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Poliovirus Receptor
Polio's first interaction with a host cell consists of binding to a
specific cell surface protein, the poliovirus receptor (PVR). The PVR,
a cell surface sialylated glycoprotein, is a member of the immunoglobulin
superfamily. The defining feature of this superfamily is a "loop" in the
protein structure called the Ig domain. PVR has three Ig loops (which are
outside the cell), numbered 1-3 starting with the loop farthest from the
cell surface. Polio appears to bind to its receptor on loop 1. The receptor
molecule binds to a "canyon floor" on the virus particle. The poliovirus
receptor is expressed in many human tissue types, including tissues such
as kidney that are not normal sites of poliovirus replication. Why doesn't
polio replicate in these cells? Possible reasons include a block of viral
replication after entry, lack of exposure to those tissues during an infection,
or an unknown secondary mediator of viral entry.
Protein Synthesis
(Diagram
displaying rates of protein synthesis in infected and uninfected cells.
The rise in protein synthesis after 2 hours in infected cells is due to
viral protein production. Fundamental Virology, p.505)
The poliovirus genome is made of positive sense single stranded RNA.
The genome encodes a single 'polyprotein' of between 2100-2400 aa's. Both
ends of the genome are modified, the 5' end by a covalently attached small,
basic protein VPg (~23 AA's), the 3' end by polyadenylation. In a series
of cleavages, viral proteases cleave themselves out and break down the
polyprotein into 10 separate gene products involved in replication and
packaging.
The viral proteases 2A cleaves the p220 subunit of the cap binding
complex (eIF4F subunit of eIF4G), making host cell mRNA unrecognizable
to ribosomes. As a result, the 2A protease abrogates most of the host cell's
own protein synthesis. Doing so increases the number of free ribosomes
to translate viral RNA and insures that the cell will break down, releasing
progeny viruses. Viral mRNA relies on a 5' UTR containing a 'clover-leaf'
secondary known as an internal ribosome entry site (IRES) that serves as
a ribosome docking site to the 40S subunit of ribosomes.
Fundamental
Virology, p.482
The 5' methyl-guanosine cap attached to host mRNA for ribosome recognition
is unnecessary in poliovirus, allowing for viral RNA translation. The primary
attenuating mutation in the Sabin vaccine is located in the IRES.
While Sabin poliovirus can replicate efficiently in gut epithelial tissues
(primary site of replication), it is unable to replicate efficiently in
the nervous system.
RNA Replication
Replication occurs entirely in the cytoplasm. In addition to serving
as a template for protein synthesis, the positive sense strand genome is
utilized as a template for the synthesis negative sense strands. Host cells
lack the necessary machinery to replicate RNA. Poliovirus uses a viral
RNA-dependent RNA polymerase to produce RNA molecules of the opposite polarity.
Viral protein VPg covalently attached to uridine (VPg-UUU) serves as the
primer. The first round of replication produces a single antisense molecule.
This antisense template is then used to produce sense copies of the original
genome that can be packaged into viral capsids prior to viral release.
Antisense genomes can translated as well.
Packaging and Release
After the virus has translated its RNA to produce the necessary proteins
and replicated its genome, it needs to package the newly synthesized RNA
molecules inside capsids. A complete virus consists of the RNA packaged
inside the capsid. The capsid proteins self-assemble into an immature capsid,
a structure which contains all of the necessary proteins, but which has
not finished cleaving them into their final form. The mature poliovirus
capsid has icosahedral symmetry and contains 60 copies of each viral capsid
protein (VP1,2,3,4) The viral RNA enters the incomplete capsid and is secured
inside when the viral proteases make the final cleavages. Once the genomes
have been packaged into mature virions, the virus particles await the cell's
lysis in order to be released. As many as 100,000 virions can be released
from a single infected cell.
Uncoating
Binding between the virus and its receptor leads to conformational
changes in the capsid. VP4, an internal capsid protein detaches from the
capsid. The capsid swells and the poliovirus genome is susceptible to degradation.
When VP1 is released, the genome is released onto the cytoplasm of the
cell. The viral entry strategy is very inefficient; only 1% of the viruses
initiate an infection.
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Time after Infection
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Event
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~30 min
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cellular protein
synthesis declines sharply (SHUTOFF)
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~1-2h
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Sharp decrease in
cellular macromolecular synthesis; margination of chromatin (loss of homogeneous
appearance of nucleus)
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~2.5-3h
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Start of viral protein
synthesis; vacuolation of cytoplasm, beginning close to nucleus & spreading
outwards
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~3-4h
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Permeabilization
of plasma membrane
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~4-6h
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Virus assembly in
cytoplasm (crystals sometimes visible)
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~6-10h
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Cell lysis; release
of virus particles
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 |
Scanning Electron
Microscopy of surface alterations of HEp-2 cells
Koch, The Molecular Biology of Poliovirus,
p.231 |