k? (L20-21) Flashcards
polio replication polio vaccination
Category of baltimore scheme for herpes
class I (double stranded dna genome) All herpes viruses become latent after infection
Picornaviruses
Picornaviruses (Picornaviridae)
Literally pico = small, RNA viruses
Generic characteristics:
- Single stranded positive sense RNA (Baltimore Class IV)
- Non enveloped icosahedral particle, 60 copies of VP1 - 4
- RNA typically 7-8 kb, single open reading frame
- Genome has covalently attached protein at 5’ end (VPg)
- Cytoplasmic replication
- Replication typically cytopathic
- RNA-dependent RNA polymerase
human picornaviruses: Historic classification by disease caused
- Hepatitis A: acute viral hepatitis
- Rhinoviruses: Common cold - mild URT disease
- Polioviruses: Paralytic Polio - acute flaccid paralysis
- Coxsackie A & B viruses: human gut isolates causing skin rashes (A) or affecting heart, liver etc (B)
- Echoviruses: “no disease” (enteric cytopathic human orphan)
what viruses are groupes as enteroviruses
Polio, Coxsackie and Echoviruses grouped as Enteroviruses
More recently isolated examples simply ‘Enterovirus (EV) XX’
because of what/how they infect- replicate in the gut (even with diff pathologies)
[[ Rhinoviruses group in the same place as enteroviruses (in terms of genome sequence) despite their different biology
Rhinovirus causes the common cold - extremely mutative ]]
Picornavirus classification (family, genus, species, serotypes)
Family = Picornaviridae
Genus = Enterovirus, Hepatovirus, Parechovirus, (others)
Species = Enterovirus A – J; Rhinovirus A - C
of which Enterovirus A – D = human enterovirus A – D
Further subdivided into serotypes Unique groups neutralized by type-specific antisera Poliovirus type 1, type 2, type 3 Echovirus type 7 Rhinovirus type 14
Total of ~100 human enteroviruses and probably ~200 human rhinoviruses
how may serotypes can we split polio into? (also explain serotypes)
3 different serotypes of polio
Serotypes have to do with the antibody response that the virus elicits and the extent to which that response cross reacts with other viruses
So if 2 viruses in the same serotype - the antibody response to one will neutralise the activity to the other one
If the antibody of the first virus do not destroy the infectivity of the other one, then they are very separate serotypes
From a functional disease point of view, the serotypic distinction will determine if the virus will infect us
steps of Poliovirus replication cycle
Attachment Entry Uncoating Gene Expression and Genome Replication Assembly Release
Identifying Poliovirus Receptor
PRIMARY DETERMINANT: virus receptor
Cell infection restricted to human and primate cells
Virus replicates in mouse cells but cannot infect them
cDNA library transfer screen in murine cells led to PV receptor isolation:
- CD155 (a.k.a. PVR)
- Transmembrane anchor
- Ig superfamily member
use of polio receptors
Level of infectability of a microorganism depends on if they have the right receptors on its surface
Viruses use that receptor to attach to the cell’s surface and it will allow it to enter the cell
We can turn some non-infectious viruses infectious by introducing the right receptors on their surfaces
CD155 is the human receptor that allows the polio virus inside our cells
it is a transmembrane protein, and each loop is an immunoglobulin like fold
poliovirus receptor has immunoglobulin domains on its extracellular parts
cDNA library transfer screen
cdna library transfer screen for every cdna coming out of the human cell, so every mrna copied into a DNA form, can the cdna molecule infect some mouse cells that are not usually infected by this virus?
use of CD155
CD155 renders mouse cells permissive
we’ve got a time course of infection here in the open symbols of what are human cells in the lab
here we have some mouse cells that have been persuaded to express the human protein using the clone DNA from the experiment
and they too are able to support a productive infection by poliovirus
there is no control of mouse cells without the CD155 to price that the virus cannot enter those cells
Virus particle with bound receptors
how does the CD155 interact with the polio virus particle?
particle with three different surface proteins VP1 2 and 3. is icosahedral and what that means is there are dotted around its surface 12 5 fold axis of symmetry, which are effectively the vertices of the icosahedral particle
the 5 fold axis of symmetry is seen in the red circle looking like a star
that’s the pointy bit of the particle formed by VP1
in yellow we can see the receptor interactions with the particle. so the receptor binds at 5 equivalent sites around each of the vertices of the particle
Receptor Binding Site: Canyon and Pocket
TOO MANY NOTES… GO TO L20 S12
Receptor-induced conformational change
Receptor binding displaces pocket factor
Increases flexibility of VP1; allows VP4 to interact with membrane
Probably needs interaction with multiple CD155 molecules
this shows the binding event, receptor and the particle with its vertex
the confirmation of the vertex changes to open up a channel which allows the RNA of the particle to pass across the membrane into the cytoplasm of the cell
Virus interaction with CD155 steps (3)
Attachment Conformational change (Irreversible commitment) Entry at cell surface (Delivery of genome & Usually in a pit)
LONG SUMMARY IN L20 S14
The poliovirus genome and proteins
KEY POINTS SINGLE ORF POLYPROTEIN PROCESSING BY PROTEASES TO LIBERATE THE FINISH PRODUCT CAPSID PROTEINS
LONG AF NOTES IN L20 S16
IRES element role
IRES element directs polyprotein translation
IRES – internal ribosome entry site:
a highly structured piece of RNA at the 5’ end of PV genome / mRNA
- Non-canonical translation initiation mechanism ….
- Allows virus to disrupt cap-dependent translation
3C protease cleaves key cap recognition factors - Turns cell into virus protein factory
how does translation initiate on that rna molecule to produce that body protein?
On the right - shows protein synthesis in infected cells and then 1/3/5/7h after (this is radio labelling where every protein that is produced incorporated radioactivity and produces a black band on the film)
0h - all the proteins of diff sizes are being synthesised
1h - not much difference
5h - background of cellular activity has largely reduced and has been replaced by the synthesis of the viral proteins like the precursors and the finished products
→ so virus takes over the cell, they are cytolytic and destroy the cell
5 CAP recruits ribosome onto its rna - viral RNA doesn’t have a Cap only has VPG. but VPG fulfils the other role of the cap of protecting the RNA from degradation but it does not recruit ribosomes. it has a different mechanisms to recruit ribosomes.
in the untranslated region it has an IRES. it’s a place on the rna that allows the ribosomes to bind so that they can move along and find aug.
mRNA and Genome Synthesis
Replication complexes develop from cytoplasmic cis-Golgi membranes
(+) sense and (-) sense RNA made by same mechanism
Both have VPg at 5’ end: acts as primer for RNA synth
Far more (+) RNA made than (-) RNA
Only (+) RNA released from replication complex
this shows the flow of information that has to occur
the incoming RNA is positive sense so we need to produce a negative sense copy first, student form copies of positive sense by base pairing replication
you can have amplification in each step
in the infected cell we have a lot more positive sense RNA than negative sense. this initiates the hydroxyl group in the VPG.
Polymerase starts at the 3’ end, produces copies
Poliovirus reverse genetics
DIAGRAM IN L20 S19
the fact that the poliovirus RNA is all that is needed to initiate infection, is the reason why it is possible to have reverse genetics in the poliovirus (taking the genetic material of the virus and introducing it into the cell artificially, the cell will become infected and produce progeny virus)
Poliovirus Pathogenesis 1
transmission, initial replication, virus drains into?, natural human infections
Transmission primarily faecal-oral
Possibly by respiratory droplets where hygiene levels are high
Initial replication in the oropharyngeal and intestinal mucosa
Follicle-associated epithelium (M cells and Peyer’s patches)
Virus shed in faeces (major transmission route: faecal-oral)
Virus drains into cervical and mesenteric lymph nodes and then to the blood
Transient viraemia
Most natural human infections (~99%) end at this stage
Minor disease with non-specific symptoms
E.g. malaise, sore throat, fever
Poliovirus Pathogenesis 2
this is the other 1-2%
Transient viraemia established as before
Viraemia allows replication at extraneural sites (reticuloendothelial tissue, brown fat, muscle)
Virus enters the central nervous system (CNS)
- Replication in motor neurons in spinal chord, brain stem or motor cortex leads to paralysis
- Withered/deformed limb(s)
