters by receptor mediated endocytosis at sites called caveolae
2) Virions pass into lumen of endoplasmic reticulum
3) VP2 interacts with ER and releases chromosome with VP1 to cytoplasm
4) VP1 has nuclear localization signal that facilitates entry into nucleus
5) One step growth experiment:
SV40 infects a cell not in a dividing state and actually forces that cell to go through an s phase (unscheduled s phase).
First, T-ag increases during EE, then unscheduled s phase during LE, then viral dna during L, then released virus still during L
When a cell becomes transformed into a cancer cell, just continues to divide when it shouldn’t (unscheduled s phase helps cause to divide- causes tumor). just does it because it needed to have an s phase of some sort, not “intentionally” causing tumors. True of all dna viruses that cause cancer
What is step 6? of infection features
Progeny virions assembled in nucleus (also papilloma, aden, and herpes)
Five VP1s plus one VP2 or VP3 creates either (VP1)5 -VP2 or 3
Pentamer that travels through nuclear pore, combines with SV40 chromatin (packaging signal near ori) and the virions are assembled
7) Progeny virus released by cell death and lysis
Gene Expression
Refer to diagram if necessary
Short early leader peptide- no other function other than to be made. Important in regulation.
Little t-Antigen produced, in early phase, splicing event removes termination codon, then can proceed on this spliced version of the RNA. Large T antigen produced by protein in that region. First 80 aa in little t and large T antigen are the same. That’s early early transcription.
More spliced RNA; more VP1 over VP2 and 3
LE starts more to the right (SELP). Late early mrna spliced and codes for small t antigen. Also very similar, in terms of splicing, to mrna. Translated to produce large T-antigen.
90% of the time, all that’s made is short leader peptide- can’t get past this. 2nd aug not used 90% of the time. 10% of the time, leaky scanning leads to large and small t antigen produced in LE.
90% skips over to vp1, x% skips to vp2, leaky scanning just gets vp3
Spliced version of other rna, takes out augs for both vp2 and vp3. most of the time, spliced version translates to produce vp1.
Leaky scanning for late: translation initiates, only 10% of the time makes agno: by leaky scanning, will make vp2
Regulation
ibp: initiator binding protein
3 ibp sites
EE and LE mRNA overlap, LE has more genes than EE
TATA box
3 T-Ag binding sites for large t antigen
In the absence of large T-antigen, early mRNA synthesis begins at EE mRNA start site and goes to the left.
Synthesis of full length late mRNA requires full length replication because of ibp. bound to three Ibp sites, when bound to even one site this is sufficient to block rna polymerase from moving fully down. If all three are occupied, LE mrna repressed and late transcription stimulated at various sites.
binding of IBP changes to late transcription
two sites bound: represses EE transcription
There are a series of start sites for initiating transcription of late mRNAs.
3 binding sites for large T antigen. Links late transcription to DNA replication. Concentration of large T antigen is high, it’s also high enough to cause replication of SV40. DNA titrated out. Limited amount of ibp protein. Eventually new molecules, not enough ibp to bind here.
Dna replication has also started, will accumulate a lot of sv40. Titrates out what little ibp is there, once dna titrates out all ibp, remaining molecules can be transcribed to produce full length mrna.
This is what links dna replication to late stage transcription
when site one and two are bound, large t antigen acts as a repressor and shuts off EE transcription. Only then is late mrna made.
Eventually occupies site three, all three represses initiation of transcription at LE rna, activates transcription of late mrna
relative affinities of three sites for large t antigen: site one has most and site three has least. Site one becomes occupied first because it has the highest affinity. Site three occupied later. Look at summaries and diagrams if this is confusing later.
DNA replication
Site 2 for large t antigen binding = origin of replication, place where large t antigen binds and forms two hexamers
large concentration of T antigen required to assemble double hexameter at SV40 origin of rep
T antigen double hexameter is DNA helicase. Binds to a series of repeated sequences in center of ori.
This is the reason replication is bidirectional from origin- because each goes in separate direction. Takes ATP (helicase requires it) and RP-A (single strand binding protein, ssb). Binding proteins sit behind helicase and move along with it.
DNA pol alpha primase/topoisomerase 1 required as goes along.
Eventually two replications forks going in opposite directions. Function of large T antigen: has helicase activity that requires it to be assembled into this hexamer.
SV40 is a circle, two divergent replication forks go around.
No divergent sequences, they can run into each other at terminus, but when replication forks come together that is the end of the process and you end up with two separate ds dnas.
Nascent chain extended by pol delta. Okazaki fragments initiated by DNA pol alpha primase. No specific terminus on circular genome DNA.
Domain organizations and functions of T-Ag
1) induces unscheduled S phase
2) binds to SV40 ori, unwinds DNA for initiation of DNA replication
3) provides DNA helicase for SV40
4) represses early transcription
5) activates late transcription
Infection of non permissive mouse cells
entry and uncoating, early transcription normal, T-ag (t-ag) accumulates in the cell normally, virus induces unscheduled s phase
BUT
no viral dna replication, no virus production, cell can become transformed
permissive monkey cells, bad non permissive cells
sequence of events of infection up through unshed s phase occurs, but no viral production or any steps beyond
SO cells can be transformed. One prereq for transformation is integration of dna
