Picornaviruses

Plus Strand RNA Virus Families

PICORNAVIRIDAE• More than 200 viruses prevalent world-wide.• cause many serious diseases of animals and man.• Foot and mouth virus first animal virus described (1898).• Poliovirus is an important model:

- first virus purified and crystallized.- first inactivated vaccine used (Salk 1950’s).- first picornavirus to be sequenced.- first infectious cDNA clone of an animal virus.- first picornavirus structure to be solved.

PICORNAVIRUS PROPERTIES

• Capsids are small unenveloped 25 -30 nm Capsids are small unenveloped 25 -30 nm icosahedra.icosahedra.• Resistant to pH 3 to 9 (except for Resistant to pH 3 to 9 (except for Rhinoviruses).Rhinoviruses).• Plus sense single stranded RNA genomes Plus sense single stranded RNA genomes

((~~7400 bases).7400 bases).

• Genome is monopartite.Genome is monopartite.• RNA 5’ end has a covalently attached VPg (22-RNA 5’ end has a covalently attached VPg (22-24 aa) 24 aa) and 3’ end is polyadenylated.and 3’ end is polyadenylated.

• The 5’ end contains a highly structured (The 5’ end contains a highly structured (~~740 740

nt) nt) untranslated region that contains untranslated region that contains several AUG’s.several AUG’s.• The naked RNA is sufficient for infection.The naked RNA is sufficient for infection.• The RNA is translated into a polyprotein that is The RNA is translated into a polyprotein that is cleaved cleaved into enzymatic and structural into enzymatic and structural proteins. proteins. •The Virus replicates in the cytoplasm.The Virus replicates in the cytoplasm.

Hepatitis A Simian hepatitis A

Echovirus 22

Evolutionary distance

Hepatoviruses

FMDV-AFMDV-O

EMC

TMEV (Theilers)Aichi

Aphthoviruses

Cardioviruses

Kobuvirus

Coxsackie B3Swine vesicular disease

Enterovirus 70

Poliovirus type 1

Coxsackie A16

Bovine enterovirus

Human rhinovirus 14Human rhinovirus 2

Human rhinovirus 89

Enteroviruses

Rhinoviruses

00.5

Parechovirus

A Phylogenetic Tree of the Picornaviridae

Members of the Picornaviridae Cause Many Serious Diseases of Man and Animals

Genus Enterovirus

- Poliovirus type member, 3 major types cause paralysis.

Genus Rhinovirus

- cause respiratory tract infections, acid labile, cause

colds in humans (110 types) and pigs.

Genus Hepatovirus

- Hepatitis A, contagious liver infections.

Genus Cardiovirus

- EMC group, cause heart and brain inflammation acid labile, source is a rodent reservoir.

Genus Apthovirus

- Foot and mouth disease, most destructive in Africa.

PICORNAVIRUS STRUCTUREPICORNAVIRUS STRUCTURE• The Basic capsid building block is a protomer thatconsists of one copy each of VP1, VP2, VP3 & VP4. •VP1, VP2 & VP3 are on the virion surface, with VP4 being internal. •VP1, VP2 & VP3 have no sequence homology, but have the same topology.

• Five protomers assemble into a pentamer.• The icosahedron is formed by 12 pentamers.

VP1VP2VP3

5-fold Axis

3-fold Axis

2-fold Axis

Capsid is a pseudo T=3 icosahedron consisting of 60 identical asymmetric protomers arranged as 12 pentamers.

Each protomer is composed of a single copy of each of the four capsid proteins, VP1, VP2, VP3 and VP4.

VP4 is located on the inner surface of the protein shell formed by VP1, VP2 and VP3.

All three proteins contain a central –barrel jelly roll.

The jelly roll is a wedge-shaped structure that consists of two antiparallel -sheets.

Picornavirus Capsid Structure

Picornavirus Members Bind to Many Different Host Receptors

Scr-short consensus repeat

LDL-low density lipoprotein

T/S/P threonine/serine/proline

IG Like - Members of IG Like - Members of the immunoglobulin the immunoglobulin family of receptors family of receptors found on cells of the found on cells of the immune system.immune system.

A single type of receptor mediates virus Human Rhinovirus binding and entry

The integral membrane protein, intercellular adhesion molecule 1 (Icam-1) was identified as the receptor for the major group of human rhinoviruses. Icam-1 is a member of the immunoglobulin superfamily. Icam -1 is found on the surface of many tissues, including nasal epithelium and lung epithelium. The normal function of Icam-1 is to bind a ligand on the surface of lymphocytes and to promote immunological and inflammatory functions. This host response accounts in part for cold symptoms.

LIFE CYCLE OF RHINOVIRUS

Picornavirus Attachment To the Icam-1 Receptor

Antibody molecules can also penetrate into the canyon and antibodies that bind to the virus in this manner neutralize infectivity by blocking entry of the receptor into the canyon. However, viruses quickly mutate to change the shape of the canyon to prevent antibody binding.Some antiviral drugs (WIN

compounds) can irreversibly replace the lipid that lines the tunnel and inhibit uncoating and in some cases attachment of the virus.

A depression or canyon is formed at the junction of VP1 and VP3 in the capsids of rhinoviruses and some enteroviruses like poliovirus.The canyons are the sites of interaction with cell surface receptors.As the picornavirus binds to Icam-1, the host receptor penetrates into the viral canyon and this causes a change in conformation of the capsid to permit virus entry.

Polyproteins are Proteolytic Processed During Translation

During translation, the 2A proteinase cleaves at its amino terminus immediately after it is translated. The 2A cleavage releases the structural protein (P1) that is the precursor to the individual viral capsid proteins. The Proteinase 3C and its precursor 3CD carry out the remaining polyprotein cleavages.

The 2B protein is a host range determinant that is also involved in RNA

synthesis. The 2C protein is very

highly conserved and is also involved in RNA

synthesis. 3D is an RNA dependent RNA polymerase and has

GDD polymerase motif. The 3B protein is the VPg.

More Ribosomal Scanning!!!!!!!More Ribosomal Scanning!!!!!!!

• As 40S subunit scans the untranslated Region (UTR) a number of eIFs (eukaryotic elongation factors) including eIF-3, eIF-2, eIF-4 & eIF-5 become associated with the subunit. • eIF4G, a component of the eIF4F cap binding complex, acts as a bridge between the cap structure and the 40S subunit.• The helicase, eIF4A, associates with eIF4F to unwind secondary structure in the UTR.• The 40S subunit plus the eIFs increase subunit size progressively.• eIF-5 associates with the subunits to promote 60S subunit assembly and recognition of AUG Kozak Sequences(5’-GCA/GCCAUGG-3’).

Picornavirus Translation: Picornavirus translation is cap independent

Picornavirus mRNAs have no cap (pUp) at their 5’ terminus and no VPg.

The genome has a (743 nt) long UTRuntranslated leader sequence that

contains 8 upstream AUGs preceding the translational initiation site.

Internal ribosomal binding occurs at an Internal Ribosomal Entry Sequence (IRES).

The IRES consists of a high level of secondary structure in the UTR leader

sequence that mediates ribosome 40 S subunit binding and initiation.

eIF-3, eIF-4G and eIF-4a promote IRES assembly. A host factor X is also required.

The first step in replication is to translate the viral RNA.

Picornaviruses replicate using an RNA copying mechanism.

The replicating RNA has a VPg, but the mRNAs lack this protein.

Replication of Picornavirus RNA

gRNAgRNA

- RNA- RNA

mRNAmRNA

PP