BIOC20 Lecture 7-12 New Lecture Notes 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$on 1: - Picornaviruses are small (pico) RNA genome viruses: Ø Naked icosahedral capsid (non-enveloped) Ø Diameter ~30nm Ø Hundred of known viral species that infect humans, mammals, birds, etc. - Linear ‘+’ sense ssRNA genome ~7-8.9kb with a single ORF encoding a polyprotein Sec$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$ons - A polyprotein is generated and post-transla$onally cleaved by host signal pep$dase and furin protease Structural proteins are palmitoylated (adding faj acids) in the Golgi A capsid protein domain binds to the packaging signal on the genome RNA PE2: Precurosr to E2, palmitoylated and glycosylated Capsid: forms nucleocapsid E3: Amino-terminal part of PE2 containing the signal sequence E2: Carboxy-terminal part of PE2, a component of virus envelope projec$ons 6K: membrane associated; palmitoylated E1: Component of virion envelope projec$ons; type I transmembrane glycoproteins Sec$on 3.4 - Capsid proteins interact with the cytoplasmic tails of envelope proteins studding the plasma membrane - Virus exits cell by budding Lecture 8 Key Points 1. Filoviruses 1.1 structure 1.2 Disease and spread 1.3 Genome, replica=on and transla=on 1.4 RNA edi=ng and protein func=on 1.5 Virus entry and exit 1.6 Latent infec=ons 2. Inﬂuenza viruses (introduc=on): 2.1 types 2.2 Disease and spread 2.3 Genome and proteins Sec$on 1.1: - La$n ﬁlum (ﬁlament or thread like) - Filamentous, enveloped par$cles. Diameter 80nm, length of 800nm or more - Helical nucleocapsids with nega$ve sense ssRNA genome - Genome size ~15-19kb Sec$on 1.2: - First outbreak of Marburg virus was in Marburg and Frankfurt, Germany in 1967 Ø Laboratory workers processing monkey $ssues- 32 cases, 7 died - A similar virus emerged in 1976 by two independent epidemics in Caire and Sudan - Re-emerging viruses that cause severe, oben fatal disease. It could reoccurred with the pa$ent who had it before - More recent EBOV outbreak in 2014-15 was the largest outbreak in history Ø Zaire EBOV is the most fatal type Ø Despite public percep$on, ebola is unlikely to spread disease with large numbers of casual$es - Two genera: Marburgvirus and Ebolavirus. Ø Ebolavirus species are named aber their original site of discovery Ø There is a single Marburgvirus species: lake Victoria Marburgvirus Ø There viruses are probably transmijed to primates from fruit bats (zootonic spread) Ø Infec$ons with Marburg and Ebola viruses cause servere haemorrhagic fever> major symptoms are blood cloqng, etc. (lethality is up to 90%) Ø Local outbreaks are oben followed by lengthy absence of disease, disease is oben self-limitng (that means they due not spread easily because:) >> spread of ﬁlovirus infec$ons among humans is limited to close contacts such as 1) aerosolized human-human spread is low risk, 2) person-person transmission is mediated by physical contact with secre$on/excre$ons. 3) close family members and medical staﬀs are the most at-risk >>Epidemics usually are self-litmi$ng, as virus transmission period is transient and generally ineﬀec$ve Sec$on 1.3 - Linear, nega$ve snes, single-stranded RNA genome - Genome length ~15-19kb - - Seven genes, transcribed in series form 3’ end (if it wrijen as 3’-5’- its indicate a nega$ve genome) of genome by viral RNA polymerase Most proteins are packaged in virion: (red=associated with genomic RNA and/or nucleocapsid)(black=associated with the viral envelope) 1) Nucleocapsid protein (NP) 2) RNA polymerase cofactor(VP35) 3) Matrix Protein (VP40) 4) Envelope glycoproteins (GP, cleaved into GP1, GP2, or sGP) 5) Minor nucleocapsid protein (VP30); 6) Membrane protein (VP24) 7) RNA polymerase (L) Ø Ebola makes addi$onal secreted glycoproteins (sGP, delta-pep$de) Most genes produce a single mRNA and a single protein Each genes (NP, VP35, etc.) codes for a single pro$ens, except for GP> gets cleaved into GP1 and GP2 or sGP (secreted GP, out of the cell later on) All genes ﬂanked by conserved sequences that signal transcrip$on terminal, polyadenyla$on and reini$a$on Ø Individual trancripts made for each viral protein (except GP) Ebola virus has mRNA edi$ng, while Marburg virus don’t. Replica=on - Transcrip$on and replica$on occurs in cytoplasm similar to other ‘-‘ sense RNa viruses. The template for mRNA synthesis is the ‘-‘ sense RNA genome - 3’ leader contains promoter for viral RNA polymerase and packaging signal for assembly of nucleocapsid - - 1. Viral RNA polymerase begins transcrip$on at 3’ terminal of the genome 2. When lijle or no free NP protein (during ini$al infec$on) is present> RNA polymerase transcribes a short sequence, then terminates to release a free leader RNA and then scans for nearby mRNA start site and re-ini$ates transcrip$on at the next gene (re-ini$a$on is not 100% eﬃcient) 3. L-protein adds methylated 5’ cap and poly(A) tail like cellular mRNAs. 4. Once suﬃcient NP made, genome replica$on can begin (replica$ons can occur when there is suﬃcience NP due to the need of packaging the genome) Genome replica$on occurs via synthesis of an$genome RNA (‘+’ sense)> serves as template for synthesis of new nega$ve-strand RNA genomes: Ø Viral RNA polymerase changes it mode of synthesis: no longer recognizes termina$on-poly-a reini$a$on sites Ø RNA pol extends the growing chain to end of template without stopping (NP bind to each protein= number of NP decreases) The template for both mRNA synthesis and genome replica$on is the ‘-‘RNA genome Ø The cytoplasm of infected cells contain inclusion bodies (aggregates of (viral) proteins/nucleic acids within the host cell) which contain viral nucleocapsid Ø Genome RNAs are packaged into nucleocapsids during replica$on> replica$on only occurs when there is enough NP to encapsidate the genome Sec$on 1.4 - Ebola virus uses RNA edi$ng to make two glycoproteins from the same gene: Ø sGP (~80%): a shorter protein that does not include the transmembrane domain at the C terminus and is secreted. Add$onally, yield delta-pep$de upon proteoly$c cleavage Ø GP(~20%): RNA polymerase “stujers” and adds an extra ‘A’ residue to change the reading frame to yield full length GP (frameshib muta$on) - The edi$ng site contains a stretch of 7 ‘U’s (it’s U because is RNA)> transcribed into A’s in ‘+’ sense mRNA - Occasionally, the viral RNA polymerase will ‘stujer’ over this stretch of ‘U’s and add addi$onal ‘A’> changes Thr to Asn and changes the reading frame downstream. In some cases, it may be 6 or 9 ‘A’ producing a smaller sGP(ssGP) - Edi$ng of Ebola gene product can produce sGP(most common), GP, ssGP(least common) Sec$on 1.5 - GP is synthesized as a precursor protein, inserted into the lumen of ER: Ø An N-terminal signal sequence is cleaved oﬀ during inser$on in ER Ø Protein undergoes extensive glycosyla$on in ER and Golgi> transport to plasma membrane - Cellular furin protease then cleaves GP into the ectodomain GP1 (N-terminal) and transmembrane GP2 (C-terminal) Ø GP1 and GP2 held together by a disulﬁde bridge between two cysteine residues (cysteine bridge) Ø GP forms trimers at plasma membrane and in the envelope (virion surface) - Filoviruse GP mediates ajachment and entry (by fusion) GP mediates binding to mul$ple cellular receptors: Ø Asialoglycoprotein receptor: Liver-speciﬁc, binds to and internalizes glycoproteins that lack terminal sialic acid Ø Folate receptor-alpha: binds to folic acid Ø Integrin: cell-surface proteins that interact with extracellular adhesion proteins and ini$ate intracellular signalling Ø DC-Signs: type II transmamebrane proteins that bind mannose and involved in interac$on with T-cells - Most binding experiments have been performed using replica$on-deﬁcient pseudotypes (recombinant/pseudo virus) Ø Study which proteins bind to which receptor (psuedotypes) Ø Filovirus pseudo types have GP incorporated into the envelopes of unrelated recombinant viruses> can be used to study viral proteins. However, this study can be problema$c because by using diﬀerent virus, results that we got could not be accurate for other viruses Ø Genera$ng pseudotypes requires various vectors that are co-transfected Ø We use diﬀerent virus instead of the speciﬁc virus because due to safe$ness - Virus is taken up via micropinocytosis and using its fusion pep$de (N-terminal of GP2) it fuses within vesicles to enter cells (probably a low pH trigger) Protein func=on - sGP is released from infected cells and is found in serum of infected pa$ents> can be used as a biomarker and as a poten$al vaccine/an$viral target Ø func$on is not en$rely clear: 1. Considered non-structural but may subs$tute as a structural protein by forming a complex with GP2 2. Some pseudotypping data shows that it may limit GO cytotoxicity> more eﬃcient replica$on and inefec$vity 3. Act as a soluble factor that targets elements of the host defence system e.g. binding to an$bodies and contribute to immunosuppression - Possible func$ons of sGP: 1. Act as a decoy an$gen 2. Prevent ac$va$on of neutrophils 3. Reduce the produc$on of pro-inﬂammatory cytokines 4. Or inhibit immune cell chemotaxis 5. A combina$on of inac$vated neutrophils and reduced levels of pro-inﬂammatory cytokines may aid in restoring endothelial barrier func$on - Minor nucleocapsid protein VP30 ac$vates viral mRNA synthesis in Ebola virus: Ø A stem-loop structure at the beginning of the NP gene inhibits RNA polymerase from inita$ng mRNA synthesis. VP30 reverse this inhibi$on Ø However, if stem-loop is experimentally disrupted by muta$ons, transcrip$on is not dependent on VP30 Ø Stem-loop structure can be present in the genome or NP mRNA Ø VP30 is present in nucleocapsids but mechanisms of ac$on is unclear - VP40 is the most abundant viral protein and is associated with viral envelope: Ø Located at cytoplasmic side of plasma membrane and/or inner side of viral envelope Ø Like matrix proteins of other enveloped viruses, it bridges envelop GP’s to nucleocapsids Ø Expression of VP40 in mammalian cells result in virus-like partciles that bud from the plasma membrane Ø Cellular proteins that are involved with traﬃcking and sor$ng of intracellular vesicles may interact with VP40 to form virions at plasma membrane >> Mul=vesicular bodies- cytoplasmic vacuoles containing many small membrane vesicles and enzymes Sec$on 1.6 - In those who survive Ebola virus infec$on: Ø Observed periodic spikes in EBOV an$bodies occurring months aber clinical resolu$on of symptoms> suggests latent infec$on and poten$al disease recrudescence that could trigger outbreaks >>Recrudences: recurrence of disease/symptoms aber a period of inac$vity Ø Scien$sts suggest vaccina$ng survivors of Ebola virus may help prevent disease recrudescence> to boost protec$ve an$body responses in survivors Sec$on 2 - Family Orthomyxoviridae from Greek ortho (correct/normal), myxa (mucus)> based on ability of virus to ajach to mucoproteins on cell surface Ø Myxoviruses split into paramyxo and orthomyxo, with diﬀerent structure and replica$on cycles - Enveloped par$cles, quasi-spherical or ﬁlamentous> envelope is derived from hostmembrane by budding. Diameter ~80-120nm - Compact helical nucleocapsid Sec$on 2.1: - Mu$ple subtypes within each virus type dis$nguished by varia$ons in the surface glycoproteins-hemagglu$nin (HA) and neuraminidase (NA) Ø Can highly pathogenic and low pathogenic avian inﬂeunza Ø Orthomyxoviridae have two addi$onal genera: 1) Thogotovirus (transmijed by $cks), 2) Isavirus (infects ﬁsh, par$cular salmon) - Symptoms: high fever, sore throat, cough, headache, muscular pain - Most fatal in elderly, infants and chronically ill, oben by secondary bacterial infec$ons Sec$on 2.2: - Inﬂuenza virus infec$ons of the respiratory tract can lead to secondary bacterial infec$ons by providing easier access: Ø Virus infects an causes a loss of the ciliated epithelium and disrupts mucocilliary ﬂow- ﬂow of mucus and debris along the epithelial lining of the respiratory tract> moved by cilia on epithelial cells Ø Virus induces produc$on of interferons and cytokines> local and systemic inﬂammatory response Sec$on 2.3 - Segmented nega$ve sense ssRAN genome (608 diﬀerent segments), mul$ple helical nucleocapsids> single virion includes a ‘complete set’ of genome fragments. - Genome size (total)~10-15kb Segmented nega$ve ssRNA genome where 8 of the inﬂuenza virus genome segments code for a total of 11 diﬀerent viral proteins> 9 of the proteins are packaged into viral par$cles Lecture 9 Key points: 1. Inﬂuenza viruses 1.1 Genome and proteins 1.2 Membrane fusion 1.3 RNA replica=on and synthesis 1.4 Assembly and release 1.5 An=genic change 2. Retroviruses 2.1.1. Genome and proteins 2.1.2. Entry and early life cycle 2.1.3. Diseases Sec$on 1.1 - Uni-12 and Uni-13 are highly conserved sequences (universal primers) that are selfcomplementary - ssNCR= segment speciﬁc non-coding region - Genome segments 1-6 each encode for a single protein: three are RNA polymerase subunits (PA, PB1, PB2), envelope glycoprotein HA, neuraminidase (NA) In some inﬂuenza viruses, a 2nd reading frame on segment 2 codes for a shorter protein PB1-F2 (once it is inside the cell, it localizes to mitochondria and enhances apoptosis) Alternate splicing can occur in mRNA transcribed from segments 7 and 8> diﬀerent proteins >>Segment 7: Matrix M1 and envelope protein M2 >>Segment 8: Non-structural proteins NS1 and NS2 Viral Hemagglu=nin (HA) binds to cell surface receptors and mediates fusion with endosome Ø HA binds to sialic acid residues on cell surface receptors of various cell types (mucoproteins) Ø HA is type I transmembrane protein Ø HA forms trimers on virus surface mediate fusion of viral envelope with the endosomal membrane - N-terminal is exposed to the outside of the virion with a hydrophobic transmembrane domain near the C-terminal. Fusion is facilitated by: 1. Ac$va$on by cleavage by cellular proteases (cellular furin or trypsin) >> cleavage of HA by cellular proteases into 2 subunits: 1) HA1 is the surface subunit, binds to sialic acid; 2) HA2 is anchored in the viral envelope, contains the hydrophobic fusion pep$de 2. Conforma$onal changes aber acidiﬁca$on in endosome >> The viral protein M2 forms an ion channel that facilitates release of nucleocapsids from the virion> allows protons to enter the interior of the virus (virus acidiﬁca$on) weakening the interac$on of M1 (matrix protein) with the nucleocapsids >>M2 is a good target for an$viral drugs (e.g. Amantadine)> issue of drug resistance due to muta$on in virus Ø Fusion pep$de will get inserted into the cell when the conforma$onal changed or pH changed Sec$on 1.2: - M2 is a rela$vely small protein ~97AA> forms a tetramer> creates a small pore in envelope Ø External N-terminal domain, a transmembrane domain and a larger internal domain (base) Ø Trytophan (W41) ‘gate’ is locked through molecular interac$on with Asp (D44): opened by protona$on of a his$dine residue (H37)> conforma$on change> H+ conductance Ø Ø Once the gate opened> nucleocapsids enter the nucleus where mRNA synthesis and RNA replica$on occur Sec$on 1.3: - Nucleocapsids enter the nucleus where mRNA synthesis and RNA replica$on occur Ø NPs (green spheres) wrap the RNA into an unusual twin helical conforma$on with a central loop Ø Nucleocapsids also contain a trimer of RNA polymerase proteins: PA, PB1, PB2 >>PB1 binds conserved sequences at 5’ and 3’ ends> ~12-13 nt sequences that are highly self-complementary; which means at the 3’ end and 5’ end, the sequence can base pairs with one another - Nps and RNA polymerase proteins contain nuclear localiza=on signals that interact with cellular impor$n-a> nuclear entry - Unlike other RNA viruses, orthomyxociruses replicate in the nucleus, complica$ng the machinery required for viral replica$on It’s complicated because they need to go back and forth between cytoplasm and the nucleus Impor=ns 1. Import of nucleocapsid into the nucleus through nuclear pore complex 2. Viral mRNA synthesis 3. Export of viral RNA and transla$on in cytosol 4. And 6. Import of newly synthesized viral proteins (early and late proteins) 5. Replica$on to an$genome and genome 7. Export of new nucleocapsid to the cytoplasm - (maintaining the concentra$on between cytosol and nucleus by RanGDP and RanGTP) Nucleocapsids are exported from he nucleus in a complex with matrix protein (M1) and NS2> Newly made M1 is imported into nucleus and forms a complex with newly made nucleocapsids (6)> NS2 binds to M1 NS2 contains a nuclear export signal> recognized by expor=ns> export of compleexes to cytoplasm (step 7) How an=genome and genome replicate inside the nucleus of Inﬂuenza virus - Capped 5’ ends of cellular pre-mRNAs are used as primers for synthesis of viral mRNAs Ø Viral transcrip$on machinery cannot make mRNA on its own> uses cellular premRNAs Ø PB2 recognizes capped cellular pre-mRNAs Ø PB1 acts as a nuclease AND polymerase> cleaves bound pre-mNRA at an ‘A’ or ‘G’ 1013nt from the 5’ cap and adds complementary nt Ø Capped fragment is used as a primer for synthesis of viral mRNAs - Viral mRNAs synthesized in this way have heterogenous sequences for the ﬁrst 10-13nts Ø No viral capping enzyme is encoded Ø 5’ end sequence will depend on the cellular pre-mRNA used Ø “Cap-stealing” mechanism is used by other orthomyxoviruses - Replica$on of inﬂuenza virus is blocked by inhibitors of cellular RNA synthesis - Each genomic RNA segment contains a stretch of poly-U (15-22 nt from the 5’ end of the viral genome RNA) - Viral mRNAs terminate in poly(A) tail generated by “stujering” transcrip$on Ø Aber reaching poly-U, RNA polymerase pauses and stujers> reads through poly-U mul$ple $mes> generates poly-A tail - PB1 remains bound> prevents further transcrip$on - - - Transcrip$on generates a set of 8 viral mRNAs: Ø Segments 1-6 are exported directly to cytoplasm Ø Segment 7 and 8 undergo alterna$ve splicing in the nucleus> have splicing consensus sequence (sequence that are most commonly found in that posi$on) recognized by cellular splicing machinery Ø Only a frac$on of these two viral mRNAs are spliced> ra$o of unspliced to spliced mRNAs is 9:1 Ø Unspliced RNAs yield M1 and NS1>abundant Ø Spliced RNAs create M2 and NS2> less abudant Genome replica$on creates ‘+’ strand (an$genome) complexed with NP> produces nucleocapsids with an$genome RNA> no ‘stujering’> creates full-length copy (mechanism poorly understood)> copied to genome RNA in a similar mechanism - Amount of NP present in the nucleus plays a role in balancing transcrip$on and replica$on Ø NP binding to growing RNA chains> [NP] drops in the nucleus Ø Drop in [NP]> more mRNA synthesis to make more NP> imported into nucleus Ø Possible mechanism: NP does not bind to capped mRNA with cellular 5’ sequences but does bind to uncapped genome RNA Sec$on 1.4: - Viral envelope proteins transverse through ER and Golgi to assemble in the plasma membrane and direct budding of virions - HA, NA, M2 are directed to plasma membrane via Golgi network and accumulate in lipid rabs - Cytoplasmic tails of HA, NA and M2 interact with M1 that is on nucleocapsid - One copy of each genome segment is packaged Ø Viral proteins recognize and interact with speciﬁc RNA sequences in the nucleocapsid> packaged into bundles in the virion> budding and release (including envelop acquisi$on) - - Pinching involving NA cleaves sialic acid, the cellular receptor that binds to viral HA. NA is a type II transmembrane protein> short cytoplasmic N-terminal, membrane spanning domain, and a long C-term domain extending outward from viral envelope> reverse orienta$on of HA (type I transmembrane protein) Evidence indicates M2 is involved in ‘pinching’ the budding virions from the plasma membrane Sec$on 1.5: Why did we have to take vaccine every year? Because it’s mutate - An$genenic change: gene$c variability generates new virus strains that can poten$ally cause pandemics Ø An$genic change prevents life$me immunity and can occur in two ways 1. An=genic dric: slow, con$nuous accumula$on of (point) muta$ons 2. An=genic shic: results from reassortment (exchange) of genes during a mixed infec$on with two or more subtypes> can poten$ally create a new subtype that has a poten$al for pandemics Sec$on 2 - From reverse transcrip=on- making DNA copy of an RNA molecule - Spherical enveloped par$cle with diameter ~80-100nm - Envelope assembled at plasma membrane - Icosahedral or conical capsid - Contains reverse transcriptase - Retroviruses were reclassiﬁed into two subfamilies and eleven genera Sec$on 2.1 - Likely a relic of RNA world- reverse transcriptase genera$ng DNA copies of RNA genomes - Linear single-stranded posi$ve sense RNA (+ssRNA) Ø Dimer of two iden$cal molecules, 7-10kb, packaged in virions >> Complexed with nucleocapsid protein Ø 5’ cap and 3’ poly(A) tail - Once inside the cell> makes dsDNA from ssRNA genome (RNA>-ssDNA> dsDNA) Ø DNA enters nucleus with the viral enzyme integrase> directs integra$on of viral DNA into host cell DNA> viral genome is now treated like a cellular gene - Retroviruses have a unique replica$on cycle based on reverse transcrip$on and integra$on of their genomes> RT produces a DNA copy of the RNA genome - Viral proteins derived from the gag, pol, and env genes are incorporated in viriond Ø Gag (group-speciﬁc an$gen) proteins: MA (matrix), CA (capsid), NC (nucleocapsid) Ø Env (elope) protein: SU (external surface protein, also called gp120) and TM (transmembrane protein, also called gp24) Ø Pol (ymerase) proteins: involved in genome replica$on and integra$on- PR (protease), RT (reverse transcriptase), IN(integrase) Ø Each (gag, pol, and env) made as a polyprotein that gets cleaved into mature proteins Ø Adjacent to R, there are unique regions designated U5 (untranslated 5’, 80-200 nt) and U3 (untranslated 3’, 240-1200 nt) Ø A speciﬁc cellular tRNA is bound to genomic RNA by base pairing with primer binding sequences (PBS) downstream of U5 Ø Y packaging sequence Ø 5’SS and 3’SS are splice sites. Ø Ppt is polypurine tract. Used during reverse transcrip$on Sec$on 2.2. - Retroviruses enter cells by the fusion pathway and tropism is based mainly on interac$on of SU with cellular receptor - Envelope either fuses with plasma membrane or is endocytosed, followed by pH dependent dusion releases viral core into cytoplasm Ø Conforma$onal shib in SU that exposes the hydrophobic N-terminal of the TM protein> inserted into cell membrane - Early phase includes entry, making a DNA copy of its RNA genome (reverse transcrip$on), and integra$ng it into cellular genome - RT is a dimer with two enzyma$c ac$vi$es Ø RNA-dependent DNA polymerase Ø Ribonuclease H (RNAse H)> a ribonuclease that speciﬁcally degrades the RNA part of the RNA:DNA hybrid - DNA polymerase ac$vity is facilitated by a virus associated cellular tRNA (gets inserted into the virion)> acts as a primer Sec$on 2.3 - Retroviruses can cause severe diseases e.g. AIDS and various type of cancers - HIV is a type of len$virus- from La$n len1s (slow), due to slow progression of the disease Ø Spherical enveloped par$cle, diameter ~100nm. Conical capsid with icosahedral symmetry Ø Linear single-stranded RNA, posi$ve sense genome (+ssRNA) Ø Two iden$cal genome RNAs in each virion Ø Cellular tRNA molecules packaged in virions used as primers for reverse transcrip$on - HV replicates in and kills lymphocytes and macrophages (both cells are CD4+, glycoprotein found on immune cell surface) Ø Infec$on results in deple$on of CD4+ T cells, rendering the host immunocompromised Ø Pa$ents suﬀer from mul$ple infec$ons from other pathogens> oben results in death - Acute infec$on of HIV: ﬂue like symptoms, infec$on of gut associated lymphoid $ssue (GALT)> signiﬁcant drop in CD4+ cells Clinical latency: cytotoxic T lymphocytes (CTLs) and an$bodies respond to infec$on but virus replica$on persist in lymph nodes resul$ng in gradual deple$on in CD4+ AID: acquired immunodeﬁciency syndrome An$viral drugs can control HIV-1 infec$on and prevent disease progression, but no eﬀec$ve vaccine just yet Lecture 10 Key Points 1. Retroviruses 1.1. Reverse transcrip=on 1.2. Integra=on 1.3. Transcrip=on and splicing 1.4. Assembly and release 2. Human Immunodeﬁciency Virus (HIV) 2.1 Genome and protein 2.2 Entry and replica=on 2.3 Latent infec=on 2.4 Transcrip=on 2.5 Protein func=ons 2.6 Assembly, budding and matura=on Sec$on 1.1 1. Synthesis of minus strand strong-stop (DNA synthesis stops abruptly in vitro) DNA. Ø Extends from tRNA primer to 5’ end of viral RNA template, short sequence of R+U5 2. Removal of template RNA Ø RNase H digests RNA in DNA:RNA hybrid Ø Frees R+U5 DNA strand Ø 3. First-strand transfer Ø DNA copy of R+U5’ transfers from 5’ end to 3’ end of genome RNA Ø R sequence from this fragment hybridizes with 3’R-sequence 4. Copying of full-length genome Ø Extended by RT to copy remaining genome RNA into a full length minus strand DNA Ø Produces long-terminal repeat. (LRT) with ends bearing U3-R-U5’ 5. Removal of template RNA> RNase H digests all RNA except the polypurine tract (ppt)> acts as a primers for synthesis of +strand DNA by RT 6. Synthesis of plus-strand strong-stop DNA> ini$ated at ppt primer, extends through the newly formed U3-R-U5 (LRT) and original tRNA (~18nts) 7. Removal of tRNA and ppt primer by RNAse H> exposes primer binding site (PBS) on +strand DNA 8. Second strand transfer> exposed PBS can hybridize with PBS’ 9. Extensions of both DNA strands by RT> linear dsDNA with LTRs on both ends called proviral DNA - Reverse transcriptase makes mistakes, it cannot remove the nucleo$de due to lacks of 3’-5’ exonuclease ac$vity> cannot proofread the newly synthesized strand (cellular polymerases have this ac$vity) Ø Leads to rela$vely high muta$on rate (~1-10 incorrect nt per proviral DNA)> retroviral popula$on is not considered uniform- quasispecies (collec$on of variants due to small muta$ons) - Linear dsDNA remains associated with components of the viral core i.e. all of this happens within the virus core Ø Preintegra=on complex: complex of retrovirus capsid proteins and newly synthesised proviral DNA> ready to be integrated into the host genome >> Too large to go through the nuclear pore> most retroviruses must wait un$l cell devides and nuclear envelope disintegrates (HIV uses a diﬀerent mechanism) Sec$on 1.2: - Integrase (IN) is packaged in the core virion> i.e., inside the preintegra$on complex; binds the two-ends of proviral DNA and brings them together - Proviral DNA gets integrated into cellular genome at random sites - Integrase removes two 3’ nts on each viral DNA strand, leaving free 3’ OH. This is important because we need 3’OH to form a bond with 5’ phosphate= phosphodiester linkages - Integrase facilitates a cleavage-liga$on reac$on to join viral and cellular DNA by bringing two 3’-OH ends of viral DNA close to two phosphodiester linkages 46 nt apart on the host DNA Ø This cuqng and joining leaves 4-6 nt single stranded gap on host DNA and a 2 nt unpaired gap on viral DNA - Host enzymes carry out repair synthesis of the gaps and removes the two unpaired nt of viral DNA> generates a direct repeat in host DNA - Once integrated, proviral DNA becomes part of the host genome> get replicated along with host DNA - Spread of infec$on can happen in diﬀerent ways; Ø Infec$on with progeny virus Ø Dividing of cells already carrying proviral DNA Ø Transmijed from parent to oﬀspring because of DNA integra$on (e.g. in germ cells) - A ‘cure’ will require removal of all integrated viral DNA from the host> not currently possible Sec$on 1.3: - Late phase of replica$on: viral RNA and protein synthesis, assembly of virions - Proviral DNA can remain laten (unexpressed) for long period of $me> need appropriate transcrip$on factors - U3 contains speciﬁc sequences that interact with cellular machinery> controls under what condi$ons the transcrip$on takes place Ø Sequence elements in LTR direct transcrip$on and polyadenyla$on by host cell enaymes - TATA box at U3/R junc$on (leb) directs transcrip$on by cellular RNA polymerase II - Poly (A) signal on the right LTR (between R/U5) directs cleavage of the transcript at R/U5 boundary and poly(A) is added by host cell enzymes Ø Generates full length RNA iden$cal to genomic RNA of the virus - All retroviruses make at least two mRNAs (but can be more complex): Ø Unspliced (full-length)> Gag and Gag/Pol polyproteins Ø Spliced> Env proteins Sec$on 1.4: - Virions mature into infec$ous par$cles aber budding from the plasma membrane: Ø Env protein is translocated into the lumen of ER then through Gogi, and directed to plasma membrane ( It is happened in the Golgi and ER because that is where posttransla$on and glycosyla$on) Ø Ø Ø Ø Gog/Pol polyproteins in cytosol initate assembly viral core Gag is targeted to plasma membrane by myristate NC por$on binds to a packaging signal (psi) on viral genome RNA Protease is ac$vated and cleaves Gag/Pol into mature single proteins aber assembly Sec$on 2.1 - Linear single-stranded RNA, posi$ve sense genome (+ssRNA) - Two iden$cal genome RNAs in each virion and cellular tRNA molecules packaged in virions used as primers for reverse transcrip$on - HIV is a fairly complex virus - HIV encode: Ø Ø Ø Ø Four capsid proteins: MA, CA, NC, p6 (budding protein, BP) Three enzymes: PR, RT and IN Two envelope proteins: surface (SU, gp120) and transmembrane (TM, gp41) Six regulatory proteins: Vif, Vpu, Vpr, Taf, Rev, Nef >>Structural proteins: Gag, Pol, Env, Vpr >>Non-structural proteins: Vif, Vpu, Tat, Rev, Nef Ø Splicing of HIV-1 primary transcript generates more than 25 mRNAs >>singly spliced (4 kb class) >>Doubly spliced (2kb class) Sec$on 2.2 - HIV-1 targets cells of the immune system by recognizing a CD4 receptor and chemokine receptors: Ø CD4 (primary receptor) is found on both T lymphocytes and monocytes/macrophages Ø A co-receptor of either CCR5 or CXCR4 is also required (R5= viruses that use CCR5, macrophage-tropic, X4= viruses that use CXCR4, T-cell tropic) Ø Varia$ons in viral SU (gp120) determines co-receptor usage Ø Ligands for these receptors can block viral entry through blocking these receptors using ligands and that will prevent viral entry - CD4 binding causes conforma$onal changes in viral gp120 (SU) Ø Exposes regions of gp120 that can recognize the co-receptor(s) Ø Expose gp41 (fusion domain) to insert in host cell membrane - Fusion occurs aber close proximity of viral and host cell membranes> releases viral core into cytoplasm - Once inside, nucleocapsid par$ally breaks down> permits access to cellular nucleo$de pool - RT replicates viral genome but generates small muta$ons due to absence of proofreading ac$vity> can aﬀect targe$ng by immune system or drugs - - Current therapies involve mul$ple drugs targe$ng various steps in virus replica$on Unlike other retroviruses, HIV-1 ac=vely directs transport of proviral DNA into the cell nucleus> produc$ve infec$on even in non-dividing cells - MA, Vpr and IN (part of preintegra$on complex) direct transport into nucleus: Ø MA has a nuclear import signal and interacts with impor$ns Ø Vpr and IN interact directly with nuclear Sec$on 2.3: - Elimina$on (cure) of HIV-1 infec$on is complicated by host genome integra$on> immune system cannot iden$gy infected cells if viral proteins are not expressed Ø Integra$on of proviral DNA is followed by either latent or ac$ve infec$on Ø Latency is regulated by transcrip$onal control elements in HIC-1 LTRs> binding various cellular transcrip$on factors (e.g. NFAT, NFkB) exist in U3 region of LTR Ø Ac$vity of these transcrip$on factors is sensi$ve to various cellular s$muli - Due to latent infec$on, pa$ents must con$nue treatment for life Sec$on 2.4 - The viral Tat (transac$vator of transcrip$on) protein is localized in the nucleus Ø Highly basic 86 AA protein produced by doubly spliced HIV-1 mRNA - Tat binds to TAR (Tat responsive element) at transcrip$on start site, increases HIV-1 transcrip$on by suppor$ng elonga$on by RNA polymerase II - Tat binds to TAR on growing nascent RNA but not on proviral DNA> one of the ﬁrst evidence that sequence elements on newly synthesized RNA can direct ac$vity of RNA polymerase In absence of Tat, RNA pol lacks processivity (ability to travel the en$re length of a gene) In presence of Tat, cellular Cdk9 (it needs cyclin to ac$vate) and Cyclin T(regulate the cell cycle due to the pH going up and down) are recruited, increased phosphoryla$on (indicated by PPP) of carboxy terminal domain in RNA pol> enhanced processivity/eﬃciency - - Ø CRS(ac$ng repressive sequences) present in gag, pol, env regions inhibit RNA transport to cytoplasm> need to have Rev response element (RRE) for transport Ø The viral Rev (regulator of expression of virion proteins) protein mediates cytoplasmic transport of viral mRNAs that code for HIV-1 structural proteins> binds to RRE> only on unspliced/single spliced mRNAs >>Doubly-spliced mRNA does not have CRS and can be transported normally Ø Rev con$nuously shujles between cytoplasm and nucleus (has both nuclear import and export signals) - Tat and Rev are essen$al for HIV transcrip$on and transport of mRNAs encoding structural proteins - Aber proviral DNA integra$on, only the doubly-spliced (2kb) RNA is transported out of the nucleus> allows for synthesis of Tat, Rev and Nef (early proteins) - Tat ad Rev are imported into the nucleus> transcrip$on of provirus DNA (Tat) and export of RRE containing mRNA (Rev) Ø Full length or single-spliced mRNA encoding other viral proteins (Late proteins) Sec$on 2.5: - HIV Vif (viral infec$vity factor) protein increases virion infec$vity Ø 193 AA protein found in cytoplasm of the infected cells Ø Dele$on of gene reduces HIV-1 infec$vity - Reuqired for counterac$ng a host cell an$viral factors> APOBEC3G cy=dine deaminase (C>U) - APOBEC3G is incorporated into virions and could deaminate residues in viral DNA> mutates viral DNA> aﬀects viral proteins> reduces infec$vity - Vif induces ubiqui$na$on and degrada$on of APOBEC3G by proteasomes> increased infec$vity - HIV Vpr (virion protein R) enhances HIV-1 replica$on Ø 100 AA protein that gets recruited into virions (interacts with gag C-terminal Ø Plays a cri$cal role in entry of PIC into the nucleus Ø Facilitates packaging of cellular Uracil DNA glycoslase (UNG)- removes uracil from DNA - - Vpr can also arrest infected cells in G2 stage of cell cycle> probably by promo$ng degrada$on of cellular proteins required to go from G2 to M phase of cell cycle> beneﬁcial for HIV-1: transcrip$on most ac$ve at G2 stage HIV Vpu (viral protein unique to HIV-1) enhances release of progeny virions form infected cells Ø 81 AA protein that gets inserted into membrane via its N-terminal domain Ø Accumulates in Golgi and endosomes CD4 in the cytoplasm can bind to and retain viral gp160 protein (precursor to SU/gp120)> reduces gp120 incorpora$on into virions Vpu in membranes can trigger degrada$on of CD4 through its binding to cellular B-TrCP> releases gp160 and increases surface expression of gp41 and gp120 (cleavage products of gp160) Ø Vpu also counteracts host an$viral factors that tether virus to host cell surface> enhances virus release from plasma membrane In absence of Vpu, virions accumulate on cell surface (par$cal budding) Expression og HIV-1 Vpu also enhances release of other unrelated viruses - Vpu induces degrada$on of tetherin (host en$viral protein; also involved in NFkB ac$va$on> inﬂammatory response) Ø Tetherin promotes endocytosis and degrada$on of virions - HIV Nef (nega$ve eﬀector) proteins is an important mediator of pathogenesis and enhances virus infec$vity Ø 210 AA protein localized to inner surface of plasma membrane through a fajy acid modiﬁca$on (myristate, 14C saturated fajy acid) at its N-terminal amino acid - Nef works to decreases surface expression of CD4 (via adaptor protein-2, AP-2_ and major histocompa$bility complex protein I (MHC1)> important mediators of immune response - Modiﬁes cell signalling in T cells> general ac$va$on of T cells without a presence of an an$gen> cannot mount an eﬀec$ve immune response Sec$on 2.6: - Genome packaging signal (psi) is encoded only on genomic RNA Ø Intramolecular base pairing (forms secondary structures) causes ‘psi’ to be inac$ve> forma$on of genomic RNA dimer exposes ‘psi’> allows interac$on with nucleocapsid protein (NC)> ini$ates assembly >> Several Gag proteins interact with NC-genomic RNA complex> transported to plasma membrane for ﬁnal assembly and budding via ESCRT complexes - Once released, the protease in virion par$cle is ac$vated> cleaves Gag and Gag-Pol polyproteins into individual pep$des> self-assemble in virion to form a conical capsid> ac$vates the virion for infec$on Lecture 11 Key Points 1. Poxviruses 1.1 Structure and classiﬁca=on 1.2 Disease and history 1.3 Genome and proteins 1.4 Assembly and release 2. Papollomaviruses 2.1 Structure and classﬁcatoin 2.2 Disease 2.3 Genome and Proteins 2.4 Entry and infec=ous cycle 2.5 Assembly and release 2.6 Diagnosis and vaccines Sec$on 1.1 - Poxviridae from English pocks (pox), referring to blistering skin lesions - Two subfamilies: 1. Chordopoxvirinae: Infec$ons vertebrates and humans 2. Entomopoxvirinae: Infects insects Sec$on 1.2 - Smallpox a was a debilira$ng and fatal disease globally. Smallpx scars on Egyp$an mummies provide evidence for early existence, wrijen records suggest that virus was endemic in Egypt and India by ﬁrst centry AD - Smallpox has been eradicated through worldwide vaccina=on - Disease progession: Ø Respiratory infec$on (spread via nasopharyngeal secre$on or scabs/pus) Ø Asymptoma$c phase (12 days) Ø Sudden onset fever, rash, vomi$ng Ø Painful rash, fever resumes, blisters covering body and airways, diﬃculty breathing and ea$ng. Most survivors with life long-scarring - Variola=on: infec$on via a non-antural route that yields less severe disease Ø Led to developing the concept of vaccina$on, which has eradicated smallpox worldwide - Variola$on was named aber the virus cause smallpox (variola virus) - Small amount of material from smallpox sores transferred to uninfected individuals Sec$on 1,3 - Linear double-stranded DNA, 150-250kb. Covalently closed hairpin ends: no free 3’ and 5’ ends with 10kb inverted terminal repeats - 150-250 genes> up to 100 proteins in the virus Ø Each gene has its own transcrip$onal promoter Ø No introns in the genome> no splicing required - Virion sec$ons show a biconceave core ﬂanked by lateral bodies (red)> trypsin sensi$ve, containing proteins (of unknown composi$on and func$on) Internal core is surrounded by core wall structure Ø Tubular structure within the core, contains the DNA Ø Contains breadth of viral enzymes for viral mRNA synthesis - Ø Two forms diﬀer in membrane composi$on, as a result of diﬀerences in morphogenesis Both forms are infec$ous, but use a diﬀerent set of viral structural proteins (surface) to bind to cellular receptors - Mature Virus (MV): Ø Has an outer envelope> enters by fusion or endocytosis Ø Ajaches to cellular glycosaminoglycans (broad host range)> polysaccharides formed from repea$ng units of diﬀerent 6-C sugars, including one amino sugar> bound to host surface proteins Ø Virion is stable and responsible for person to person transmission - Extracellular virus (EV): Ø Mature virus wrapped in an addi$onal lipid bilayer, with a dis$nct set of glycoprotein Ø Enters cells by phagocytosis> outer membrane ruptures due to decreases in pH in endosome> releases mature virus par$cle> MV fuses with vesicle membrane> virion core is released into cytoplasm Ø EV envelope is fragile> can rupture once in contact with host cell surface> releases MV par$cle> responsible for cell-to-cell (or $ssue-to-$ssue) spread within an infected person ‘ - - Poxviruses replicate in the cytoplasm> rela$vely rare for DNA viruses because it usually is in the nucleus Virus-coded enzymes packaged in the core carry out early RNA synthesis and packaging Ø Vaccinia virion can be considered a ‘mini-nucleus’, an mRNA synthesis machine Ø Viral enzymes transcribe, cap, methylate and polyadenylate mRNA Poxvirus genes are expressed in a regulated transcrip$onal cascade controlled by viral transcrip$on factos that bind to speciﬁc promoter sequences (~35 nt) in viral DNA: Ø Virus early transcrip=on factors (VETF): are packaged into virion and act upon release into cytoplasm Ø Viral intermediate transcrip=on factor (VITF) trigger intermediate gene expression Ø Viral late transcrip=on factor ac$vate late genes> encode VETF to be packaged with progeny virions Sequen$al expression ensures that enough new replicated DNA is available before structural proteins are synthesize - - Ø Genes required for DNA replica$on are early genes> expressed immediately aber an infec$on >>Expressed within virus cores> early mRNAs are translated into ‘early’ viral proteins Some of the early viral proteins dissolve the virus core and release viral DNA into the cytoplasm> further transcrip$on and DNA replica$on Viral DNA replica$on in cytoplasm forms “DNA factories” visible under EM (viroplasm) - - Ø Poxviruses produce large DNA concatemers (oligomers of genome length units) that are later resolved into monomers 1. Site-speciﬁc nick in one of the hairpins 2. Extension of the 3’ end (~100 nt) 3. Aber extension, hairpin ends are refolded 4. Hairpins from step 3 can be used for further DNA synthesis 5. En$re genome length is synthesized and then loops back through the 2nd hairpin> generates head-to-head dimer 6. Concatemers are resolved by virus-coded resolvase> makes staggered single strand breaks at the ends of concatemer junc$ons> forms DNA molecules with hairpin ends which are joined by DNA ligase Intermediate and late genes are called postreplicate genes> transcribed only aber DNA replica$on has started Ø Use dis$nct promoters and inita$ons factors Ø Inita$on of intermediate genes requires two VITFs and a cellular factor involved in mRNA metabolism Ø Inita$on of late genes requires three VLTFs and may require an addi$onal early gene product as well as a cellular factor - Postreplica$ve mRNAs have a poly(A) extensions and 3’ end heterogencity Ø A slippage mechanism during transcrip$on ini$a$on adds “poly(A) heads” Ø Heterogenous 3’ ends result from ineﬃcient transcrip$on termina$on at mul$ple sites Sec$on 1.4: - - Assembly of vaccinia virions is a complex process Rigid, cresent-shaped membrane structures are synthesized (de novo synthesis) and are des$ned to become mature viral envelope (not connected in a pre-exis$ng internal membrane) Crescent mature into spheres (i.e. immature virions, IV in ﬁgure 26.5) and enclose viroplasm> electron dense and containing viral core proteins Viral DNA enters the spheres to form discrete and dense cores (aka nucleoids) - - Ø Immature virions develop internal core and lateral bodies by rearrangement of msterial captured inside the spheres> acquires ﬁnal shape> mature virion (MV) >> Vast majority of new virions remain as intracellular MVs >> A small propor$on of MVs undergo further matura$on to become extracellular virus (EV), designed for export and cell-to-cell spread Ø MVs can be wrapped in Golgi-derived cisternae> adds addi$onal lipid layers and viral proteins Ø Wrapped viruses (WV) have two bilayer membranes and are transported to the plasma membrane b ac$n tails Ø The outermost viral membranes fuse with cell membrane, releasing extracellular virus (EV)> EV can remain ajached to cell membrane via microvilli projec$ons - - Ø Vaccinia viruses (and some others) produce ac$n ‘comer’ tails> indeed by viral proteins that promote ac$n polymeriza$on/depolymeriza$on> used for ‘propulsion’/budding out of the host cell Poxviruses make several proteins that target host defences against invading pathogeny Sec$on 2: - Papillomaviridae from La$n papilla (nipple) and Greek -oma (rumor) referring to warts (papillomasb) - Naked icosahedral capsid with a diameter of ~55nm - Circular double-stranded DNA ~8kb (similar to bacteria)> DNA is packaged as a “minichromosome” with cellular nucleosome histones Sec$on 2.2: - Over 100 known human papillomaviruses (HPV). Other hosts include cajle, dogs, deer, tabits, etc - Oncogenic human papillomaviruses are a mjor cause of genital tract cancers and can cause genital warts (condylomas), neoplasias (uncontrolled growth of cells), and invasive squamous cell carcinomas - Benign warts at speciﬁc sites (skin, mucosa, larynx) depending on virus strain> transmijed by direct contact - Some types cause cervical carcinoma, a sexually transmijed disease and a major cause of cancer in women - May cause other anogenital cancers (cancers of anus, genital organs) in both men and women, and cancers of the oral cavity, oropharynx are tonsils Sec$on 2.3: - Papillomavirus genome is circular, double-stranded DNA that contains 8-10 open reading frames (OFRs)- 6 early genes (E1,2,4,5,6,7 ), 2 late genes (L1, L2; major and minor capsid protein) Only one of the DNA strands is used for mRNA transcrip$on Ø Viral mRNAs are made from two promoters and two polyadenyla$on signals: >>Early promoter located upstream of E6 gene; late promoter located upstream of E1 gene >>Two poly(A) signals are located downstream of E5 Sec$on 2.4 - - cell is complicated the entry of HPV into host Ø The infec$ous cycle follows diﬀeren$a$on of epithelial cells Ø Virus infects the mucosal epithelium and have to reach non-diﬀeren$ated basal cells Ø Virus ajaches to heparin sulfate proteoglycans and alpha6-integrins and enters by endocytosis Ø Viral DNA is maintained in the nucleus, replica=ng modestly un=l cell diﬀeren=ates into a kera=nocyte - - Ø Viral E1 and E2 proteins bind to the origin of replica$on (ori, present in the long control region, LCR) and direct inita$on of DNA replica$on Ø LCR (also some$mes called URR-upstream regulatory region) also contains enhancer sequence(s) (may be speciﬁc kera$nocyte enhancer) Ø Late promoter is ac$ve in diﬀeren$ated kera$nocytes Ø Some of the transcripts are polyadenylated at the late site> produces abundant E1 and E2 proteins> increased level of E1 and E2 result in increased DNA replica$on, producing thousands of DNA molecules per cell - - Ø E1 is a DNA helicase that locally unwinds the DNA Ø E2 binds to speciﬁc sites near ori and forms a complex with E1> increases E1 aﬃnity for ori Ø Numerous cellular transcrip$on factors and other regulatory molecules bind to regulatory regions of papillomavirus DNA for transcrip$on Ø E7 protein is a small (~100 AA) protein that interacts with cell cycle regulatory proteins, par$cularly re$noblastoma (Rb) protein. Rb is a tumor suppressor protein which represses the ac$vity of E2F transcrip$on factos Ø E7 can also induce the ubiqui$n-mediated degrada$on of Rb Ø Rb is a tumor suppressor protein which represses the ac$vity of E2F transcrip$on factors under normal condi$ons. Rb binds to E2F> blocks ac$va$on of cell-cycle genes (involved in G1 to S phase (DNA synthesis phase) transi$on >> CDK/cyclin complex phosp

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