BIOC20 Lecture 7 Picornaviruses & Flaviruses PDF

Lecture 7 Key Points 1. Picornaviruses 1.1 Structure 1.2 Genome and proteins 1.3 Replica=on and transla=on 1.4 Virus entry and exit 2. Flaviruses (similar to the above) 3. Togaviruses (similar to the above) Sec$on 1: - Picornaviruses are small (pico) RNA genome viruses: Ø Naked icosahedral capsid (n...

Lecture 7 Key Points 1. Picornaviruses 1.1 Structure 1.2 Genome and proteins 1.3 Replica=on and transla=on 1.4 Virus entry and exit 2. Flaviruses (similar to the above) 3. Togaviruses (similar to the above) Sec$on 1: - Picornaviruses are small (pico) RNA genome viruses: Ø Naked icosahedral capsid (non-enveloped) Ø Diameter ~30nm Ø Hundred of known viral species that infect humans, mammals, birds, etc. - Linear ‘+’ sense ssRNA genome ~7-8.9kb with a single ORF encoding a polyprotein Sec$on 1.2 - Picornaviruses bind to cellular receptors via depressions or loop region on their surface - Mature virion contains 60 copies of each of the three to four proteins: VP1,2,3, and 4. Ø VP1-3 form the capsid shell, VP4 remains buried within the shell Ø All genes are translated as single polyprotein followed by proteoly$c cleavage >>one to three proteinase and six to eight replica$on proteins - Viral proteins VPg covalently bound to 5’ end of the RNA (normally is 5’ cap) which also has a short, genome-encoded poly(A) tail at 3’ end. Ø Poly(A) tail was encoded by the genome, but normally it is assed by poly(A) polymerases - Ø 5’ NCR contains an internal ribosome entry site (IRES)- allow ini$a$on of transla$on without having a 5’ cap Ø Allow for transla$on at an internal site away from the 5’ end Ø Contains extensive secondary and ter$ary structures> interact with a variety of cellular proteins Ø All IRES elements contain a pyrimidine rich trach 20-15 nucleo$de from AUG> likely func$on is to imitate transla$on at the correct spot. Ø Virions bind to cellular receptors via ‘canyons’ or loop regions on their surface. Ø VP1-4 all fold into a jelly roll, composed of an 8-stranded beta-barrel - Genome RNA may pass through pores formed in cell membrane by capsid proteins OR virion are internalized by receptor mediated endocytosis> capsid dissociates aber endosomal acifica$on Sec$on 1.3 - Most cellular RNAs use cap-dependent mechanism for transla$on near their 5’ end: Ø Eukaryo$c ini$a$on factors (eIFs) eIF-4A, eIF-4E, eIF-4G form the eIF-4F complex bind to 5’ end and recruit a 40S ribosomal. Ø 40S subunit ‘scans’ mRNA to find the start codon (AUG) Ø 60S ribosomal subunit then binds to form a 80S ini$a$on complex> transla$on can - be begin - - However, Picornavirus infec$on causes proteoly$c cleavage of eIF-4G> abolishes capdependent transla$on in host cells> promote the virus transla$on eIF-4E may be sequestered> fails to start transla$on Host cell proteins bind to IRES> helps with docking of 40S ribosomal subunit 40S subunit scans the RNA for ini$ator codon> assembly of 80S at the correct site> protein transla$on> virus hijacking cellular transla$on process> the cell will translate the viral RNA instead of the cell. Once the transla$on occurred, picornavirus proteins are made as a single precursor polyprotein that is autocataly$cally cleaved by viral proteinases Ø Polyprotein first cleaved into precursors P1,2, and 3 >> P1 then cleaved into capsid proteins >>P2 and P3 cleaved into non-structural proteins such as proteases Aber synthesis of viral proteins, viral RNA is replicated Most proteins made from P2 and P3 are involved in genomic RNA replica$on Replica$on of picornavirus RNAs is ini$ated in a mul$protein complex bound to cellular vesicles> serve as nuclea$on sites (viral factories) A full length nega$ve RNA strand (an$genome) is made and then used as a template for posi$ve-strand synthesis RNA synthesis is primed by VPg covalently bound to uridine residues (uridyla=on)> allows it to hybridize to the poly(A) tail and server as a primer for (-) strand synthesis Ø A lot more (+) strand created than (-) strand RNA because one nega$ve strand can replicate mul$ples posi$ve strands. Seciton 1.4 - Entry of poliovirus RNA into the cytoplasm aber major rearrangement (no$ce VP4)> VP4 and hydrophobic N-terminal of VP1 form a channel in cell membrane Major conforma$on changed cause VP4 to change it posi$on. N-terminal is hydrophobic - - Virion assembly involves cleavage of VP0 to VP2 plus VP4> VP0, VP1 and VP3 assemble into protomers (subunits composed of different polypep$de chains) Five protomers self-assemble into 14S pentamers Ø Pentamers and RNA assemble into provirion. Different pathways may exist: 1) Threading of RNA genome into capsid (uncommon because it has not well studied); 2) Pentamer associa$ng with genome RNA> assembly into provirion (more common) VP0 is cleaved into VP2 and VP4 to form mature virion Picornavirus infec$on inhibits several host cell macromolecular func$ons: Ø Shut off host cap-dependent transla$on and RNA synthesis Ø Induc$on of cytoplasmic vesicles Ø Altera$on of intracellular transport pathways - Newly synthesized virions are released from cell by lysis> ready to go infect other cells Sec$on 2: - La$n Flavus (yellow)> jaundice from yellow fever virus infec$on - Spherical enveloped par$cle ~50nm in diameter - Spherical nucleocapsid (25039nm) with icosahedral symmetry - No projec$ons, “golf ball” like appearance> envelope glycoproteins also arranged with icosahedral symmetry - Flavivirus genus is transmijed by arthropods (arboviruses)> uses several important human diseases; virus infects humans, monkeys, birds - Pes$virus genus causes economically importance diseases of cajle, sheet, etc. (no insect vectors, humans not infected) - Hepacivurs genus contains one single virus, Hepa$ts C virus. ( no insect vectors, only infects humans via blood transfusion, IV needles, sexual contact) Sec$on 2.1 - The flavivirus virion contains an envelope and envelope proteins are arranged with icosahedral symmetry: Ø 180 copies of M and E (envelope protein heterodimers) Ø Immature viral par$cles display spikes on surface (a) Ø Mature par$cles have golf ball-like appearance (c) Ø Sec$on 2.2 - Linear ‘+’ sense ssRNA genome ~10-11kb that is capped at 5’ end, but no poly(A) tail at 3’ end - All genes translated as a singly polyprotein followed by proteoly$c cleavage: Ø One capsid protein (C) Ø Two envelope proteins (M and E) Ø Seven non-structural proteins - Flavivirus genome organiza$on most resembles that of Picornaviruses (+ssRNA) Ø Dis$nct from Togaviruses, despite similar virion morphology, genome size, and transmission via anthropod Flaviviruses RNA is translated into a single, long polyprotein that undergoes proteoly$c processing to generate single proteins> both viral and cellular proteinanses are requires At the 3’ end, it has a lot of RNAs structures (secondary and teritary structures)> will get cleaved into non-structural and structural proteins - anchC protein- “Anchored” capsid protein. Precursor to capsid protein; signal sequence inserted in membrane is cleaved by viral proteinase - C protein- encapsida$on of genome RNA - prM protein- binds to E glycoprotein, inhibits membrane fusion during transmit through Golgi. Cleaved to produce mature M protein, releasing pr. - All three proteins above are structures proteins - NS3 protein- viral serine proteinase involved in polyprotein cleavage; RNA replicase component; nucleoside triphos phatase and helicase ac$vi$es. (non-structural protein) - NS2B protein- Part of viral proteinase that cleaves viral polyprotein - Flavivirus E protein directs receptor binding and membrane fusion: Ø E protein is a type I membrane protein and found as a dimer, lays parallel to the lipid bilayer (instead of protruding) Ø Domain II forms dimer interface and contains the hydrophobic fusion pep$de> protected from the aqueous environment by neighboring E in the dimer Ø Domain III used for receptor binding (immunoglobin-like fold) Ø Domain I joins domains II and III Sec$on 2.3 - Synthesis of non-structural protein results in establishment of ac$ve RNA replicase complexes> RNA synthesis is carried out on membranes in the cytoplasm Ø Replica$on requires synthesis of a complementary copy (minus-strand) of the plus strand RNA Ø Subsequent synthesis of new plus-strand(genomic RNA) for three different purposes: 1) Transla$on: making more viral protein 2) Replica$on: making more RNA copies 3) Packaging: making virion 4) Sec$on 2.4 - No cellular receptor has been clearly iden$fied for flaviviruses - Once they ajached, entry is mediated by endocytosis within clathrin-coated vesicles - Alterna$ve entry mechanism: Ø Virus bound by an$body can enter cells that express Immunoglobulin Fe receptors on their surface Ø An=body-dependent enhancement (ADE) cause more severe disease like dengue haemorrhagic fever (high fever, vascular damage and internal bleeding, shock> can lead to organ failure and death if not treated. Ø Ø Endocytosed vesicles fuse with endosomes> undergo acidifica$on> results to rearrangement of the E dimer into a fusion-ac$ve state to reveal fusion pep$de Ø E protein is a class II type fusion protein Ø Once the genome is in the cytosol, the RNA is bound by ribosomes and translated> produces polyprotein> cleaved to produce precursor/func$onal protein Ø Capsid protein precursor (anchC) is inserted into endoplasmic re$culum >> 20 AA signal sequence (on C terminus) is cleaved in lumen of ER by cellular signal pop=dase, releases polyprotein from capsid precursor (anchC) >> N-terminal of signal sequence on cytoplasm side is later cleaved by viral proteinase (NS2B/NS3A) to release mature C protein - Following the capsid protein is the precursor membrane protein (prM) Ø Transmembrane domain at C-terminus followed by a signal sequence> required to insert the E protein in correct orienta$on in the membrane - prM associates with E protein in ER to form a heterodimer> protects E from premature conforma$onal change and exposing the fusion pep$de (moving from the ER to the golgi, pH slightly changed which could lead to premature conforma$onal change> premature exposed fusion pep$de> fusion in the wrong membrane) Ø At a later stage prM is cleaved by cellular furin protease >> Releases ‘pr’ pep$de extracellularly >> Leaves M associated with virion >> Yields E homodimer - Virus is ready for exit by exocytosis once virus assembly takes place at intracellular membranes. Budding and envelope acquisi$on occurs at the ER-Golgi Ø Just before the membrane release, cleavage of prM (membrane anchor) occurs just before virion release by cellular furin >>converts immature par=cle to mature par=cle >>pr pep=de only released when virion is exposed to neutral pH outside cell Sec$on 3: - Several togaviruses cause disease in animals and humans> symptoms range from rashes, high fever to joint pain and encephali$s. - Infec$on in human is a dead-end> can’t easily be transmijed to other individuals but spread through mosquitos Sec$on 3.1 - La$n Toga (cloak/gown)> refers to virus envelope. - Spherical enveloped par$cle with a fringe of projec$ons (spikes)~ 70nm in diameter - Nucleocapsid and envelope glycoproteins are arranged in icosahedral symmetry Envelope: 240 heterodimers of glycoproteins E1 and E2, capsid: 240 copies of capsid proteins Sec$on 3.2 - Linear ‘+’ sense ssRNA gneome ~9.7-11.8kb and has both 5’ methylated cap and a 3’ poly(A) tail (~70nt) - Four non-structural proteins (for viral RNA synthesis) translated directly from genomic RNA as a polyprotein and then processed by host and viral proteases - Five structural proteins translated from a subgenomic mRNA : Ø One capsid protein Ø Three envelope proteins Ø A small hydrophobic protein (6K) - E glycoprotein binds to cellular receptors> receptor mediated endocytosis (e.g. laminin receptor) via clathrin coated vesicles Ø Laminin receptor: bind to laminin family of proteins> component of basal lamina Ø Heparan sulphate: sulphated polysaccharide that is usually ajached to a protein. - Once in endosome, pH drop leads to conforma$onal changes in E1/E2 heterodimer> leads to fusion aber exposure of fusion domain - Fusion of endosome membrane with viral envelope> release of nucleocapsid Sec$on 3.3 - Once inside the cytoplasm, RNA genome is released to be translated (may interact with cellular proteins to release the genome Ø nsP1: RNA capping enzyme:. Ø nsP2: cysteines proteinase, RNA helicase. Ø nsP4: RNA polymerase Ø These partly cleaved non-structural proteins catalyse synthesis of full-length an$genome RNA BLUE: If the RNA polymerase stopped at the codon, it would make protein123. If it go through, it would make protein 123+4; P4 is an RNA dependent RNA polymerase - Structural proteins are cleaved during transla$on and directed to different cellular loca$ons