Lecture 7 Flashcards
What super group of +RNA is picornavirus apart of?
Picornavirus like Supergroup I
What are the defining features of the picornavirus?
Naked virion, very stable and it is 22-30 nm in diameter
Icosahedral symmetry (60 copies each of VP1-4)
Oral-fecal transmission
Positive-strand, ssRNA, 5’ end VPg but no cap, poly(A) tail
Genomic RNA is the only mRNA, translated into a polyprotein (precursor)
IRERS to initiate translation of viral RNA
What are diseases caused by the family Picornaviridae?
1) Common cold: infection of the upper respiratory tract and accounts for 50% of common colds, cough, sneeze, runny nose
2) Hepatitis A: Acute liver infections; sporadic outbreaks via contaminated food in restaurants, vegetables
3) Heart infectious: myocarditis, dilated cardiomyopathy
4) Diabetes & pancreatic disorders: coxsackievirus, encephala-myocarditis virus
5) Foot and mouth disease: most destructive disease of cloven-hoofed livestock animas, quarantinable
What is poliomyelitis?
Most polio infections were inapparent and self-limiting
The paralytic form of polio infection was the most devastating and feared
Major infantile and childhood disease in the 1st half of the 20th century (hygiene hypothesis)
What was the US national war against polio?
In 1916: 27000 cases, 6000 deaths, a third in New York City alone
FDR declared national war against polio in 1930s
1952 epidemic: 58000 children infected, 21,269 (36%) displayed different degrees of paralysis: 3,145 (5.4%) died
What was the only treatment for polio?
Iron lung
Donated to CDC by family of polio patient, Barton Herbet who spent 50 years of his life in iron lung
How was the cure for polio discovered?
1955: Jonas Salk, created highly effective inactivated polio vaccine using cell cultures developed by John Enders and colleagues
Salk tested. vaccine on himself and family
Shown to be safe and efficacious against polio
What were the key discoveries made with picornaviruses?
FMDV: Loeffler & Frosch (1898)
Isolation of PV: Landsteiner, through transmission to monkeys (1909)
Cell culture: Enders, Robbins, and Weller (1949)
Plaque assay: Dulbecco (1952)
IPV: (inactivated vaccine): Jonas Salk (1955)
OPV: (weakened live virus): Albert Sabin
RdRP: Baltimore (1963), from polio-infected cell
Recognition of polyprotein: Summers and Maizel (1968)
Infectious cDNA clone: Racaniello & Baltimore
X-ray structure: Polio & human rhinovirus (1985)
IRES: Pelletier & Sonenberg (1988)
What is the genome structure of picornaviruses?
Genome: ssRNA, positive strand: 7500- 8450 nucleotides
5’ end: VPg (virion protein, genome-linked, 22-24 amino acids
VPN also in several families of RNA plant viruses
5’ UTR: very long, 624-1199 its, cloverleaf at 5’ end, IRES in UTR
Middle: Single large ORF encoding a single polyprotein precursor
3’ end: 47-125 nts NTR, polyA tail (essential for infectivity)
What is the process for polyprotein and proteolytic processing of a picornavirus?
There is a large polypeptide but has to go through protein processing
A single large ORF encodes a polyprotein, which is cut by proteases into 11-12 functional proteins required for replication and infection
2Aproline cuts once separating P1 from rest of polyprotein: 3Cpro cut at 8 places
Genes encoding structural proteins are located at the 5’ one third, whereas proteins involved in replication are encoded by the 3’ genome region
Why is polio so dangerous?
It shuts down translation of cellular mRNAs
Poliovirus infection cause complete shutdown of translation of cellular mRNAs
The translation machinery is re-directed for viral proteins only
Prevention of formation of pre-initiation complex at the cap of host mRNAs
Viruses highjacks cell
How is the picornavirus protein synthesized?
Cap-dependent translation
How does cap-dependent translation work?
For most eukaryotic mRNAs, the pre-initiation complex binds at 5’ cap, brings two ends of the mRNA together, scan for the first AUG, large ribosome subunit joins, translation starts (Translation starts at first AUG)
What are the proteins involved in cap-dependent translation?
EIF-4F: a tripartite structure, composed of eIF-4A, -4E, - 4G
eIF-4G: eukaryotic initiation factor for 4G
eiF-4G binds to cap structure
How do picornaviruses block transcription and translation of host mRNAs
They block both transcription and translation of host mRNAs:
–> Inhibit pre-initiation complex at the cap of cellular mRNAs via two mechanisms:
1) 2Apro (polio) or L protease (FMDV) cleaves eIF-4G, blocking formation of the pre-initiation complex at the Cap (translation cannot occur)
2) Dephosphorylation of 4E- BP1, binds eIF-4E tightly, sequestering eIF-4E
Inhibition of mRNA transcription: 3Cpro cleaves TATA-binding protein
Poliovirus 2Apro causes FMDV Pro
Deposphorylate 4E-BP1: Encephalomyocarditis virus
How are viral proteins translated?
By IRES
There are two types: Type 1 IRES (enter, rhinoviruses) and Type 2 IRES (FMDV, HCV)
How does IRES translate proteins?
Extensive secondary (stem-loop) and tertiary (pseudo knots) structures in 5’ UTR
Pyrimidine-rich region upstream of AUG:
AUG downstream of IRES
–> N: any nucleotide
–> R: either A or G
Initiation complex binds directly at IRES, landing at start codon, initiating translation, so there is no need for cap structure
IRES are sites for ribosomes to bind, so the cap is not needed
What happens in PV-infected host cells?
With TK translation is shut down, because 40S can not bind
With CAT translation does occur through IRES structure, even though there is no cap
Why is VPg so important?
They are protein primers for genome replication
It has nothing to do with translation and has everything to do with genome replication
How does the VPg work?
Viral 3AB attaches to ER membrane Tyrosine in 3B undergoes uridylation Inserted 'UU' anneals to poly(A) tail of the viral genome VPg (3B) cleaved off from 3AB by 3Cpro Synthesize negative-strand RNA Replicative intermediate (RI) Replicative form (RF) --> Serves as primer for genome replication
Where does viral replication occur for poliovirus?
On ER membrane derived vesicles
What is special about the viral replication for poliovirus?
Viral translation, vesicle formation and RNA synthesis are coupled
Explain the poliovirus replication cycle?
Attachement: Canyon: poliovirus, rhinovirus,
Surface loop: FMDV
Receptors: PVR, ICAM-1
Un-coating: Sphingosine within hydrophobic pocket of pentamer, help VP1 penetrate membrane to form pore
Biosynthesis: genome replication and IRES-based protein synthesis
Assembly/Maturation: VP0 cleavage into VP2 & VP4 (host protease)
How was the control of poliomyelitis?
Inactivated polio vaccine
Live attenuated vaccines (Sabin in 1960): used for global vaccination
How did Sabin derive type 3 attenuated poliovirus?
Type 3 polio was isolated from fatal paralytic case
21 passages in vivo (intracerebrali in monkeys)
8 passages in vitro (monkey testicle cultures)
39 passages in vitro (monkey kidney cultures)
3 plaque purifications (monkey kidney culture)
3 passages in vitro (preparative, monkey kidney culture)
Vaccine strain created
What does it mean to do a passage?
Infect cell cultures, and recover the modified virus, and then you reinfect in another passage with modified virus
What were the determinants of attenuation in the Sabin vaccine strain?
P1/Sabin
P2/Sabin
P3/Sabin
Explain the eradication of poliovirus
1990: Free in America Continent
2005: WHO initial target for global eradication, but revised target date was 2018
1950: Cases sky rocketed, in 1955, Salk inactivated vaccine brought cases down,
1960: Oral vaccine came in, Sabin’s vaccine, and cases were brought down even further
Explain the eradication of Polio in Canada
Cases spiked in 1953 IPV: 1955 (cases drastically dropped) OPV: 1962 (cases dropped even further 1977: last indigenous case reported 1994: WHO certifies Canada as polio free
What was/is the global situation of polio?
1988: 350,000 cases
2001: 470 cases
2007: 4 countries endemic
As of Oct 7, 2020: WPV: 102 cases (Pakistan, Afghanistan). cVDPV: 302 cases (among 17 countries
What was the Cutter incident?
Patient was inoculated with Salk vaccine, and developed paralysis of both legs
About 400,000 children were inoculated with the same vaccine in 10-day period. Within 2 months, 94 cases of polio among vaccines and 166 cases among family and community cases
How did vaccines help eradicate polio?
Sabin live vaccine became available in 1963, US cases dropped from 60K a decade ago to 396 due to use of vaccines
Ultimately, Sabin vaccine replaced Salk vaccine due to adverse incidents related to vaccine-derived polio infections and paralysis
How was poliovirus reconstructed?
Synthesize DNA fragments as oligos
Assembly into genome fragments
Clone into vector under T7 promoter
In vitro transcription to obtain viral RNA
Assay for infectivity in mice expressing receptor Per
