Picornaviridae

Supergroup I of (+)ssRNA - Picornaviruses

• Features

  • No 5' cap

  • 3' Poly(A) tail

  • No envelope

  • V stable

  • 22-30 nm diameter

  • Icosahedral symmetry

    • 60 copies ea of 4 structural prtns (VP1-4)

      • VP-3 on surface

      • VP-4 hidden under

  • Oral-fecal transmission

  • Genomic RNA is the only mRNA

    • Translated into a polyprtn (precursor)

  • IRES= start site for viral tsln

  • Triangulation # of pseudo 3

  • β-barrel jelly roll conserved in icosahedral viruses of plants, insects, animals, humans

    • Topological family of prtns

Diseases caused by Picornaviridae

• Poliomyelitis

  • Infantile infection w multiple epidemics in North America

• Common cold

  • Upper respiratory tract

  • ~50% of common colds

• Hep A

  • Acute liver infections

  • Sporadic outbreaks via food & drinks

• Heart infections

  • Myocarditis

  • Dilated cardiomyopathy (DCM)

    • Caused by group B Coxsackieviruses

• Diabetes & pancreatic disorders

  • Coxsackieviruses

  • Encephalomyocarditis virus

• Foot-and-mouth disease

  • Quarantinable

Poliomyelitis

• Most polio infections were inapparent & self limiting

• Paralytic form most feared

• Major infantile & childhood disease in the 1st half of the 20th century

  • The hygiene hypothesis

    • Early exposure to microorganisms helps build immune system

• United States

  • 1894

    • 1st reported case

  • 1916

    • 27 000 cases

    • 6000 deaths (1/3 from NYC)

  • 1930s

    • FDR declares national war against polio

  • National Foundation for Infantile Paralysis

    • Later, March of Dimes

  • 1952 epidemic

    • 58 000 children infected

    • 21 269 (36%) displayed paralysis of various severity

    • 3145 (5.4%) deaths

• Canada

  • 1910

    • 1st reported case (Hamilton, ON)

  • 1937

    • 4000 cases nationally (>50% in ON)

    • 119 deaths (4.7%)

    • Only single iron lung available - hospital staff rush to make more

  • 1953

    • ~9000 cases

    • 500 deaths (5.5%)

  • Panic - social distance, isolation, quarantine, etc.

• Iron lung

  • Only treatment & hope of severe polio patients w lung complications

  • Stay in lung until immune syst heals body

  • Ex. Barton Hebert

    • Stayed in iron lung for last 50 yrs of life

• Salk vaccine

  • Jonas Salk (1955)

    • 1st highly effective inactivated polio vaccine

    • Tested on self & family to convince others

    • Large clinical trials in USA

Key discoveries made w picornaviruses

• FMDV

  • Loeffler & Frosch (1898)

  • 1st animal virus discovered shortly after TMV

• Isolation of PV

  • Landsteiner (1909)

  • Via transmission experiment to monkeys

• Cell culture

  • Enders, Robbins, & Weller (1949)

• Plaque assay

  • Dulbecco (1952)

• IPV (inactivated vaccine)

  • Jonas Salk (1955)

• OPV (weakened live virus vaccine)

  • Albert Sabin & Hilary Koprowski (1960)

• RdRP

  • Baltimore (1963)

  • From polio-infected cell

• Polyprtn

  • Summers & Maizel (1968)

• Infectious cDNA clone

  • Racaniello & Baltimore (1981)

• IRES

  • Pelletier & Sonenberg (1988)

Genome structure & expression strategies

• (+)ssRNA

• 7500 - 8450 nts

• 5' end

  • VPg (virion prtn, genome-linked, 22-24 aa residues)

  • 5' UTR: v long secondary structures

• Middle

  • Single large ORF encoding 1 polyprtn as precursor

• 3' end

  • 47-125 nts UTR

  • Poly(A) tail essential for infectivity

Polyprtn & proteolytic processing

• Viral RNA is only mRNA

• Single large ORF encodes a polyprtn

  • Polyprtn cut by proteases into 11-12 functional prtns needed for rep'n

• 2A^pro cuts once

  • Separates P1 from rest of polyprtn

• 3C^pro cuts at 8 places

  • Produces all final prtns needed for rep'n

• Polio infection shuts down tsln of cellular mRNAs

  • Tsln machinery re-directed for viral prtns only

  • Prevention of PIC formation at cap of host mRNAs

• Cap-dependent tsln

  • All mRNAs, PIC assembles at 5' cap

    • Brings 2 ends of mRNA together and scan for AUG

    • Large ribosome subunit joins & tsln starts

  • Prtns in tsln:

    • eIF-4F: tripartite structure (eIF -4A, -4E, and -4G)

    • eIF-4G: euk initiation factor 4G

• Picornaviruses block tsln of host mRNA

  • Doesn't block own tsln

  • Inhibit PIC formation at 5' cap via:

    1. 2A^Pro (polio) or L protease (FMDV) cleaves eIF-4G

    2. Dephosp'n of 4E-BP1, binds eIF-4E tight, sequestering eIF-4E (used mt FMDV, not PV)

  • How PV prtns are translated

    • Secondary structures= stem-loop (5' UTR)

    • Tertiary structures= pseudoknots (5' UTR)

    • AUG Is downstream of IRES

    • Pyrimidine-rich seq'ce upstream of AUG

    • PIC binds directly at IRES -> lands on AUG -> initiates tsln

      • No need for cap

Poliovirus replication cycle

• Attachment

  • Canyon: poliovirus, rhinovirus

  • Surface loop: FMDV

  • Receptors: PVR, ICAM-1

• Uncoating

  • Sphingosine in hydrophobic pocket of pentamer helps VP1 penetrate mem (forms pore)

• Biosynthesis

  • Genome rep'n & IRES-based prtn syn

• Assembly

• Maturation

  • VP0 cleavage into VP2 & Vp4

Control of poliomyelitis

• Inactivated polio virus vaccine (Salk, 1955)

• Live attenuated vaccines (Sabin, 1960)

  • Dif Sabin vaccine strains possess mut'ns at multiple sites

The Cutter Incident

• April 1955

• Patient inoculated in buttock w Cutter vaccine

  • 9 days later, admitted to hospital for flaccid paralysis in both legs

• Stats

  • ~400 000 children given same vaccine in 10-day period

  • Within 2 months:

    • 94 cases of polio among vaccinees

    • 166 cases among family & community contacts

• 1995, Stalk succeeded with killed polio vaccine

Reconstruction of poliovirus

• US department of defence

• Syn DNA fragments as oligos

• Assembly into genome fragments

• Clone vector under T7 promoter

• In vitro txn to get viral RNA

• Assay for infectivity in mice expressing receptor Pvr

VRC: sites of poliovirus rep'n

• Viral rep'n on vesicles from ER

• Viral tsln, vesicle formation, & RNA syn are couples

VPg as prtn primes for genome rep'n

• Viral 3AB attaches to ER mem

• Tyr in 3B undergoes uridylylation

• Inserted 'UU' anneals poly(A) tail of viral genome

• VPg (3B) cleaved off from 3AB by 3C^Pro

• Syn (-)RNA

• Replicative intermediate (RI)

• Replicative form (RF)

Summary

• Picornaviridae: fam of (+)ssRNA viruses

  • Order: Picornavirales

  • Realm: Riboviria

  • Kingdom: Orthornavirae

• Belong to picornavirus-like supergroup of RNA viruses

• Members cause various diseases in humans & livestock

  • Poliomyelitis

  • Common cold

  • Heart diseases in humans

  • FMDV

• Polio

  • Effects

    • One of most damaging viral diseases of 20th century (after atomic bomb)

    • Causes epidemics of flaccid paralysis & death in infants due to lack of protecting antibodies in mothers as a result of the practice of better personal hygiene

    • Development & global use of vaccines (OPV & IPV) resulted in near worldwide eradication

  • Prototype of Enterovirus genus & family Picornaviridae

    • RNA genome:

      • V long 5' UTR

      • Single ORF

        • Tsln via cap-independent mechanism by cellular machinery using unique structure designated to the Internal ribosomal entry site (IRES)

        • Translated into a polyprtn, which is cleaved by proteases (encoded by the virus) into final prtn products

        • 2A: cleaves off P1 (b/w P1 & P2)

        • 3CD & 3C: cleave remaining sites

        • 2A & L cleave the euk tsln initiation factor (eIF-4G) -> shuts down tsln of host RNA upon infection -> infected cell now a factory for progeny viruses

      • 3' UTR

      • N-term: VPg polypeptide

        • Prtn primer for syn of gRNA & complimentary RNA (uridylylation)

• Research discoveries

  • Identification of RNA-dependent RNA Pol (RdRP)

  • Establishment of non-neuronal cell cultures

  • Plaque assay

  • Inactivated polio vaccine (IPV) (Salk)

  • Attenuated oral vaccine (OPV) (Sabin)

  • Polyprtn & proteolytic processing

  • Internal ribosome entry site (IRES) for initiation of tsln of viral DNA

  • 1st infectious viral clone for an animal virus

  • 1st synthetic polio viral clone from synthetic biology approach