BIOC20 - Lecture 7: Picornaviruses, Flaviviruses, and Togaviruses PDF

Lecture 7 Positive strand RNA viruses Picornaviruses, Flaviviruses, Togaviruses Structure Genome and proteins Replication and translation Virus entry and exit Reminder: Quiz 3 available tomorrow at 1  Picornaviruses - Structure Picor...

Lecture 7 Positive strand RNA viruses Picornaviruses, Flaviviruses, Togaviruses Structure Genome and proteins Replication and translation Virus entry and exit Reminder: Quiz 3 available tomorrow at 1  Picornaviruses - Structure Picornaviruses are small (pico) RNA genome viruses: Naked icosahedral capsid (non-enveloped) Diameter ~30 nm Hundreds of known viral species that infect humans, mammals, birds, fish, etc. Cause diseases such as hepatitis, myocarditis, common cold, polio (poliomyelitis) etc. in humans Linear ‘+’ sense ssRNA genome ~ 7 -8.9 kb with a single ORF encoding a polyprotein. 2 EM image of poliovirus Model of poliovirus, Fig 11.2  Picornaviruses – Genome and Proteins Picornaviruses bind to cellular receptors via depressions or loop regions on their surface Mature virions contain 60 copies of each of the three to four proteins: VP1, VP2, VP3 and VP4 Viral protein VPg covalently bound to 5’ end of the RNA (instead of 5’ cap) which also has a short, genome-encoded poly(A) tail at 3’Encoded end. by genome, not added by poly (A) polymerases Fig 11.4 3  Picornaviruses - Genome and Proteins VP1-VP3 form the capsid shell, VP4 remains buried within the shell. All genes are translated as single polyprotein followed by proteolytic cleavage One to three proteinases Six to eight replication proteins P1: encodes structure proteins P2, P3: non- structural proteins Fig 4 11.4  Picornaviruses - Genome and Proteins The 5’ non-coding region contains an internal ribosome entry site (IRES) – allow initiation of translation without having a 5’ cap Allow for translation at an internal site away from the 5’ end Contains extensive secondary and tertiary structures  interact with a variety of cellular proteins P1: encodes Polyprotein is structure processed by proteins viral proteases into precursor / P2, P3: non- mature structural proteins proteins 5  Picornaviruses - Genome and Proteins Example: Extensive secondary and tertiary structures in IRES elements  multiple types (I and II shown in this figure) All known IRES elements contain a pyrimidine rich tract 20-25 nucleotide from AUG  likely function is to initiate translation at the correct spot. Fig 6 11.6  Picornaviruses - Genome and Proteins Virions bind to cellular receptors via ‘canyons’ or loop regions on their surface VP1-4 all fold into a jelly roll, composed of an 8-stranded β-barrel Genome RNA may pass through pores formed in cell membrane by capsid proteins OR virions are internalized by receptor mediated endocytosis  capsid dissociates after endosomal acidification. 7  Picornaviruses - Entry Example: Entry of poliovirus RNA into the cytoplasm after major rearrangement (notice VP4)  VP4 and hydrophobic N-terminal of VP1 form a channel in cell membrane Fig 8 11.2  Picornaviruses - Genome and Proteins Most cellular RNAs use cap- dependent mechanism for translation near their 5’ end: Eukaryotic initiation factors (eIFs) eIF-4A, eIF-4E, eIF-4G form the eIF-4F complex bind to 5’ end and recruit a 40S ribosomal subunit 40S subunit ‘scans’ mRNA to find the start codon (AUG) 60S ribosomal subunit then binds to form a 80S initiation Fig 9  Picornaviruses - Genome and Proteins Picornavirus infection causes proteolytic cleavage of eIF-4G  abolishes cap-dependent translation in host cells eIF-4E may be sequestered  fails to start translation Host cell proteins bind to IRES  helps with docking of 40S ribosomal subunit 40S subunit scans the RNA for initiator codon  assembly of 80S at the correct site  protein translation Fig 10  Picornaviruses - Genome and Proteins Picornavirus proteins are made as a single precursor polyprotein that is autocatalytically cleaved by viral proteinases Polyprotein first cleaved into precursors P1, P2 and P3 P1 then cleaved into capsid proteins P2 and P3 cleaved into non-structural proteins such as proteases etc. RdRp = RNA dependent RNA polymerase 11  Picornaviruses - Genome and Proteins After synthesis of viral proteins, viral RNA is replicated Most proteins made from P2 and P3 are involved in genomic RNA replication Replication of picornavirus RNAs is initiated in a multiprotein complex bound to cellular vesicles  rapid proliferation of vesicles upon infection  serve as nucleation sites (viral factories) A full length negative RNA strand Fig (antigenome) is made and then used as a 11.7 template for positive-strand synthesis RNA synthesis is primed by VPg covalently 12  Picornaviruses – Assembly and Exit Virion assembly involves cleavage of VP0 to VP2 plus VP4  VP0, VP1 and VP3 assemble into protomers (subunits composed of different polypeptide chains) Five protomers self-assemble into 14S pentamers Pentamers and RNA assemble into provirion. Different pathways may exist: Threading of RNA genome into procapsid Fig Pentamer associating with genome 11.8 RNA  assembly into provirion 13  Picornaviruses - Assembly and Exit Picornavirus infection inhibits several host cell macromolecular functions: Shut off host cap-dependent translation and RNA synthesis Induction of cytoplasmic vesicles Alteration of intracellular transport pathways Newly synthesized virions are released from cell by lysis  ready 14 to go infect other cells Picornaviruses - Assembly and Exit Reference slide 15  Flaviviruses Latin Flavus (yellow) ➔ jaundice from yellow fever virus infection Spherical enveloped particle ~50 nm in diameter Spherical nucleocapsid (25-30 nm) with icosahedral symmetry No projections, “golf ball” like appearance  envelope glycoproteins also arranged with icosahedral symmetry! 16 EM image of Dengue virusModel of Dengue virus, Fig 12.2C  Flaviviruses Flavivirus genus is transmitted by arthropods (arboviruses)  causes several important human diseases Virus infects humans, monkeys, birds Pestivirus genus causes economically important diseases of cattle, sheep etc. No insect vectors, humans not infected Hepacivirus genus contains one single virus, Hepatitis C virus No insect vectors, only infects humans via blood transfusion, IV needles, sexual contact 17  Flaviviruses - Structure The flavivirus virion contains an envelope and envelope proteins are arranged with icosahedral symmetry: 180 copies of M and E (envelope protein heterodimers) Immature viral particles display spikes on surface (a) Mature particles have golf ball-like appearance (c) Fig. 12.2. Morphology of dengue virus type 2 virions. (a) Surface of immature virion at neutral pH has prM-E heterodimers arranged perpendicular to surface bilayer. (b) Surface of immature virus at low pH in trans-Golgi network, with orientation of spikes parallel to18 lipid surface, with pr in blue. (c) Mature, infectious virions, after release of pr peptide.  Flaviviruses – Genome and Proteins Linear ‘+’ sense ssRNA genome ~ 10-11 kb that is capped at 5’ end, but no poly(A) tail at 3’ end All genes translated as a single polyprotein followed by proteolytic cleavage: 3 structural proteins One capsid protein (C) Two envelope proteins (M and E) Seven non-structural proteins Fig 19 12.4  Flaviviruses - Genome and Proteins Flavivirus genome organization most resembles that of Picornaviruses (+ssRNA) Distinct from Togaviruses, despite similar virion morphology, genome size, and transmission via arthropod Flaviviruses RNA is translated into a single, long polyprotein that undergoes proteolytic processing to generate single proteins  both viral and cellular proteinases are required 20  Flaviviruses – Genome and Proteins For example: Yellow fever virus ‘+’ ssRNA genome 10 virus proteins (3 structural, 7 non- structural) 3 NS proteins have enzymatic activity and 3 final structural proteins included in mature virion are precursor Viral and proteins cellular proteases cleave at specific sites (anchC and prM) 21 Flaviviruses – Genome and Proteins Structural Non- structur al 22  Flaviviruses - Genome and Proteins Flavivirus E protein directs receptor binding and membrane fusion: E protein is a type I membrane protein and found as a dimer, lays parallel to the lipid bilayer (instead of protruding) Domain II forms dimer interface and contains the hydrophobic fusion peptide  protected from the aqueous environment by neighboring E in the dimer Domain III used for receptor binding (immunoglobulin-like fold) Domain I joins domains II and III Fig 23 12.3  Flaviviruses – Attachment and Entry No cellular receptor has been clearly identified Following attachment, entry is mediated by endocytosis within clathrin-coated vesicles Alternative entry mechanism: Virus bound by antibody can enter cells that express immunoglobulin Fc receptors on their surface Antibody-dependent enhancement (ADE) causes 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. DENV = Dengue Virus 24  Flaviviruses – Attachment and Entry Following attachment, entry is mediated by endocytosis within clathrin- coated vesicles Endocytosed vesicles fuse with endosomes  undergo acidification  results in rearrangement of the E dimer into a fusion-active state to reveal fusion peptide E protein is a class II type fusion protein (see lecture 5, slide 10) 25  Flaviviruses – Genome and Proteins Once the genome is in the cytosol, the RNA is bound by ribosomes and translated  produces polyprotein  cleaved to produce precursor / functional protein Capsid protein precursor (anchC) is inserted into endoplasmic reticulum 20 AA signal sequence (on C terminus) is cleaved in lumen of ER by cellular signal peptidase, releases polyprotein from capsid precursor (anchC) N-terminal of signal sequence on cytoplasmic side is later cleaved by viral proteinase (NS2B/NS3A) to release mature C protein 26 Fig  Flaviviruses – Genome and Proteins Following the capsid protein is the precursor membrane protein (prM) Transmembrane domain at C-terminus followed by a signal sequence  required to insert the E protein in correct orientation in the membrane prM associates with E protein in ER to form a heterodimer  protects E from premature conformational change and exposing the fusion peptide At a later stage prM is cleaved by cellular furin protease Releases ‘pr’ peptide extracellularly Leaves M associated with virion 27 Fig  Flaviviruses – Genome and Proteins Synthesis of NS proteins results in establishment of active RNA replicase complexes  RNA synthesis is carried out on membranes in the cytoplasm Replication requires synthesis of a complementary copy (minus- strand) of the plus-strand RNA Subsequent synthesis of new plus-strands for 3 different purposes: Translation: making more viral proteins Replication: making more RNA copies Packaging: making virions Reminder: Viral RNA is synthesized asymmetrically: 10X more 28 plus-  Flaviviruses – Assembly and Exit Virus assembly takes place at intracellular membranes Budding and envelope acquisition occurs at the ER-Golgi Particles are processed as they move through Golgi Virions exit cell by exocytosis Cleavage of prM (membrane anchor) occurs just before virion release by cellular furin converts immature particle to mature 29 Also see Fig  Togaviruses Several togaviruses cause disease in animals and humans  symptoms range from rashes, high fever to joint pain and encephalitis Infection in humans is a dead-end  can’t easily be transmitted to other individuals but spread through mosquitos remains a major public health concern (especially in tropics) Reclassified 30  Togaviruses - Structure Latin Toga (cloak / gown)  refers to virus envelope (another example of arboviruses) Spherical enveloped particle with a fringe of projections (spikes) ~70 nm in diameter Nucleocapsid and envelope glycoproteins are arranged in icosahedral symmetry Envelope: 240 heterodimers of glycoproteins E1 and E2; capsid: 240 copies of capsid protein 31 Model and EM image of Sindbis  Togaviruses – Genome and Proteins Linear ‘+’ sense ssRNA genome ~9.7 – 11.8 kb and has both 5’ methylated cap and a 3’ poly (A) tail (~70 nt) 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 One capsid protein Three envelope proteins A small hydrophobic protein 32 Fig. 13.2  Togaviruses – Genome and Proteins Linear ‘+’ sense ssRNA genome ~9.7 – 11.8 kb and has both 5’ methylated cap and a 3’ poly (A) tail (~70 nt) Four non-structural proteins (for viral RNA synthesis) translated directly from genomic RNA as a polyprotein and then processed by proteases Five structural proteins translated from a subgenomic mRNA One capsid protein Three envelope proteins A small hydrophobic protein 33  Togaviruses – Genome and Proteins E glycoprotein binds to cellular receptors  receptor mediated endocytosis (e.g. laminin receptor, heparan sulphate) via clathrin-coated vesicles Laminin receptor: bind to laminin family of proteins  component of basal lamina Heparan sulphate: sulphated polysaccharide that is usually attached to a protein Once in endosome, pH drop leads to conformational changes in E1/E2 heterodimer  leads to fusion after exposure of fusion domain 34  Togaviruses – Genome and Proteins Once inside the cytoplasm, RNA genome is released to be translated (may interact with cellular proteins to release the genome) For example: In Sindbis virus, NS proteins are translated as a P123 polyprotein  P1234 is only made after a readthrough of the stop codon (10-20% of the time) Further cleaved to produce various non-structural proteins Fig. 13.3 35  Togaviruses – Genome and Proteins Partly cleaved non-structural proteins catalyse synthesis of full-length antigenome RNA Early, full length (-) sense antigenome RNAs are synthesized by P123 and nsP4 (RdRpnsP4 in this figure) Later, P123 is proteolytically processed leading to the switch to ‘+’ RNA synthesis Also see Fig. 13.3 on next slide 36 Togaviruses – Genome and Proteins Fig. 13.3 37  Togaviruses – Genome and Proteins Structural proteins are cleaved during translation and directed to different cellular locations A polyprotein is generated and post-translationally cleaved by host signal peptidase and furin protease Structural proteins are palmitoylated in the Golgi A capsid protein domain binds to the packaging signal on the genome RNA Fig. 13.4, Sindbis virus 38 Togaviruses – Genome and Proteins 39  Togaviruses – Assembly and Exit Capsid proteins interact with the cytoplasmic tails of envelope proteins studding the plasma membrane Virus exits cell by budding Fig. 13.4, Sindbis virus 40  Togaviruses – Life Cycle Life cycle of a togavirus: Can you identify all the step 41  Coming up…. Negative strand RNA viruses Filoviruses Influenza viruses Quiz 3 – Available Wed night (Oct. 16, 10 PM) – Friday night (Oct. 18, 10 PM) Covers lectures 5 and 6 Format similar to other quizzes 42