Lec 5 PICORNAVIRUS Flashcards
PicoRNAvirus examples
Enterovirus
-Polio virus: polio has been around for eons (paralysis)
-Coxsackie A and B
-Enterovirus D68 (taken in through mouth, moves into neurons,
Rhinovirus ( 100 different types, cause common colds)
Cardiovirus
- Mengo virus
- Encephalomyocarditis (EMC)
Apthovirus
-Foot and Mouth Disease (FMDV)
Hepatovirus
-Hepatitis A virus. Bloodborne and spreads
Poliovirus
Polio Vaccines
Getting closer to eradication
Sugar cubes are the archaic way.
Children in Canada get the 5 in one vaccine
DTaP-IPV-Hib: Diphtheria, Tetanus, Whooping Cough, Polio, Hib
In 1988, 350,000 cases whereas in 2012 243 cases
Originally the Salk or Sabin vaccines:
- SALK is INACTIVATED (Killed) poliovirus. Heat treated or formaldehyde destroys viral infectivity but leaves antigens intact. Came first.
- SABIN is an ATTENUATED viral vaccine. Passage virus through different cells until it can no longer cause symptoms in humans. Virus still replicates therefore creates more antigens before immune system causes more powerful response. Better immunogenicity. Came second.
Polio is 99% eradicated due to the vaccines
No known natural virus reservoir except humans
Picornavirus Virus Structure
Capsid proteins
VP0 = VP? + VP?
NAKED ICOSAHEDRAL virus 22-30 nm
Pentamers form a perfect spherical shape.
Capsid composed of 60 molecules of each of 4 proteins (240 proteins)
VP1, VP2, VP3, and VP4
VP1, VP2, and VP3 form OUTER SURFACE with binding groove (CANYON)
VP4 is INTERNAL and acts as RNA BINDING PROTEIN.
VP0 precursor is cleaved to VP2 and VP4 during VIRION MATURATION
CANYON: host receptor binding site. Found in-between the pentamers.
Picornavirus Viral Genome Structure
positive sense SS-RNA genome 2.4 X 10^6 da, 7-8 Kb
Relatively small. Similarities to mRNA.
3’ end poly A tail
5’ END NOT CAPPED but COVALENTLY LINKED to a 2 kD protein (VPg) through a pUp ester linked to TYROSINE
Secondary structure INTERNAL RIBOSOME ENTRY SITE (IRES) found in 5’-untranslated region. Important in mRNA function for NONCAP-TRANSLATION, host translation still works.
Picornavirus Viral - Host Genome Interactions
5’ non-coding region
Capsid Proteins
IRES Sequence
+ve sense RNA viruses all start with vRNA serving as mRNA
VPg attaches to rRNA, 3’ non-coding region help the virus enter host cell w/ VP4
5’ non-coding region
- internal ribosome entry site
- Binding site for replicase complex
Capsid proteins
Nonstructural proteins
- Protein processing
- Host shutoff
- RNA replication
3’ non-coding region
-Binding site for replicase complex
Poly A tail
INTERNAL RIBOSOME ENTRY SITE (IRES) SEQUENCE forms secondary structure that allows viral genome to attach to ribosome.
2 regions: Py rich, A/C rich and GNRA regions on 5’ end help 5’ end bind
ORF codes for only 1, big polyprotein.
Picornavirus Life Cycle
- Receptor binding
2 routes for entry, depending on how the virus enters. Enter by Direct injection or endocytic uptake, uncoat and release mRNA into cytoplasm.
IRES driven translation creates polyprotein which is processed to create several effects.
Polyprotein processing forms pro-apoptotic effects AND anti-apoptotic effects to prevent the host from performing apoptosis.
This also shuts off host cell transcription via stopping 5’ cap dependent translation by destroying eRF4G
The virus RNA forms on internal membranes to form - ssRNA, to make + ssRNA packaged into virions.
Picornavirus Attachment to host cell
Very specific receptors limit polio infections to humans, can be induced to grow in PRIMATE CULTURES
Polio binds to 2 members of Ig-superfamily:
CD155 or
Pvr (polio virus receptor)
Different receptors for Coxsackie and Rhino viruses, some require DUEL RECEPTORS
CD55 receptor for coxsackievirus A and B, echovirus, some enterovirus, but many require CO-RECEPTORS
Many picoronaviral receptors belong to Ig-superfamily and integrin proteins also help the virus enter the cell.
Picornavirus Penetration and Uncoating
Binding of virus to receptor initiates altered particle or A particle formation, structural change, VP4 lost.
Rhinovirus and foot and mouth virus use RECEPTOR MEDIATED ENDOCYTOSIS, drop in pH in ENDOSOME causes final CHANGE in VP4, OPENING HOLE through which RNA exits (LOW 1% success rate).
FUSION REGION in VP4 and VP1 may allow RNA release from phagolysosome or plasma membrane (VP1 and VP2 also suspected)
RNA sensitive to RNAase in 30-60 min, if you set up an infectious system. This means when the virus is coated, it is protected from RNAses, until its protected.
Model for entry of POLIOVIRUS RNA into the cytoplasm
Attach to surface receptors, canyon binding leads to a a major rearrangement in capsid proteins VP 1,2,3, then VP4 is EXTERNALIZED and the hydrophobic N termini of the 5 VP1 proteins INSERT INTO THE MEMBRANE and FORMS A CHANNE,L through wihich the viral RNA can pass into the cytoplasm.
VP1’s N terminus holds VPg, which holds vRNA
Picornavirus Viral Replication
Initiation of protein synthesis
This virus is an effective pirate
+ ssRNA viruses all start with vRNA serving as mRNA
Viral protease cleaves the cap binding complex ELF-4G. Holds the initial Met (AUG). Scans vRNA until the 80S ribosome is assembled.
Picorna virus RNA is not capped and so need a different mechanism of translation initiation
The virus has a protein that destroys the host’s normal EIF-4G factor so the host 5’ capped mRNA will no longer be able to be translated. The IRES takes over the 5’ non-capped part and the host’s 4G factor attaches to viral IRES to assemble the ribosome!
Initiate via Internal Ribosome Entry site.
Host cell proteins bind and dock the 40s Subunit
Picornavirus Protein translation
Picorna vRNA yields a LARGE POLYPROTEIN that is cleaved to give both NON-STRUCTURAL AND STRUCTURAL PROTEINS. Cleaved in a TEMPORAL manner.
Polyprotein contains 3 regions
5’ end (P1) codes for VP1-VP4 (CAPSID)
Middle (P2) codes for regulatory proteins
2BC allows for RA binding, vesicle formation
3’ end (P3) codes for functional proteins
3AB allows Rmembrane association of replication complex
VP0 cleaved at maturation to form VP2 and VP4
Picornavirus Protein functions
P1 produces VP1,VP2, VP3, and VP4 the capsid proteins
P2 produces 2A a nascent PROTEASE that makes the first cut FREEING P1 and then CLEAVES CAP BINDING PROTEIN TO SHUT OFF PROTEIN SYNTHESIS from capped mRNA,
2A and2B involved in RNA replication
P3 produces 3A, 3B the VPg protein,
3C the PROTEASE involved in CUTTING MOST OF THE POLYPROTEIN, and
3D the RNA dependent RNA polymerase
Protein synthesis required for RNA replication
Picornavirus RNA replication Requirements
Requires 3B (VPg) as PRIMER (VPg-pUp-) 3D as RdRp RNA dependent RNA polymerase 2B and 2C as regulators Host factors and associated with host membranes
Host-cell transcription is also dramatically reduced by several picornavirus infections. Transcription by all 3 types of eukaryotic RNA polymerase is terminated, creating a disproportionately higher number of viral RNA molecules in the host cell. This effect seems to be mediated by the 3C protease, which cleaves several cellular transcription factors between Gln–Gly pairs
POLYMERASE COMPLEX copies vRNA to complementary RNA(cRNA) and then cRNA to make vRNA multiple times
Polymerase make ~ 5% cRNA and >90% vRNA
vRNA has 2 functions
- early vRNA serves as mRNA for protein synthesis
- Serve as new viral genome
Reaches peak 3-4 hours post infection
Proposed mechanism for replication of Picornavirus RNA
2B, 2C and 3A participate in induction of vesicles
Host protein PCBP2 binds to PBG at cell membrane surface (made by virus) has poly U surface. This tail binds to poly A tail and it w/ PBG is cut off to create REPLICATIVE INTERMEDIATE to serve as PRIMER.
3AB anchors Complex to membrane 3B portion identical to VpG is covalently attached to 5’ ends of genome and antigenome
PROTEIN BOUND URIDINES hybridize to the polyA tail and serve as a primer to initiate RNA synthesis
3D PROTEIN is RNA polymerase, reads 5’ U’s to create progeny vRNA
PCBP2: Host polyC binding protein 2
Cre cis-acting replication elements
Picornavirus Post RNA replication protein synthesis
Production of multiple copies of genomic + ss RNA allows for FURTHER PROTEIN SYNTHESIS
As CAP binding protein is now destroyed FURTHER TRANSLATION MUST OCCUR on CAP negative RNA
Therefore VPg is CLEAVED from SOME of the +ve RNA and IRES drives translation
