Picornaviridae

1. Picornaviridae Molecular Virology

2. 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.

3. ss, positive sense RNA, icosahedral capsid, 25-30nm, and no envelope PicoRNAviruses Pico= small [Greek] Positive sense ICOsahedral RNA genome 4 coat proteins VP1, VP2, VP3,VP4 simple capsid : 60 capsomere (composed of VP1,2,3) The single-strand genome of 7500–8500 nucleotides

4. Properties • Picornaviruses are among the smallest pathogens of vertebrates and are responsible for many important diseases in humans and animals. • Picornaviruses are responsible for a wide range of clinical diseases resulting from multiple factors such as receptor specificity, tissue-specific susceptibility, virulence and the mechanisms of transmission. • Picornaviruses bind to cell specific surface receptors and this interaction is an important factor in determining host and tissue specificity of each virus.

5. Properties • replicate in cytoplasm • Positive sense genome (genome RNA is infectious) directly as a messenger RNA to template • encode viral polymerase • mature viral proteins generated through proteolytic cleavage • virions released on cell lysis

6. Properties • Picornaviruses cannot gain entry to cells by membrane fusion, as is the case for enveloped viruses, and require special mechanisms to breach cellular membranes and safely deliver the genome into the host cell. • Genome replication occurs in association with virus modified cellular membranes. • Viral RNA templates complementary negative strand molecules which in turn template multiple positive strand copies. • The synthesis of all RNA molecules is initiated by an uridylated peptide primer. • The mechanisms of transmission of infection play key roles in the epidemiology of picornavirus infections.

7. Evolutionary relationships

8. Evolutionary relationships 7 Genera Recognized acid resistant acid sensitive

9. Classification

10. Virion structure • Naked icosahedral virus, 22-30 nm in size, with a dense core of about 16 nm. sedimentation value is 160S • The capsid consists of a densely-packed icosahedral arrangement of 60 protomers, each consisting of 4 polypeptides, VP1, VP2, VP3 and VP4. • VP4 is located on the internal side of the capsid. • RNA is single stranded RNA ： 1. 35S, 2.4 X 106 daltons, 7-8 Kb, 30% of virion mass 2. 3' end 90 poly A nucleotides 3. 5' is not capped but terminated in pUp that is covalently linked to a 2 kD (VPg) protein joined through an ester link to tyrosine

11. Virion structure (a) Basic ‘jelly roll’ fold of proteins VP1–3. (b) Poliovirus VP1. (c) Poliovirus VP2. (d) Poliovirus VP3. (e) Icosahedron showing locations of 2-, 3- and 5-fold axes of symmetry and VP1–3. (f) Cryoelectron microscopy-derived reconstruction of poliovirus. (g) Cryoelectron microscopy-derived reconstruction of complex of poliovirus and soluble form of cell receptor. Courtesy of Dr J Hogle.

12. Genome

13. Genome • linear, ssRNA(+) genome of 7.1-8.9 kb, polyadenylated, composed of a single ORF encoding a polyprotein. • Viral genomic RNA has a viral protein (VPg) at its 5’ end instead of a methylated nucleotide cap structure. • The long UTR at the 5’ end contains an internal ribosome entry site (IRES). The IRES allows direct translation of the polyprotein. • The P1 region encodes the structural polypeptides. • The P2 and P3 regions encode the nonstructural proteins associated with replication. • The shorter 3’ UTR is important in (-)strand synthesis. • L is an additional N-terminal leader protein present in some genera that can either be a protease (aphthoviruses, erboviruses) or have other function (kobuvirus, cardiovirus).

14. Genome polyprotein processing

15. Attachment Attachment. 1 Capsid RNA

16. Receptor proteins IG Like - Members of the immunoglobulin family of receptors found on cells of the immune system. Figure 4. CAR, coxsackie/adenovirus receptor; PVR, poliovirus receptor; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vitronectin cellular adhesion molecule 1; DAF, decay-accelerating factor; HAVcr-1, hepatitis A virus receptor 1; LDLR, low-density lipid receptor; 2 1, integrin; v 3, integrin; CBV, Coxsackie B virus; PV, Poliovirus; HRV, Human rhinovirus; EMCV, Encephalomyocarditis virus; HAV, Hepatitis A virus; EV1, Echovirus 1; FMDV, Foot-and-mouth disease virus and CAV9, Coxsackie virus 9. Courtesy of Dr D Evans.

17. 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.

18. Attachment To the Icam-1 Receptor • A canyon is formed 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. • Ab：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. • antiviral drugs (WIN compounds) ：inhibit uncoating and attachment of the virus.

19. Penetration and Uncoating 1 Attachment. Capsid Nucleus RNA Host cell Cytoplasm 2 Entry and uncoating. Uncoating releases viral RNA and proteins. • Receptor mediated endocytosis, fusion with lysosome, lowered pH, loss of VP4 • Loss of VP4 opens hole in centre of pentamer for RNA release • Release of RNA into cytoplasm (about 1% of virus succeed) • Fusion area in VP4 may create pore in phagolysososme to allow RNA out.

20. Entry into Cells • Some non-enveloped virus inject their genome into the host cytoplasm through creation of a pore in the host membrane. This is mediated by a viral pore-forming peptide associated with the viral capsid. • The icosahedric capsid of picornaviridae is able to create a pore either at plasma or endosomal membrane to inject their genomic RNA.

21. Entry into Cells • During interactions of poliovirus with its receptor major conformational rearrangements occur in the virus particle. • The particles lose VP4 and the hydrophobic N-terminus of VP1 is displaced to the virion surface • N-termini of VP1 forms a pore in the cell membrane through which the RNA is released into the cytosol. • Some evidence suggests that virus particles may undergo endocytosis in some cell types.

22. Replication Attachment. 1 Capsid Nucleus RNA Host cell Cytoplasm Entry and uncoating. 2 RNA replication by viral RNA- dependent RNA polymerase. 3 Uncoating releases viral RNA and proteins. – strand is transcribed from + viral genome. Viral genome (RNA) Viral protein mRNA is transcribed from the – strand. ssRNA; + or sense strand; Picornaviridae

23. Replication Cytoplasmic • VPg is removed from the viral RNA, then translated into a processed polyprotein. • In entero-, rhino, and aphtoviruses, shutoff of cellular cap-dependent translation through the cleavage of the translation initiation factor eIF4G by viral protease. • Replication of viral RNA takes place on membrane vesicles derived from the ER. • A negative-sense complementary ssRNA is synthesized using the genomic RNA as a template. • New genomic RNA synthesized is packaged into preformed procapsids.

24. Replication initiates by formation of a Covalent Bond • Polioviral RNA is linked to the 22 aa VPg via a uridine-tyrosine phosphodiester bond. • During replication, mRNAs are produced by cleavage of this phosphodiester bond by a cellular enzyme to produce viral mRNAs containing a 5’ terminal Up. • Genomic RNAs incorporated into virus particles contain the 5’ VPg. • These steps occur in vesicles derived from the smooth endoplasmic reticulum.

25. Replication • vRNA translated to form a large polyprotein which is then cleaved to yield 3 major segments • P1- the capsid proteins are at the 5' end • P2- regulatory protein in the centre • P3-growth functional proteins at the 3' end • Polyprotein is cleaved in a cascade process with early cleavage by protein 2A (a nascent protease), intermediate cleavage by 3C, maturation cleavage of V0 by VP2 to yield VP2 and VP4 .

26. Replication 1. 2Apro • Poliovirus genome contains a single ORF, which encodes a 247 kD polyprotein. • It has VPg at its 5’ end and a poly(A) tail at its 3’ end • Processing occurs in 3 steps. • The first is to cleave the P1 capsid protein precursor, which is catalyzed by 2Apro • The second step is to process the noncapsid and the capsid precursors catalyzed by 3Cpro and 3CDpro • The third step is the processing of VP0 into VP4 and VP2

27. Replication • The organization is as follows:

28. Replication Polyproteins are Proteolytic Processed During Translation • Protein synthesis is required for RNA synthesis • RNA replication requires: • 3B (VPg) as a primer (VPg-pUp-) • 3D to serve as the polymerase • 2B and 2C for vRNA synthesis • host factors • synthesis takes place on host membranes • Polymerase complex copies +vRNA to -cRNA and then -cRNA back to +vRNA • Polymerase makes 5-10% cRNA and >90% vRNA • Most early vRNA ends up as mRNA and don't have VPg associated; 50% of later vRNA is packaged and has VPg • Late mRNA lacks caps because the 2A protein cleaves the cap binding protein of ribosomes so host synthesis is blocked but naked vRNA is translated • Translation reaches peak 3-4 hours post infection

29. Polyproteins are Proteolytic Processed During Translation

30. Replication Attachment. 1 Capsid Nucleus RNA Host cell Cytoplasm Entry and uncoating. 2 RNA replication by viral RNA- dependent RNA polymerase. 3 Translation and syn-thesis of viral proteins. 4 Uncoating releases viral RNA and proteins. – strand is transcribed from + viral genome. Viral genome (RNA) Viral protein Capsid protein + strand mRNA is transcribed from the – strand. ssRNA; + or sense strand; Picornaviridae

31. Replication Attachment. 1 Capsid Nucleus RNA Host cell Cytoplasm Entry and uncoating. 2 Maturation and release. 5 RNA replication by viral RNA- dependent RNA polymerase. 3 Translation and syn-thesis of viral proteins. 4 Uncoating releases viral RNA and proteins. – strand is transcribed from + viral genome. Viral genome (RNA) Viral protein Capsid protein + strand mRNA is transcribed from the – strand. ssRNA; + or sense strand; Picornaviridae

32. 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 UTR untranslated 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.

33. A Host Protein is Required for Synthesis of Polio Virus RNA A highly based paired structure formed from the 108 nucleotides at the 5’ end of the (+) strand RNA of poliovirus, which forms a cloverleaf like structure. The viral protein 3CD and a PCBP a host Cellular protein, (PolyrC Binding Protein) interact with different loops of the cloverleaf. This interaction brings the 5’ end close to the 3’ end. At this point VPg priming can occur. Formation of this ribonucleoprotein complex is required for synthesis of both plus and minus strand RNA.

34. Steps in the Assembly of Poliovirus P1 Peptide Folded P1 5S Unit • After Translation and release by the 2A protease, P1 folds and is cleaved by the 3CD protease to form the 5S protomers. • assemble into a 14S complex. • The 14S complex may then form a 75S empty capsid that assembles with the RNA to form a noninfectious 150S provirion. • Some researchers argue that the 14S subunits assemble in a series of steps with the viral RNA to form provirions. • The provirion is converted to an infectous form by internal cleavage of VP0 molecules to VP4 plus VP2. Assembly steps are not reversible

35. Poliovirus Life Cycle • The capsid binds to host receptors and releases Viral RNA into the cell. • Translation of the released RNA produces a polyprotein that is processed by the 2A and 3CD proteinases to form the viral gene products. • RNA synthesis is initiated by the 3AB proteins and elongated by the 3D protein. • As proteins viral capsid assembly begins in the cytoplasm from P1 products.

36. Laboratory Diagnosis of Infections • Most common method: isolate virus from stool samples or throat samples from the mouth • Rarely isolated from CSF • Grows well in any human or monkey kidney cell lines (causes good CPEs). • Identify serotype with neutralization assays • Nucleic acid methods: genomic sequencing to determine if the infection is caused by vaccine or wild-type virus.