Lecture 25 Flashcards
(47 cards)
Give families of (+)ssRNA viruses?
- Picornaviridae
- Coronaviridae
- Flaviviridae
- Togaviridae
Why are (+)ssRNA viruses so cool?
- One piece of RNA must do it all (template for translation, replication and packaging)
- Must be able to fold in different structures to recruit the different proteins of the different stages of the viral life cycle
Historical importance of Picornaviruses?
- Polio in Egyptian steele
- Major outbreaks only started in early 1900s because of modern sanitation, more hygiene, only contract polio later in life when not protected by mum’s Ab
- Example of how human behaviour shapes viral outbreaks and behaviour
Clinical feature of poliovirus infection?
- Transmission: fecal-oral route
- Symptoms: fever, headache, vomiting, diarrhea, neck stiffness, pain in the arms and legs, muscle weakness, paralytic poliomyelitis (2% of cases), in some cases respiratory arrest and death
- iron lung chambers used to help breathe
The poliovirus vaccines, how they were made and how they work?
- Jonas Salk vaccine: inactivated poliovirus vaccine (IPV) developed in HeLa cells and delivered by injection, inactivated with formalin, removing infectivity without destroying antigenicity, high potency, quite safe, but must be refrigerated, hard to distribute vaccine that must be refrigerated
- Albert Sabin vaccine: oral polio virus vaccine, live attenuated virus, several strains passaged through animal models and cell cultures, and plaque purified assays used to try to select for viruses that had altered properties or attenuated properties, so they are not neuropathic. Three types of viruses mixed together for the oral polio vaccine that did not require cold storage to transport and could be given orally, much easier to distribute
Epidemiology following distribution of polio vaccines?
Dramatic reduction of polio cases per year in the USA, but some vaccine-related cases from the Sabin vaccine, since it was live attenuated, could mutate enough to cause polio but not poliomyelitis, just mild gastroenteritis. Pre-vaccine 1 in 200 cases developed poliomyelitis, post-vaccine it was 1 in 2.4 million cases. Sabin vaccine discontinued, but polio free USA/Canada shortly after.
Why is polio a good candidate for eradication? And how is that going?
- No animal reservoir
- Plan to eradicate polio by WHO, most countries have completely eliminated it, only a few countries still endemic for polio (Afghanistan, Pakistan, Mozambique and Malawi)
- Sadly waste water polio discovered in the UK and USA and one case of vaccine derived polio in New York, need to double down on efforts to eradicate polio globally
Morphology and etymology of the Picornaviridae family?
- Pico (spanish = small) and RNA = RNA
- type virus: poliovirus
- Non-enveloped viruses, can have “pseudo envelopes” although no true envelope because no envelope proteins
- Icosahedral capsid
- Smallest of +ssRNA viruses with only 25-30 nm in diameter, about the size of a bacterial ribosome
- Monopartite (only one piece of RNA), linear, 7-8.5 kb in length
Some players of the Picornaviridae Family and the three important ones (besides polio)?
- Enteroviruses (polio, coxsackie, enterovirus 70)
- Rhinoviruses
- Apthoviruses (FMDV)
- Cardioviruses
- Hepatoviruses
- Three main dawgs: Foot and mouth disease 1st virus discovered in 1897, severe veterinary importance, highly contagious disease that affects all clove and hooved ruminants (cattle, swine, sheep, goat, deer, elephant, antelope, giraffe), severe significant economic impacts
Hepatitis A virus concern if travel to the Caribbean, associated with contaminated food, acute infection that lasts 6 weeks or so, vaccine like twinrix (protects hep A and B) outbreak in 2017 San Diego, mostly in homeless population, cuz no access to clean bathrooms also sprayed detergent in the streets
Rhinoviruses 40% common colds in humans
Clinical facts about HAV?
- liver infection
- transmission through faecal oral route, direct contact, food and beverages, cups and spoons, any other objects handled by the infected person
- debilitating but most recover
preventable with careful hand washing, keeping toilets and bathrooms clean, avoiding infected water sources - Symptoms: nausea and vomiting
Genome organization and proteins
- VPg (viral protein genome linked) at 5’ end, followed by a cloverleaf element and a viral IRES (internal ribosomal entry site) (all in 5’ UTR), a single poly protein followed by 3’ UTR and polyA tail is encoded in viral genome, not added after
- Polyprotein subsequently cleaved into mature viral proteins
P1 (capsid) includes VP1,2,3,4 (all capsid proteins)
P2 and P3 (non structural proteins)
P2 (2A protease, host protein synthesis shutoff, 2B cell permeability, 2C vesicle formation)
P3 (3A cellular transport, 3B VpG, 3C protease, host transcription shutoff, 3D RdRp
Picornavirus capsid structure
- VP1,2,3 on the outside, VPg inside with genome, VP4 internal scaffold for the other capsid proteins, 60 copies of each of the VP proteins, canyon between VP1,2,3 this is where receptor binds
Cellular receptors for picornavirus
- Often integrins, non specific binding, velcro, pan tropic
- CD155 receptor for polio (bonds to canyon)
How to picornaviruses enter the cell?
Depends on the virus
- Some use receptor-mediated endocytosis (rhinoviruses, FMDV)
- Others inject their viral RNA directly across the plasma membrane (poliovirus), dramatic rearrangement of the capsid following binding to receptor, VP4 moves from inside the capsid to outside the capsid, and then genome can be injected, sphingosine molecule on the receptor binds to negatively charged pocket in capsid, when it pulls out, causes the massive rearrangement as the pocket collapses, a drug has been developed to bind to this pocket, inhibiting sphingosine binding, rearrangement and therefore injection of genome
How does cap-independent translation work for picornaviruses?
- no 5’ cap
- use IRES which allows translation during host protein shutoff bcs polio turns off host cap dependent translation
- IRES can recruit the cleaves eIF4G/A bound to the rest of the protein synthesis machinery (no need for eIF4E)
how does polio turn off host protein synthesis?
Protese 2A cleaves eIF4G which links eIF4E (cap binding protein) to the rest of the translation machinery, so no translation
Describe and explain the results from the SDS PAGE gel of protein synthesis of a cell infected by polio?
- 0 hour => big black smear (lots of host proteins)
- hour 5 and 7 only viral protein
- later on no protein because cell dies without its own protein synthesis
How is the poly protein processed?
- 2A cleaves the P1/P2P3 boundary
- 3C cleaves VP0/VP3/VP1
- 3C cleaves 2A/2B/2C/P3
- 3C cleaves 3AB/3CDpro
- 3C cleaves 3A/3B 3Cpro/3Dpol
- Unknown cleavage between VP4/VP2 inside the virion when no proteases (2A and 3C) are present since non-structural proteins, so very odd
What is the dilemma of (+) ssRNA viruses?
- Ribosome 5’ to 3’
- Polymerase 3’ to 5’
Can’t start RNA synthesis unless translation is off, because only get half a protein and half RNA
Must have a mechanism to turn off translation in order to start RNA synthesis, not really understood in most viruses, but yes in picornaviruses
How does polio solve the (+)ssRNA dilemma?
Recruitment of the poly RC binding protein and poly RC binding protein 2 which bind to the cloverleaf and IRES of the genome helping to recruit ribosomes. When enough protein is made, 3CD protease cleaves polyRCBP so it loses its affinity for the IRES and can no longer help recruit ribosome, but it can still bind to the cloverleaf and can interact with the polyABP, so 3CD (has protease and RdRp activity) can bind to the cloverleaf as well and the circularization of the genome allows the polymerase to be loaded onto the 3’ end, translation off, RNA synthesis on.
How does RNA synthesis happen in picornaviruses?
There is a CRE (cis acting RNA element which recruits 3CD and VPg to it, and the polymerase can catalyze uridylation of VPg which can then be used as a primer for RNA synthesis. A (-) strand is made which can then serve as the template for RNA genome synthesis or for more mRNA to be made.
Where does picornavirus replication occur?
Virus-induced cellular vesicles which are induced by viral proteins 2B, 2C and 3A. Vesicles are absolutely required for viral RNA synthesis and serve as the nucleation site for the viral replication complex or viral replication organelle. Stain positively for dsRNA since that’s where RNA synthesis happens.
How do does picornavirus sequester these vesicles?
- In early infection, single-membrane vesicles (SMVs) containing dsRNA are derived from the Golgi and ER because secretion is inhibited
- Later on, the vesicles wrap around each other to form double-membrane vesicles (DMVs) which serve as the site of replication and assembly
- The viral proteins must be tethered to the membrane in some way, do this through the 3AB protein that binds to the membrane and the PCbp binds to the genome.
How does polio get packaged?
The P1 precursor polypeptide gets cleaved into the different VP proteins, making a 5S promoter which then assembles to make a pentamer which can then condense around the RNA to make a virion either collect around the RNA and get built on it or a prolapsed is made and then the RNA genome is somehow inserted into it.
