Chapter 5 - Picornaviruses part 1 Flashcards
Should we be concerned about poliovirus
Most are vaccinated in Canada, but international travel is common and easy for someone to travel to an area where polio is an endemic and come back to infect people
Endemic = virus has a continued presence in the community, usual.y a few case throughout the year
History of Poliovirus Infections
Improvement in sanitation increased in incidence of polio infections
Because polio virus can only replicate in human and spread from human to human
By being cleaner, less immunity cuz not exposed (hypothesis)
Poliomyelitis
Inflammation of the Grey matter of spinal cord
Caused by virus which can infect many types of cells in human and result acute infection
- any cells with CD155 can be infected
Most cases (>95%) are limited to the throat and GI epithelial cells
Only small number of cases did the virus penetrate the CNS causing major illness
- When the virus infects CNS it made a mistake
- no benefit for virus infecting CNS, it cannot be transmitted to another person
- Virus can spread
Infection route and types of polio
Abortive:
- Ingestion of poliovirus in food/water
- infection of epithelial cells of throat and intestine
- Replication of virus
Result in Abortive Polio (95% of cases)
The virus also excreted in feces
Rarely during replication, it can spread to the bloodstream
If involve CNS becomes paralytic polio (0.1-0.5%),
if doesn’t involve CNS becomes nonparalytic polio (1-2%)
Paralytic
Infection of motor neurons of the spinal cord, brain stem, or motor cortex
Non Paralyrtic
Local inflammatory response, self-limiting infection of meninges
Abortive Polio
Many cases are asymptomatic
Other cases present as mild cold for about 72 hours
- Slight fever, sore throat, headache
Accounts for 95% of infections
Most cases are undiagnosed at the time of infection
Non Paralytic polio
Present with mild influenza like symptoms
Back pain, muscle stiffness and tenderness,
Headache moderate fever
Excessive fatigue, irritability
Vomiting diarrhea, skin lesion
Symptoms last 1-2 weeks
Accounts for 1-2% of infection
Paralytic polio
Fever for 5-6 days before other symptoms
Other symptoms develop fast
Abnormal sensation in an area
Muscle cramps or spasm in calf neck or back, stiff neck and back headaches
Muscle pain and weakness that is only one-sided
- Location depends on where spinal cord is infected
- Worsens into paralysis
Sensitivity to touch, mild touch may be painful
Difficult breathing if respiratory muscle involved
In very severe cases: death if breathing muscle paralyzed
Not too severe:
- paralysis of the muscle
- intense physiotherapy can regain some use of a limb
- loss of surviving motor neurons with age
- can lead to post-polio syndrome
Post polio syndrome (PPS)
PPS develops 30-40 years after initial polio illness
Symptoms include
- New muscle weakness: common in muscle thazt had a nerve damage from polio
- fatigue
- muscle joint pain
- Muscle affected by polio tend to be weaker than normal other muscles having to work harder putting extra wear and tear on the muscle
PPS progresses slowly
What causes PPS
PPS isn’t caused by reactivation of the virus
- Poliovirus causes an acute infection
- As an RNA virus, it cannot persist in the cell for decades
- no evidence of poliovirus circulating in North America
Caused by:
- surviving motor neurons may sprout new dendrites to promote use of these muscle
Over the years, this stress may be more than the neuron can handle
The dendrites deteriorate and eventually so does the neuron itself
Who does poliovirus affect
Mainly affect children under age of 5
- These children may not have been exposed to virus developed immunity
However older children teenagers and even adults can be infected and suffer the effects of polio
If you have not been vaccinated you are at risk especially if travel to endemic areas
As you get older, you are more likely to get paralysis polio and the effects of it also get more severe
- In children if infect CNS, just get non paralytic polio
Who does poliovirus affect
Several key factors have been identified as increasing likelihood of paralysis
- Pregnancy
- Removal of tonsils
- Immune deficient
- Intramuscular injection
- Injury
- Strenuous exercise
Why do we study poliovirus
Simple replication path
Humans are the only known natural host
Poliovirus known to infect monkey kidney cell line
- This monkey cell line is used to manufacture polio vaccine
- Cell lines are not normal cells
Poliovirus is still a problem in many countries, but endemic in only Pakistan and Afghanistan
Why poliovirus is still a problem
Vaccine programs have lapsed
- virus has been re-introduced into a country where it had once been eradicated
Some areas may be geographically difficult to reach
Political unrest with military actions
Local populations refusing vaccination - distrust of western societies
Until the virus is eradicated globally, it is a problem for all countries in the world
Just because it is currently circulating in North America does not mean that it cannot return –especially if the number of individuals that are not vaccinated continues to increase
Generation of Polio Vaccines
Infected human cells
- -> wild poliovirus
- -> Passage through monkey cell line
- monkey cell line must have a structure that virus can bind to - might not be as effective as human cells but enough to initiate genome entry
- -> Mutations
- -> Reduced virulence to human cell line (attenuated)
- ->–>
1. prepare as oral vaccine or
2. chemically inactive –> prepare as injected vaccine
Polio Vaccine - I
Infect human cell like with wild type poliovirus
–>propagate poliovirus, will find mostly wild type progeny but there will be mutants
~ 1 in 10 viruses progeny will produce a single mutation in the genome
Mutation may be neutral.
If harmful it may no longer be replicated
Or good mutation - strain can infect different species of host cell
Polio Vaccine - II
Take those virus progeny (along with mutated ones) and infect monkey cell line
–> propagate polio virus
–> Mutant viruses are able to infect and replicate the non-human cell line,
the wild type strain does not infect and replicate as well
- the mutated viruses are selected for further propagation
aka now mutated virus more fit to monkey cell line aka _attenuated for human cell line
Polio Vaccine - III
Using those mutated polio virus only infected monkey cell line
–> propagate poliovirus
–> Possible that some virus mutated back to wild-type (virument ) form - a potential problem in OPV
antibody against the mutated virus should bind to wild type virus
Repeat the passage of polioviruses on the non-human cell line several times
- This allows for the mutate viruses to acquire additional mutations that allow them to replicate well in the non-human cell line
- the wild type viruses are diluted out of existence cell culture
Polio vaccine
The virus strain has accumulated many mutations relative to the original wild stain
- it may not loner be able to infect human cell line
- it may be able to infect human cell, but not replicate as well within it
Now we made attenuated virus may be used as vaccine.
However some virus particles may revert and mutate back to wild type regaining ability to infect humans cell well
Salks’s Vaccine - Injected Poliovaccine
IPV:
virus is inactivation, no viral replication, cannot spread to other susceptible people
The virus is injected into muscle (booster vaccine)
- get humoral response
- IgG immunity in blood
- but not gut immunity, could still get infection in gut
- could spread to other susceptible people
- no concern if virus enter blood stream
Sabin’s Vaccine - Injected Poliovaccine
OPV
virus can replicate in throat or gut:
- might spread to bloodstream (viremia)- systemic immunity (IgG in bool –> immunity)
- or if infection of motor neurons –> paralytic polio - Local immunity - IgA secretion in gut –> immunity
- Excretion from body
- contamination of food and water
- possibly infect other susceptible people
Poliovirus vaccines
Both Salk’s (IPV) and Sabine’s (OPV) vaccines work well
- The evidence is the dramatic decline in poliovirus infections post-immunization
If you grew up in North America, you likely received the IPV as a child
In the early to mid-1950s, IPV was used initially, then it was replaced with the OPV. Currently, the IPV is the vaccine routinely used
Terminology:
-
Viremia: presence of virus in the bloodstream
Primary viremia: spread of virus into the blood from the initial site of infection
- Eg polio from throat then to blood
Secondary viremia: spread of virus to other organs that come in contact with the blood where the virus replicates then enters the bloodstream once more
Poliovirus Serotypes
Stereotypes refer to distinct variations within a subspecies of viruses
There are 3 serotypes of poliovirus
- the different serotypes have slightly different versions of the the capsid protein (VP1/2/3) that can be distinguished by different antibodies
All 3 serotypes can cause paralytic polio
- serotype 1 (PV5) seems to be the most virulent and cause most polio these days
- serotype 2 and 3 is declared eradicated in 2015, 2019
Poliovirus Serotypes
Different serotypes have different epitopes
- thus need different antibodies and immunity
Wild poliovirus type 1 in endemic in countries - afghanistan, pakistan
cVDPV - vaccine derived poliovirus in non-paralytic polio or paralytic polio is not the problem in many countries
- small percentage from OPV
Of case reported doesn’t tell us
- how many vaccine doses were administered
- How many people were protected due to vaccination
- how many were abortive or non paralytic
- aka data is skewed
VAPP
Vaccine-associated paralytic polio is a rare adverse reaction following OPV
- (IVP) Inactivated poliovirus vaccines do not contain viruses that can replicate, so they cannot cause VAPP
The mechanism of VAPP is believed to be a mutation or reversion of the vaccine virus to a virulent form
Reversion is believed to occurs in almost all vaccine recipients, but it only rarely results in paralytic disease
- Resulting in paralysis is identical to that cause by wild type virus and may be permanent
VAPP is not a bigger problem than infection with natural poliovirus
- worth the risk, it is ethical to continue OPV
Poliovirus as therapeutic
Poliovirus can be used as a therapeutic –PVSRIPO immunotherapy
In a clinical trial (phase II), patients with a glioblastoma that received an intratumoralinfusion of a modified viral chimera combining the polioand rhinoviruses(PVSRIPO immunotherapy) compared to patients receiving standard treatment at the same institution
he CD155 protein that poliovirus uses as a virus receptor that is virtually universally expressed in malignant cells of solid neoplasia. The modified virus does not attack motor neurons
Now a clinical trial using PVSRIPO immunotherapy in combination with Pembrolizumab (a monoclonal antibody that blocks a protective mechanism of some cancer cells) is in progress
Model of Poliovirus
Naked - ~30nm, roughly icosahedron in shape
Contains a single piece of + sense RNA
capsid = nucleocapsid
Capsid has 20 capsomeres made of 3 triangular subunits
Each triangular subunit is made of 3 proteins: VP0, VP1, VP3
Poliovirus
Has 2 forms:
Immature form:
- non infectious
- 3 different polypeptides (vp0, vp1, vp3)
Maturations: RNA genome inserted into capsids, cleaves VP0 into VP2 and VP4
Mature form:
- infectious
- 4 different poly peptides: VP1,2,3,4
RNA genome acts as ribozyme: RNA molecule with enzyme activity
How many protein molecules are in an immature poliovirus particle?
2033 = 180
60 copies of VP0,1,3 each
In mature cirus:
2034 = 240 proteins
Poliovirus Structure:
VP1/2/3 are visible from the viral surface
VP4 is not visible from the surface as it is completely internal to the capsid
VP1 is the virus’ anti-receptor and the “canyon” is the region that interacts with the host cell’s “virus receptor”, a protein known as CD155
Poliovirus Genome
Poliovirus genome is made of positive sense ssRNA about 7500 nucleotides long
Both ends are modified
- 5’ end by a covalently attached small basic protein VPg
- 3’ end is poly A tail
- there is no 5’cap
The genome encodes a single ORF which encodes a 247 kDA polyprotein
Challenge In replication - 1
Poliovirus is an RNA virus that replicates its genomes in the cytoplasm
Since genome is + sense, the RNA can be translated right away
- RDRP must be encoded in its genome
- Because the host cell lacks a polymerase that can read RNA as a template and synthesize genome RNA and mRNA
- (for progeny virus)
(cant transcribe using RNA template)
Challenge In replication - 2
Poliovirus RDRP require a primer - a molecule that can provide an 3’-OH group to add the next nucleotide
Solutions: it encodes a gene for VPg
- VPg is a small protein modified by adding Uracil nucleotides to it - thus providing the required 3’OH group
- VPg - UU-3’OH
Challenge In replication - 3
Eukaryotic ribosomes can translate monocistronic mRNA but polio has several proteins thus genes (polycistronic)
Solution:
Virus RNA has a single start and single stop codon in a frame
After assembly the ribosomes translate the entire thing into 1 big protease - polyprotein
Challenge In replication - 4
Eukaryotic ribosomes recognize mRNAs with a 5’cap and a polyA tail
Poliovirus genome (+sense RNA) lacks a 5’ cap
Solution: RNA has a secondary structure to interact with ribosomes subunit
- IRES allows the poliovirus to interact with the host cell proteins to assemble on the ribosome
- IRES structure: a secondary structure in the RNA that can interact with the small ribosomal subunit and other cell proteins resulting in the full assembly of the ribosomes
VPg gets in the way with translation, therefor it is cleaved off before translation
- only useful in transcription
Cap-dependent Translation in cells - normal eukaryotic cell
- A set of proteins associate with the 5’cap of the mRNA
- Protein eLF - 4G - A different set of proteins associate with 40s ribosome subunit then bind ribosomal unit comes in
- Some of the proteins interact with the ribosome assembles on the mRNA
Cellular mRNA has a 5’ cap
Protein eLF4G binds near the 5’cap as part of the cap binding complex
- small ribosome subunit with elF3 and Met tRNA bind to CBC
- large ribosomal unit binds
Cap-independent Translation in cells - poliovirus
Poliovirus is an example of virus that uses cap independent initiation of translation
IRES recruits the cellular initiation factors to allow for ribosomal assembly
IRES is upstream (before) of AUG
Once translation is initiated, the rest of the process is the same as translation with cap
ITAF = IRES trans-acting factor
- various host proteins exploited by poliovirus to mediate translation
The expression of these proteins in some cell types means that the cells are permissive to poliovirus
notes that some proteins are the same for cap-dependent and cap-independent translation
Challenge In replication - 5
Virus must compete with the cells mRNA for access to ribosomes
The cells ribosomes are better suited for assembly on cellular mRNA than the poliovirus genome
- There are many more copies of cellular mRNA that poliovirus genomes, so virus mRNA at disadvantage
Solution:
- it has a protease that damages part of the cap binding complex that cellular mRNA needs to assemble onto ribosome
- host cell protein synthesis is impaired - probably cause lysing of host cell when virus assembly is done
(recal how eLFG is used in cap dependent)
Protease from the poliovirus polyprotein damages the CBC -specifically eLF4G
Because elF4G is damages, it can’t bind to ElF3
The ribosome cannot assemble efficiently on the host cell mRNA
Eukaryotic mRNA does not have IRES, no secondary structure (eg IRES) to compensate
Challenge #6:
The host cell has RNases that degrade RNA molecules
This is needed to recycle cellular mRNA, tRNA and rRNA
Solution:
Replicate the genome in compartment made from host cell membranes- protect the genomes from the RNases
- RDRP replicates the viral genomes only - not host cell mRNA
Binding and Entry of Poliovirus Genome
Interaction of host cell’s virus receptor CD155 with canyon of the VP1 of poliovirus
- binding initiates infection of host cell
Genome Entry:
When VP1 binds to host cell’s CD155, it induces conformational changes in capsid of virus particle
The N-terminal end of VP1 protein is inserted into the cell membrane to form a pore
VP4 which was inside of the capsid prior to binding is now on the outside and may form part of the pore
The RNA passes through the pore and into the cells cytoplasm
It is unclear whether the particle enters the cell or just the genome
- may depend on strain
Translation of polio virus genome
- Release of VPg by cellular protease leaving the 2 Us
-
VPg
No role in translation of RNA
It was needed as a primer for transcription to make new RNA strand
Prior to assembly on ribosome, one of the cellular protease cleaves it off
- For newly made +RNA that are used as mRNA, the VPg is removed (translation)
- For newly made +RNA that serve as a genome, the VPg is not removed (transcription)
Plus sense RNA is translated several times, yielding many copies of the polyprotein
- Includes many copies of VPg and RDRP and other proteins
VPg is a small protein to which uridine is covalently attached
It is part of the primer from RNA synthesis - both + and - sense
A primer is a starting molecule to which nucleotides are added to build nucleic acid
(at first +sense RNA doesnt have VPg, but translates the protein VPg then it modified the RNA for transcription?)
Viral Replication
There are 2 rules that apply to all polymerases
- They read template 3’ to 5’
- They synthesize complementary strand 5’
- Original + sense RNA template transcribed and makes - sense RNA templates
- RNA copies each act as a template to make more - RNA
- New - RNA template made from each mRNA copy make more + RNA
over n over
Translation: Synthesis of Several Polyproteins
The first translation of the + RNA is challenging for poliovirus - it has to compete for access to ribosomes and has a disadvantage
One product of the polyprotein is a protease that damage the cap binding complex (CBC) of the host cell mRNA
This eliminates the competition for the ribosomes and allows the poliovirus mRNA translation to become very efficient
Polio Replication
Each poliovirus capsid to be constructed requires 60 copies of each of the protein VP0, VP1 and VP3
If one infected can result in 1000 poliovirus progenies being made
Means that 60*1000 copies of VP0/1/3 will need to be made
Assembly of Poliovirus Procapsid
VP0/1/3 first form to assemble the triangle subunit
Then 3 subunits assemble to form capsomere
Then 20 capsomeres assemble to form pro-capsid
- no genome in capsid yet
Assembly of Poliovirus Procapsid
After procapsid is assembled, the poliovirus genome is inserted
How Does it know whether it is inserting its own genomes and not cellular mRNA or whether they are putting in + or - sense RNA
Some proteins in the capsid recognize sequence in nucleic acid
If they accidentally package - sense RNA instead of + sense
Won’t be able to transcribe cuz no RDRP, not able to replicate
RNA would not be able to cleave VP0, so the capsid would remain immature (non infectious)
because + RNA acts as a ribozyme, - RNA doesn’t so it cannot cleave VP0
Maturation of Polio Virus
Poliovirus genome has ribozyme (only the + sense)
- it recognizes a site on the VP0 protein and cleaves it to form VP2 and VP4
The ribozyme would recognize an aa sequence in the VP0 protein
After cleaving the VP0, there would be conformational changes that:
- seals up the capsid, protecting the RNA from damage
- Reveal the VP1 antireceptor structure on the virus surface
Release of Polio virus
One of the proteins that is made by poliovirus is a viroporin
- helps destabilize the membrane
The only progeny virus particle that will be infectious are those that have completed assembly and maturation before the host cell lyses
Hypothetically, if a particle packaged with (-) RNA somehow became a mature infectious particle, the virus could bind to a host cell and release its genome into the cell’s cytoplasm (because the capsid proteins are “normal”), but nothing after that. Why not?
Genome cannot be translated. No RDRP packaged in the capsid. No viral replication
