Lecture 13- Poliovirus Flashcards
Comparing RNA phage and poliovirus
RNA phage regulates EVERYTHING; leaves nothing to chance. Poliovirus replicates by polyprotein
pico RNA virus =
picornavirus
Pico stands for small RNA virus
Corna = animal virus
Basically animal viruses with small genomes
Picornavirus groups:
1) enteroviruses (polio)
2) Rhino viruses (cause of the common cold)
3) Hep A
4) cardiovirus (EMC, encephalomyocarditis virus)
5) aphthovirus (foot and mouth disease virus) (first animal virus- discovered in 1898)
The virion
VP1, VP2, VP3 on surface of virion (VP= virion protein)
VP4 buried inside, associated with RNA
T=1, so capsid contains 12 pentamers (simple capsid structure)
Each of the 5 protomers of a pentamer contains one each of VP1, VP2, VP3, and PV4
Genome: + ss RNA 7433 nucleotides (w/ 5’ VPg protein) (twice as many as MS2 genome)
Small protein w/ 22 aa covalently bound to 5’ end of RNA (g stands for associated with genome)
5’ VPg similar to adenovirus (that protein served as a primer)
VP4 somewhat similar to a protein
Poliovirus genome:
VPg on 5’ end, ntr, then P1, P2, P3, 3’ end, ntr, short polyA tail
P1
-codes for all four structural proteins
-VP0 becomes VP4 and 2
P2
-codes for a protease (2A pro)
-2A protein cleaves for protease
-Also codes for 2B and C (cleaved into these two): RNA synthesis induces cytoplasmic vesicles
-these proteins, particularly 2C, are important
P3
-codes for 3A and VPg (cleaved into these two)
-codes for 3Cpro protease
-codes for 3Dpol
-3CDpro is int, cleaved into above two
The infection cycle- A. Adsorption
Adsorption, entry, and uncoating (rhinoviruses enter by rec mediated endocytosis
1) Receptor is Ig-like protein in plasma membrane (Pvr or CD155). VP1 is virion attachment protein.
2) Binding of virion to receptor triggers release of VP4 and extrusion of the hydrophobic N-terminus of VP1 to the surface of the particle. The fact that VP4 is on the inside means there must be some kind of rearrangement.
3) Hydrophobic region of VP1 inserts into the plasma membrane and generates a pore for RNA entry into cytoplasm of the cell. Hydrophobic N terminus of VP1 is extruded outside of capsid. Capsid stays on the outside. Sounds like a mech for entry by bacteriophages- very unique method of entry (poliovirus) b/c most animal viruses do not leave their capsid on the outside when they infect.
The infection cycle- B. Translation of polio genome
Exclusive translation of polio genome; shut off of host translation
Time course of events during infection (one step growth experiment)
-poliovirus enters, causes shut off of all host proteins. everything now available for poliovirus to use
-host protein synthesis rapidly shut off, mrna synthesis also shut off quickly after
-different protein for shut off of each
-methylated g residue constitutes cap of 5’ rna
-cap binding subunit recruits 40s subunit and binds it, so without CBC no translation. polio is a 2a protease- degrades P220 (one of the subunits of the cap binding complex). CBC can’t recruit 40s, can’t find AUG
-bit of a lag period before viral protein and rna synth takes off
-3Cpro degrades 1+ subunit of host rna pol
What happens to the rate of different types of synthesis as time passes during an infection?
Poliovirus enters. First, host protein synthesis shuts off. Then, 3C protease degrades RNA pol subunits and host RNA synthesis stops quickly after. Mean while, viral protein and RNA synthesis begin while those are declining. Eventually, extracellular synthesis increases.
What is the role of Polio 2A protease in infection?
It halts host translation.
There is a methylated G residue, which constitutes the cap of the 5’ RNA or the cap binding complex. CBC recruits 40s ribosome subunit which binds and scans for AUG, then recruits 60s subunit. Without CBC there would be no translation. Polio 2A protease degrades P220, a subunit of the CBC. It can’t recruit 40s and can’t find AUG to translate as a result.
What does 3C protease do?
Degrades at least one subunit of host RNA polymerase.
The infection cycle- C. Viral protein synthesis
1) Removal of VPg and internal initiation of translation
2) Processing of polyprotein
3)
Removal of VPg and internal initiation of translation
VPg uses tyrosine residue to bind to RNA. Tyrosine residue is linked to 5’ nucleotide through phos bond (b/c uses oxygen- one of the 3aa)
Cellular enzyme Tdp2 removes VPg. Cleaves VPg off the RNA so there is no VPg on the end. Note: the very 5’ end of genome is two U’s
Very long and non translated region between 5’ end and first aug. Non translated region is important because it has IRES (internal ribosome entry site). 40s can enter at IREs and scan for “first aug” (not really the first aug, but the first aug that matters- No cap, so no machinery to recruit 40s, so doesn’t matter that there are other
AUGs in that region)
Cap independent translation occurs by 30-40 ribosomes at any one time.
Produces a polyprotein
Processing of polyprotein
Processing by proteases begins before translation is complete- can never find long polyprotein in cell. Being cleaved while it is still a nascent chain.
KNOW: poliovirus codes for 4 proteases (3Cpro, 2Apro, 3CDpro, maturation protease (embedded in VP0, capable of cleaving self)
3D pol must be cleaved away from 3CD Protease to be active
Many instances where partial proteolytic portions are active. Still associated with polyprotein, but functional. In many cases, proteolytic cleavage intermediates themselves have a function or are active.
Remember: 2A pro, which cleaves between itself and N terminus (cleaves right before self) and 3C pro which cleaves in the middle of 2A pro and 3C pro. 3C pro also cleaves right before self once cleaved into P3. Cleaves again between 3AB and 3CDpro
What does 2A Pro do?
Cleaves between self and N terminus to give P1 and P2/3.
P1 gives rise to all four VPs
What does 3C pro do?
Cleaves between 2A pro and 3C pro to give P2 and P3. Cleaves in middle of P3 to give 3AB and 3CDpro
Cleaves again in 3AB and 3CDpro to give 3A and VPg, and 3Cpro and 3D pol. Also cleaves P2 to give 2A Pro, 2b and 2c
