MICR Midterm PDF

Lecture 1: Introduction Smallpox -400K deaths towards end of 18th century, 300M deaths since 1900 -highly fatal (80% in children, 20-60% in adults) -global vaccination 1966-1977, eradication 1979 -Pharaoh Ramses V, short lived king had pock marks (died of smallpox?) -could have been used as a biowea...

Lecture 1: Introduction Smallpox -400K deaths towards end of 18th century, 300M deaths since 1900 -highly fatal (80% in children, 20-60% in adults) -global vaccination 1966-1977, eradication 1979 -Pharaoh Ramses V, short lived king had pock marks (died of smallpox?) -could have been used as a bioweapon in the French-Indian wars (1754-1763) Influenza: annual epidemics and periodic pandemics -first recorded by Hippocartes in 412 BC -ancient zoonotic disease likely originated in domesticated animals ~6000y ago -50-70K deaths in the USA from seasonal flu, mostly from pneumonia -The great flu pandemic (1918-1919)=known as the spanish flu Poliomyelitis and infantile paralysis -common infectious disease in kids, paralytic form was most feared -transmission via oral-fecal, infection of CNS leading to death/paralysis -most devastating in early 20th century, related to personal hygiene -iron lung was the only treatment for patients w/ lung complications -Salk vaccine was the inactivated polio vaccine using cell cultures (1955) Ebola hemorrhagic fever -high fever, headache, muscle pain, diarrhea, vomiting, hemorrhage -transmission via direct contact with bodily fluid/contaminated objects -recovery largely depends on clinical support and immune system -origin: likely fruit bat/primates (bushmeat) HIV/AIDS -32M deaths since outbreak, 38M living with HIV -attacks immune system, death due to secondary infections -transmission via body fluid Cold sore (human herpesvirus 1) -fever blister caused by DNA virus -most common viral infections, re-activation is triggered by stress factors -HPV1 is a linear dsDNA virus, follows a cyclic pattern of infection of epithelial cells and latency Infectious mononucleosis (mono) -common infections in teens, epstein-barr virus is the common cause (DNA virus) -implicated in multiple autoimmune syndromes Human papillomaviruses -cause of most prevalent STD, genital HPV -prevalence of high-risk HPV types, can cause cervical cancer Plant viruses -papaya ringspot virus= devastation caused (solved by transgenic papaya) -grapevine leafroll associated viruses Are all viruses pathogenic? -all deadly viruses responsible for human disease originated from animals and crossed the species barrier -humans have been the biggest and most effective vector for emerging and re-emerging viruses -most of the viruses are not lethal to their hosts but find a way to co-exist through genetic mutation/ co-adaption Many good things come from viruses -DNA (phage) or RNA (TMV) serves as genetic material -viruses as vectors in gene therapy and cancer treatment -the revival of phage therapy to control bacterial infections with mult-drug resistance -protein expression, oncolytics, recombinant vaccines, functional genomics, nanoparticles Lecture 2: Discovery, morphology, composition and structure Key factors responsible for breakthrough discoveries in science -the availability of new tools and associated tech for research: ↳culturing microbes on artificial media (petri dish for bacteria/fungi) ↳bacteria-proof filters (eg. Chamberland filter) ↳light microscope TMV: first virus discovered Adolf Mayer: 1879: - extracts from tobacco with mosaic disease were infectious - Fungal pathogen not involved, failure to culture on petri dish 1882: - Initial conclusion that its a soluble, enzyme-like contagium, because it wouldn't culture D. Ivanovsky: 1892: - Passing tobacco extract through bacteria-proof filter, filtrate still infectious, said the filter was defective M. Beijerinck 1898: - Filter extract→dilute filtrate→inoculate healthy tobacco→replenish - Filter extract diluted because diluted bacterium will not affect healthy tobacco, but healthy tobacco was still affected, must not be a new bacteria that is unfilterable Stanley 1935: - Crystallization of TMZ particles purified from tobacco, concluded that virus is proteinaceous in nature Bawden and Pirie 1936: - TMV particle also contains RNA (5%) Helmut Ruska 1939: - Revealed first TMV using EM FMDV: the first animal virus discovered Loeffler and Frosch (1898): filterable virus from cattle with foot-and-mouth disease remained infectious -FMD is a highly contagious of cloven foot animals, causes high fever, reduction of milk/meat, infertility, fatal in calves Yellow fever virus: first virus discovered in humans (1901) -responsible for fatal endemics in the tropics/subtropics -natural host=monkeys/mosquitos -slave trade brought it to america -damage to liver, jaundice -caused Philadelphia epidemic in 1793 (killed ~15% of city population) -human volunteers recruited for vector transmission studies -caused by vector-borne filterable virus Discovery of viruses infecting bacteria: bacteriophages 1915: Twort - Repeated attempts to grow vaccinia - Contamination of petri dish and glassy transformation 1915: d’Herelle - Invisible antagonistic microbe of dysentery (Shigella) among soldiers responsible for killing bacteria and plaque formation 1939: Delbruck and Ellis - One step growth experiment (doermann’s experiment) 1940: Luria and Delbruck - Established ‘phage group’ to study page and E.coli (set foundation of bacterial genetics) DNA as the genetic material in phage T2 1952: Hershey and chase experiments - Nobel prize (1969) with Delbruck and Luria on discoveries concerning the genetic structure of viruses -discovered that it is the protein interior (not the shell) -labelled T2 with sulfur (contained within protein) and phosphorous (contained within nucleic DNA) Label →mix with bacteria →blend →grow culture →centrifuge →measure radio-acivitiy -it was found that phosphorous was present in cells after centrifugation, shell remains in supernatant (radioactivity in pellet) -proves that DNA gets in and replicates during infection not protein Chemical composition of viruses Nucleic acids: RNA/DNA as the genetic material, the majority of viruses discovered so far contain RNA as genomes (none use both) Proteins: structural and non-structural (some part of the capsid) Lipids: for enveloped viruses only (naked virses lack lipids) derived from cellular lipid bilayer Carbohydrates: in glycoproteins and glycolipids; involved in recognizing cell receptors and attachment to host cells Types of viruses 1. (+)ssRNA genomes - 3-31kb, linear, naked - Mostly infect plants 2. (-)ssRNA genomes - Helical symmetry, linear, pathogenic viruses to humans 3. dsDNA genomes  - Mostly infect vertebrates 4. ssDNA genomes - 2-9kilo bases, naked, circular genome, icosahedral symmetry except Inoviridae Non-enveloped (naked) vs enveloped virus Naked: - Have no coat protein, have capsid protein instead - Capsid can be RNA and DNA (nucleic acid) Enveloped: - The lipid bilayer is the envelope with glycoprotein spikes - Called nucleocapsid not capsid - Shorter lived, can be destroyed by UV, desiccation, detergents Viruses of plants vs vertebrates -on vertebrates have dsDNA (RT) which uses reverse transcriptase to convert RNA to DNA to complete the replication process=retrovirus -in plants there are no true dsDNA viruses, only dsDNA (RT) Morphology and dimensions 1. Rigid rods/flexous filaments -14x71nm to 80 x 14,000nm -ie. TMV, Filoviridae (ebola),Closteroviridae 2. Spherical (or isometric) -1-300nm in diameter -ie. Parvoviridae, Picornaviridae, adenoviridae, herpesviridae 3. Irregular and complex morphology: T-even bacteriophages (ie. T2) -has a spherical head (containing DNA) and a tail (helical sheath, tail fiber, and tail baseplate) 4. Irregular and complex morphology: baculoviruses of insects -two distinct virion forms: a. Occluded virion (OC): survival when released into environment b. Budded virion (BV): for spread within insects 5. Brick shaped viruses: vaccinia and pox viruses Capsid design -viruses use multiple copies of smaller subunits to construct capsids required to package the genome inside Helical symmetry -subunits form a sheet that is folded into a spiral that holds DNA/RNA -rod-shaped and filamentous viruses have structures built based on helical symmetry -in capsids with helical symmetry, the CP subunits have equivalent bonding relationship to one another, except those at both ends of the virion -open structure (can extend it) with unlimited packing capacity (for insertion of foreign DNA as in viral vectors) -P (pitch of the helix) = u (# of subunits per helical turn) x p (the axial rise per subunit) Icosahedral symmetry -basic design of spherical capsids/nucleocapsids: composed of 20 triangular faces -is closed structure, allows packaging of limited genome sizes -all subunits in the capsid are equivalent (identical) bonding relationship 3 pillars of icosahedral symmetry: 1. Triangulation number (T) -defines the possible icosahedral surface lattice, how many smaller triangles fit within a triangular face -if T=1 all of the triangles are equivalent (60 CPs) -only certain multiples of the amount of subunits are allowed (ie. 60 subunits; T=1,3,4,7..) -can calculate the CPs by T x 60 2. Quasi-equivalence -defines the nearly identical bonding relationship among the subunits of a spherical capsid -if T>1 then its quasi-equivalent 3. Spontaneous self-assembly -of individual CP subunits into virus-like particles (VLPs) to identify the assembly process 3 types of rotational symmetry: -viral capsids/nucleocapsids with icosahedral symmetry have 20 triangular faces and 12 pentagonal vertices -twofold symmetry at each of the 30 edges, threefold symmetry at each of the 20 equilateral triangular faces, and fivefold symmetry at each of the 12 pentagonal vertices CPs of diverse viruses share 3D structure: the B-barrel jelly roll fold -shared by viruses with icosahedral symmetry, these viruses infect different types of organisms as their hoists -made of many antiparallel beta strands -similar fold is also present in the storage protein (phaseolin) of beans Interactions among CP subunits -CP subunits are small, the number of subunits will have to increase to allow them to exist in a quasi-equivalent position in viruses with large capsids -CP subunits spontaneously assemble into larger structures, such as structural units and intact capsid shells, with or without the help of the viral genome -the CP subunits are stabilized by the max number of non-covalent bonds between them, leading to the lowest free energy state -all subunit-subunit and subunit-RNA bonds are weak (hydrophobic, van der walls) -secondary structures in viral RNA serve as packaging signals Architecture of complex viruses: phage lambda -icosahedral head -helical tail composed of tail cone (sheath), tail plate and attachment fibers -each part is assembled individually, followed by assembly into an intact virion Structurally complex virus: Adenoviridae -T=25, 1500 copies of subunits, actual is 780 copies Protein II: 720 copies/virion forming hexons Protein III: 60 copies, forming pentons Lecture 3: Classification, Taxonomy and Nomenclature Homes’ 1st attempt at classifying viruses using Linnaean system (1948) Order: Virales Group 1: phaginae (viruses of bacteria) Group 2: phytophaginae (viruses of plants) Group 3: zoophaginae (viruses of animals) -issue is that the classification makes no distinction between the disease and the virus Ie. the hepatitis virus causes different types of hepatitis, mosaic virus affects different plants Classification and taxonomy (1930-1966): - more emphasis given to virus over disease, based on morphology, capsid structure, chemical composition, and type of genome -all viruses classified into groups: 1. Herpesvirus group -dsDNA, icosahedral capsids, large virion with envelope 2. Poxvirus group -dsDNA, complex and irregular virion, 200 nm or larger, enveloped 3. Myxovirus group - (-)ssRNA, spherical virion, helical nucleocapsids, enveloped (1966-now): -ICTV considered 2 approaches to classification: 1. Monothetic -based on one characteristic at a time (ie, nature of genome, symmetry of capsid, ect) Problem: assumes all members of a group originate from the same ancestor 2. Polythetic -consider a set of multiple properties and characteristics -individual of a taxa share most of a set of common characteristics -not assume that all viruses share the same ancestor -defined by relatedness in sequences of gene, set of gene, or genomes Taxon (taxa)> order, family, (subfamily), genus, and species, evolves quickly Quasi species> a term used for RNA/retroviruses, a population structure of viruses with a large number of variant genomes. Occurs when a virus has a high error rate and is therefore very prone to mutations (such as HIV) How are viruses named Bacterial: based on a specific code Plant: refers to plant host the virus was first identified and descriptor Mammal: based on disease and symptoms Insect: based on latin name of host and effects of infection -some viral species named after place where prototype was identified or properties of virions Characteristics for classifying genus/family -nature and organization of genomes ↳DNA/RNA and strandedness (ss or ds) ↳polarity (+/-) ↳segmented or non-segmented -segmented with genomes in separate virions=polypartite, single virion=monopartite ↳topology (linear/circular, closed/open circle) -virion morphology, structure of capsid/nucleocapsid ↳helical, icosahedral, complex ↳shape, size and surface features of virion ↳envelope -genome structure, strategies of genome replication expression -enzymes (polymerase, RTase, protease, integrase ect) Characteristics used to define species 1. Natural host range 2. Cell and tissue tropism (liver, GI tract, phloem ect) 3. Pathology (in host) and cytopathology (in cell culture) 4. Mode of transmission (air borne, pollen/seeds, fecal-oral ect) 5. Physico-chemcial properties of virions (sedimentation coefficient, inactivation temp and time) 6. Antigenic properties of viral proteins (antigenicity, cross reactivity, serotypes) 7. Sequence relatedness of individual genes and whole genomes Phylogenetic analysis phylogeny> the prediction of evolutionary relatedness among viruses based on comparison of their sequences -based on nt sequence, AA sequence, or both -different methods can be used in generating phylogenetic trees 1. Neighbour joining (NJ): fast 2. Maximum likelihood (ML): more reliable 3. Maximum parsimony (MP): bayesian inference Lecture 4: Virus Replication Cycle (pt1) Limitation of research using lab animals -prior to 1949 virus research relied only on lab animals -its impossible to dissect the mechanistic details that govern viral replication cycle and pathogenesis using animals -there has been a shortage of animals -bioethical considerations Cell culture system Enders, Weller, Robbins (1949): - Primary embryonic (non-neuronal) cell cultures (from mouse) for measles virus and polio - Used in research and vaccine development - Cultured on plastic surface (research), or as liquid suspensions (bioreactors) - Large quantities, enabling studies of structure, composition, development of vaccines Dulbecco: - Mechanism of host cell transformation by oncoviruses (tumor viruses) - Plaque assay and the one step growth experiment Baltimore and Temin: - Discovery of reverse transcriptase 3 types of cell cultures 1. Primary -derived from live tissues/organs, comprising multiple cell types -finite capacity in cell division (5-20 times) Ie. monkey kidney for polio vaccine 2. Diploid cell strains -single cell type, mostly epithelial/fibroblast -normal morphology and number of chromosomes -cell division up to 100 times Ie. WI-38 from female embryonic lung 3. Immortal (continuous) cell lines -homogeneous in cell type, infinite capacity of cell division -abnormal chromosome morphology and number -loss of contact inhibition (detach from surface) -undergo genetic changes to prevent cell aging process -source= tumors, transformed cell strains, cells mutated - tumorigenic, produce tumors when implanted in lab animals Ie. HeLa, cervical tumour Cytopathic effects (CPE) ↳morphological alterations of a cell due to viral infection -this is how you assess the effects of virus on cell culture, there are signs of CPE -cell death -rounding up: detach from cell surface, and become spherical -syncytium: large, multinucleate cell bodies due to cell fusion (giant cell containing many nuclei) -abnormality in morphology and number of chromosomes -inclusion bodies (ie. negri bodies in rabies) ↳negri bodies can be seen under microscope, where genome transcription and replication occur One step growth cycle -synchronous infection of all cells with virus, high multiplicity of infection (MOI)=5-10 -monitor virus growth at a set of intervals (time course experiment) Eclipse period: from adsorption to appearance of the 1st intra-cellular virion Latent period: time between adsorption and 1st extracellular virion Burst size: the sum of virions produced in a single cell -specific curve depends on virus, DNA virus takes longer (and has a larger sum of virions) High MOI is key to achieve synchronous infection and one step growth analysis MOI=number of infectious virions added per cell -not all cells in a petri dish receive the same number of viruses -MOI of 5-10 are commonly used to achieve synchronous infection (see what is happening at each time point) -allocation of virions among cells is calculated by Poisson distribution (due to random collision between viral particles and cells) Replication cycle 1. attachment -collision between viral particles and cells via Brownian motion -weak contact via interaction between viral particle and (-) charges on cell surface -specific attachment is achieved via interactions between: a. Attachment protein on the virus -ie. HA in influenza, fiber in adenovirus, surface structure ‘canyon’ in poliovirus b. receptor(s) on the surface of the host cell -ie. Sialic acid for HA, Ican-1 for rhinoviruses -attachment is strengthened when more interactions occur between the protein and receptor On the PM and surface of eukaryotic cells -there are up to 500k potential receptor molecules per cell -PM contains lipid rafts with distinctive structure and functions -viruses hijack surface molecules and lipid rafts for entry and replication Where are proteins attached -some proteins are attached to membrane via TMD (transmembrane domain) -some proteins are anchored indirectly to the PM via fatty acids/alcohol 3 types of anchors for proteins to indirectly associate with PM: 1. Myristic acid: 14C fatty acid 2. farnesyl : 15C alcohol 3. Glycosyl phosphatidylinositol (GPI)-linked proteins (occurs only on the external surface of PM) Attachment protein and receptors for naked viruses 1. Attachment via surface features on the virion - Poliovirus and rhinoviruses have a canyon (depression) surrounding each pentamer, serving as the attachment site for receptors - Body can produce immune bodies (IgG) that can bind to the depression which neutralizes the virus and cause antiviral compoints to enter the viral port and remove the lipid (making virus inactive) 2. Attachment via fibers on virion surface (adenovirus) - Fiber is a homo-trimer of protein, at each of the 12 penton bases - Terminal knob on fiber with depression, attachment site for ‘car’ -cell receptors for picornaviruses and adenoviruses: Attachment proteins and cell receptors: enveloped viruses -glycoproteins on viral envelope are responsible for attachment ↳hemagglutinin of influenza A and B binds to sialic acid ↳surface glycoprotein (HIV-1) binds to CD4 of Th cells and macrophages, needs a co-receptor for viral entry -if the co-receptor is ɑ-chemokine receptor, HIV1 binds to T cell tropic strain -if the co-receptor is 𝛃-chemokine receptor, HIV1 binds to macrophage tropic strain Recap on receptors -have been identified for a small subset of high-impact viruses -many viruses share the same type of receptors -viruses with carbohydrate receptors tend to have broader host range because there are a lot of carbohydrate attachment sites -presence of receptor and co-receptor determines the host range and tissue tropism of a virus *know the highlighted ones Lecture 5: Virus Replication Cycle (pt2) 1. Attachment (previous lecture) 2. Entry and uncoating 3 basic tricks viruses use to enter a host cell 1. The simplest case: drilling a hole at PM (eg. picornaviruses) 2. Fusion between viral envelope and PM - Occurs at neutral pH but requires fusion proteins/peptides - (eg. paramyxoviruses, retroviruses, coronaviruses, baculoviruses ect) 3. Receptor-mediated endocytosis, followed by uncoating at an intracellular membrane (endosomal/nuclear) - Require low pH condition: endosome, lysosome - (eg. orthomyxoviruses, adenoviruses, ect) Models for poliovirus entry -can enter through the PM or endosomal membrane -steps in entry and uncoating: 1. Attachment to multiple receptor Pvr 2. N-terminus of VP1 exposed, inserted into PM, forming channel 3. Viral RNA released into cytoplasm Entry and uncoating at PM -exposure of fusion peptide, 2 alternative ways to trigger the release of fusion peptide Fusion peptide> segment of 20-30 hydrophobic AA residues, normally hidden -HIV-1 viral entry requires binding to receptor and co receptor, triggers exposure of fusion peptide and fusion between viral envelope and cell membrane Receptor-mediated endocytosis -cellular function is selective important of extracellular molecules (ligands) into a cell through receptor and membrane invagination -formation of clathrin-coated pits requires ATP hydrolysis -gradual decrease in pH from early endosome to late endosome, to lysosome Receptor-mediated endocytosis and uncoating at endosomal membrane INFLUENZA -HA attaches sialic acid, receptor bound virus enters through clathrin-coated endocytosis -vesicle sheds coat and fuses with early endosome, causing low pH (fusion peptide emerges from HA under low pH) -H+ into virion via M2 channel, matrix layer dismantles and viral envelope fuses with endosomal membrane, releasing RNPs into cytosol and RNP enters nucleus Receptor-mediated endocytosis and stepwise uncoating ADENOVIRUS -fibers bind to ‘car’ receptor, the penton base interacts with integrin, leading to endocytosis -fibers are lost in the endosome, penton bases dismantle in the late endosome -broken virion releases to cytosol, carried by microtubule and docks at nuclear pore -’tug of war’ between dynein and kinesin releases viral DNA, which enters nucleus Mechanisms of entry into the nucleus by different viral families -DNA viruses and retroviruses must enter the nucleus to complete replication cycle Adenovirus: partially disassembled virion transported by microtubules, dock onto nuclear pore, pulling by kinesin break up virion, DNA enters Herpesviruses: DNA genome injected into the nucleus due to the release of pressure in the interior of capsid Polyomaviruses: remodeling of nuclear envelope and lamina enables entry of viral DNA HIV-1: pre-initiation complex docks onto nuclear pore due to NLS on both CA and integrase, followed by integration into host chromosome Entry of phages with dsDNA genomes: T4 -t4 completes replication in less than 30 mins -tail fibers recognize and attach to receptors (LPS and Ompc) on bacterial cell surface -induction of conformational changes in base plate -tail sheath contracts to expose the inside tube -lysozymes carried in phage particle dissolve cell wall -DNA injected into cell cytoplasm Routes of entry of plant viruses into plant cells -most plant viruses don't require receptors to enter, usually enter by: ↳mechanical transmission through minor wounds and abrasions ↳vertical transmission via reproductive organs (pollen/seeds to offspring) ↳vegetative propagation materials  ↳through grafting between rootstock and scion ↳insect vectors 3. Biosynthesis ↳the synthesis of all viral components required for building the next generation of viruses transcription> production of mRNA from the genome (DNA or RNA) Reverse transcription> generation of cDNA using RNA as template, only in retroviruses translation>production of polypeptides using mRNA, relies on cell translation machinery Genome replication> production of nascent viral genomes (DNA or RNA) The revised baltimore system -recognized that viruses can be divided into 7 groups based on pathways that lead to mRNA and protein synthesis Group1: dsDNA - Generates mRNAs use RNApoly enzyme from the host cell nucleus Group 2: (+)ssDNA - Only way to transcribe to RNA it needs to convert its genome to dsDNA, using DNApoly that can be transcribed (same way as group 1) Group 3: dsRNA - Replicate genomes by encoding for RNA dependent RNApoly (RdRp)=same as group 4 and 5 - Translation occurs by converting to (+)ssmRNA via RdRp Group 4: (+)ssRNA - Can be directly translated by RdRp, usually ORF translated only - Converted to (-)ssRNA and then RdRp converts to (+)ssmRNA Group 5: (-)ssRNA - Converted to (+) ssmRNA via RdRp Group 6: (+)ssRNA (retrovirus) - Package reverse transcriptase (RT) and converted to (-)ssDNA converted to dsDNA in the nucleus by DNApoly, integrated into genome - dsDNA converted to (+)ssmRNA via RNApoly Group 7: dsDNA (pararetroviruses) - Converted to (+)ssmRNA via RNApoly - Genome replication via (+)ssRNA intermediate RT Cellular sites for biosynthesis depend on the type of virus Gene expression in DNA viruses -immediate early (IE) genes: expressed right after infection, functions: a) Rendering cells to enter S phase b) Induce expression of other viral genes c) Inhibit host defense mechanism and biosynthesis Early (E) genes: enzymes and accessory factors required for genome replication Late (L) genes: structural proteins required for assembly, some are very late DNA viruses -infect cells that are either dividing or they force quiescent cells to enter S phase -many have the potential to be oncogenic Exception: poxviridae=replicates in cytoplasm RNA viruses -all stages of the replication cycle occur in the cytoplasm -must associated with a type of intracellular membranes (+)strand RNA viruses -transcription and replication done by RdRp -gRNA is the 1st or only mRNA, it can translate directly -the 5’ ORF is translated directly on gRNA -ORF1 encodes 126kD protein with MTR and helicase domains -ORF2 encodes RdRp as part of 183kD proteins via suppression of leaky stop codons (-) strand RNA viruses -exists as a ribonucleoprotein (RNP) complex and not as naked RNA, must be transcribed before translation occurs -influenza viruses: RNP enter the nucleus wherin they replicate and transcribe ↳requires splicing of transcripts made from some genome segments Retro/pararetroviruses -2 identical (+)RNA molecules, reverse transcription starts in virion on route of entry into host cell -cDNA inserts into host chromosome, becoming part of host genome, establishing latency and source of persistent infections 4. Assembly of replication cycle 1. Formation of individual structural units from a single/several structural proteins 2. Assembly of capsid shell with structural unit ↳ large DNA viruses requires packaging protein to spool viral DNA into procapsids 3. Selective packaging of viral genome + other components 4. Acquisition of a lipid envelope (mostly from PM) 5. Exit the infected cell  6. Maturation of virions ↳applies to certain viruses, requires proteolytic cleavage Assembly of structural units -de novo process=spontaneous molecular interaction determined by the primary structure -formation of structural units results from interactions among capsid/nucleocapsid subunits -interlocking among units dictated by AA Adenoviruses: formation of penton requires fiber and penton base Poliovirus: a single polyprotein precursor and cleavage Adenoviruses: formation of hexon trimer by protein II assisted by chaperone Assembly of capsid shells -for RNA viruses, gRNA is involved in virion assembly ↳TMV and many other (+)ssDNA ↳poliovirius RNA may be involved in assembly and final cleavage during virion maturation -in large DNA viruses and phages, procapsids are first formed with scaffold proteins ↳followed by collapse and removal of scaffold structures then gDNA inserted into procapsid ↳process requires input of energy from ATP hydrolysis 5. Egress (exit) of replication cycle -naked viruses (polio, HAV, rhinovirus, adenovirus) ↳lysis of infected cells, cytopathic effects -enveloped viruses ↳budding and pinch-off at PM Lecture 6: TMV The 3 supergroups of (+)ssRNA viruses -most of these are plant viruses -divided based on the phylogenetic relationship of RdRp: 1. Alphavirus-like: contains the animal virus genus (alpha virus) 2. Picornavirus-like: best studied (polio research) 3. Flavivirus-like: (largest ssRNA group) TMV transmission -causes necrotic local lesions, easily transmissible, persists for decades -stable in dead plant debris, rod shaped structure allows it to be stable -transmitted mechanically (leaves touching) -remains infectious after processing (ie. in cigars) -transgenic plants expressing TMV CP (coat protein) exhibited delayed onset of disease= coat mediated protection Key discoveries from research on TMV 1898: TMV as the first filterable virus conceptualized (contagious living fluid) 1935: Stanley (Rockefeller) crystallization (Science 81:644) 1938: Ruska obtained the first EM graphs of TMV particles 1954: Helical structure of TMV particle (J. Watson) 1955: Fraenkel-Conrat TMV self-assembly proving RNA as the genetic material 1956: Franklin describing RNA helical structure within TMV particle Genome structure and expression strategies -small (+) ssRNA genome (6.4kb) -gRNA is the 1st mRNA to translate replication-related enzymes -5’ end cap structure, 3’ end tRNA-like structure (aminoacylated with His) -3’ UTR: expensive secondary and tertiary structures (3 pseudoknots) -ORF1 will be translated from RNA, when UAG is reached it might reach through and translate ORF2 (leaky stop) Reconstitution experiment and proof of RNA as genetic material -found that a capsid alone assembles into virion=not infectious, when RNA with capsid/alone it is infectious -therefore the RNA is the genetic material (nt are the units of heredity) -used 2 strains of TMV, removed proteins from RNA genomes and generates hybrids (one had RNA from strain A and protein from strain B) Local lesion assay to quantify TMV -spread abrasives on surface of leaf, rub leaf with dilution of viral stock -wait for infection, count the number of lesions -calculate the titer of original viral stock Assembly of capsid and genome packaging -structural unit of TMV is CP double disk -16.3 copies/turn -5’ genome segment threats through the interior of the elongating helix 3 stages of TMV movement within an infected plant 1. Intra-cellular movement -upon entry into plant cell, viral replication complexes form in association with ER, which move to other locations of the infected cell, producing multiple VRCs in the same cell 2. Iter-cellular (cell to cell) -VRC docs at plasmodesmata, traverses it with the help of movement protein (MP) and completes new replication cycle in neighboring cells ↳plasmodesmata has a size exclusion limit (1-7kDa) 3. Long-distance movement -virions gain entry into the sieve elements of the phloem, move rapidly through it to reach distal parts of the plant causing systemic infection Common properties of movement proteins -binds to RNA to form thin and long RNPs -N-term region of TMV MP increases SEL of PD -interacts with ER, actin filaments and microtubules -associates with peripheral ER (seen by gfp tagging MP) -MPs encoded by non-related viruses may complement defective MP TMV cell to cell movement, the earlier model -MP complexes with TMV RNA to form a thin thread, moves on actin filament, interacts with resident protein (p38) increase SEL, squeezes through central cavity of PD -phosphorylation by host kinase releases MP, frees viral RNA in 2nd cell for translation Composition and structure of VRC (viral replication complexes) -replicase proteins, MP, viral gRNA, ER membrane -helicase domains self interact, forming hexameric ring-like structures when over-expressed in bacterial cells -they bind ssRNA -they act as ATPase and helicase, hydrolyzing ATP and unwinding dsDNA -5:1 ratio of 126kD/183kD molecules that make VRCs -126kD align on and moves along actin filaments TMV cell to cell movement recent research/current model -TMV moves as a VRC and not as the thin MP-RNA threads (as originally thought) ↳much shorter time is required to complete the same process in neighbouring cells TMV as vectors for VIGS and proteins expression in plants VIGS= virus-induced gene silencing -RNA-based defence system for gene regulation and defence against viruses -both PSY (phytoene synthase) and PDS (phytoene desaturase) protect chlorophyll -TMV was engineered to produce mRNA for PDS/PSY= triggers RNA silencing in tobacco Lecture 7: Poliovirus and Picornaviridae family Defining features -5’ end has NO cap, polyA tail at 3’ end -no envelope, stable, icosahedral symmetry -oral-fecal transmission -gRNA is the only mRNA, translated into polyprotein (precursor) -IRES=internal ribosome entry site (for translation) -VP1 to VP3 on surface, VP4 hidden under -beta barrel jelly roll conserved -causes poliomyelitis, common cold, HepA, heart infections, diabetes, foot and mouth disease Genome structure and expression strategy -(+)ssRNA, VPg (virion protein genome linked) 5’ end -5’ end UTR=very long -middle has a single large ORF encoding one polyprotein as precursor -3’ end UTR and polyA tail Polyprotein and proteolytic processing -single large ORF encodes polyprotein which is cut by proteases into 11-12 functional proteins required for replication -2A pro=cuts once, separating P1 from the rest of the polyprotein -3Cpro=cut at 8 places to produce all final proteins required for replication -sequences encoding structural proteins are located at the 5’, non-structural proteins involved in replication at the 3’ end Polio infection shuts down translation of cellular mRNAs -infection causes shutdown of translation of cellular mRNAs -very late protein translation re-directed for viral polio proteins only -prevention of formation of pre-initiation complex at the cap of host mRNAs Protein synthesis in eukaryotes requires the cap: cap-dependant translation -for almost all eukaryotic mRNAs the pre-initiation complex assembles at the 5’ cap, brings 2 ends of the mRNA together, scans for the first AUG, large ribosome subunit joins, translation starts -proteins involved in translation: elF-4F: the bridging point elF-4G: eukaryotic initiation factor 4G Picornaviruses block the translation of host mRNAs but not its own RNA -inhibits formation of preinitiation complex at the 5’ cap of cellular mRNAs via: 1. 2Apro (polio) or L protease (FMDV) cleaves elF-4G, blocking formation of the pre-initiation complex at the cap 2. Dephosphorylation of 4E-BP1, binds elF-4E, sequestering it But how were PV proteins translated then? -extensive secondary (stem-loop) and tertiary (pseudoknots) structures in 5’ UTR (because UTR is so big) -AUG shortly downstream of IRES -pyrimidine-rich sequence upstream of AUG -initiation complex binds directly at IRES, landing at start codon, initiating translation -no need for cap structure Replicative intermediate (RI) vs replicative form (RF) -RF is in dsDNA, must produce (-)RNA -RI has connective strand to the template, and the RdRp will bind to the 3’ end, can make (+)RNA Poliovirus replication cycle Attachment: Canyon: poliovirus, rhinovirus Surface loop: FMDV Receptors: PVR, ICAM-1 Uncoating: Sphingosine within hydrophobic pocket of pentamer, help VP1 penetrate membrane to form pore. Biosynthesis: genome replication and IRES-based protein synthesis Assembly: -proteolytic cleavage of VP0 is the last step (maturation) renders the virion infectious ↳provirion is not infectious until VP0 cleaved into VP2 and VP4 Maturation: VP0 cleavage into VP2 & VP4 Control of poliomyelitis -inactivated poliovirus vaccine (Salk, 1955) -live attenuated vaccine (Sabin, 1960) ↳live is more effective (replicate effectively) but not used because it can switch back to wt Lecture 8: The family Flaviviridae (yellow fever, Hep C ect) Classification/taxonomy -represents the 3rd supergroup of (+)RNA viruses -phylogeny based on the helicase domain encoded by protein NS3 ↳grouped into 3 clusters based on vector (arthropod/no arthropod) ↳Pestivirus=infects animals, no arthropod ↳Hepacivirus=infects humans, no arthropod ↳Flavivirus=infects both, arthropod vector (replicate and transmit in mosquits, ticks ect) Defining features -have 3 distinct layers: 1. Envelope protein layer (E and M proteins) 2. Lipid bilayer (viral envelope) 3. Nucleocapsid core=icosahedral (T=3=RARE) -(+)ssRNA genomes -they all produce 1 ORF and one polyprotein -3’ UTR, no polyA tail, stem loops and pseudoknots -no cap at 5’ end, use IRES to initiate translation -structural proteins at the 5’ end, non-structural at the 3’ Yellow fever -flu symptoms, fulminant infections, jaundice fatal -attenuated vaccine strain 17-D, isolated via passages in monkeys and primary cell cultures Dengue fever -most prevalent vector-borne viral infection in the world -4 types: DENV-1,-2,-3,-4, vary by 20-40% in E protein -secondary infection with a different serotype is worse -proposed that cross-reactive antibodies assist with secondary infection entry -live attenuated vaccine approved in philippines (Dengvaxia) West Nile -infection of CNS, causing encephalitis, paralysis, death -main transmission between mosquitoes and birds, humans and mammals are dead end carriers Zika virus -causes birth defects Hep C(HCV) -transmission via body fluid, transplants, piercings/needles -discovered through molecular cloning and sequencing -infects hepatocytes and lymphocytes -7 genotypes, infection of 1 does not confer immunity for another -causes liver disease/cirrhosis/liver cancer -antiviral drugs used, no cure (interferon and ribavirin) ↳early responce predicts success or failure -DAA drugs developing to target viral enzymes (protease and RdRp)=sofosbuvir (SOF)=effective The liver and hepatocytes -function in detoxification, break down bilirubin -active in protein synthesis, storage, metabolism -hepatocytes have a lifespan of 6 months Virus replication cycle Attachment: E protein binds one or more receptors Entry: receptor-mediated endocytosis Genome uncoating: low pH dependant membrane fusion with endosome membrane Protein synthesis: polyprotein associates with ER, cleavage into multiple functional proteins RNA synthesis: in small ER-derived vesicles Assembly and release: on the cytoplasmic side of the ER, bud into ER lumen, exit via exocytosis, fusion between transport vesicle with PM releases virions Terminology/Important terms: viron> the complete viral particle (virus particle) Capsid (coat)> the protein shell encasing the viral genome Nucleo-capsid (core)> nucleic acid + protein, referring to the discrete substructure within the virion of viruses with envelopes subunit> a single, folded polypeptide, refers to individual CP Structural unit> the basic unit for building capsid/nucleocapsid, can be single or multiple subunits envelope> lipid membrane enclosing the nucleo-capsid Structural protein> proteins that are part of the virion structure Non-structural protein > a protein encoded by a virus but not part of the virion, enzymes required for viral replication, movement, and infection taxonomy> the systematic grouping of viruses using a set of taxa based on natural and evolutionary relationships between viruses Linnaeus system of classification>3 domain system today is bacteria, archaea, and eukarya (viruses don't fit) Host range> the range of hosts that can be infected by a given virus (natural vs expected host range) Tissue tropism> the preference of a given virus for certain types of cell and tissue in its host Susceptible cells> cells that allow attachment and entry of a give virus due to presence of suitable receptor(s), may/may not support viral replication Permissive cells> cells that permit the replication of a given virus, these cells contain all factors required for viral replication, may/may not be susceptible Arboviruses> viruses of humans and animals that are transmitted by, and replicate in arthropod vectors Zoonotic> transmitted disease from animals to humans (or vice versa) Enzootic> disease that regularly affects animals in one area Fusion peptide> segment of 20-30 hydrophobic AA residues, normally hidden Abbreviations CNS=central nervous system HPV=human papillomavirus TMV=tobacco mosaic virus OC=Occluded virion BV=Budded virion CP=capsid/coat protein T=triangulation number ICTV=international committee o n taxonomy of viruses NJ=Neighbour joining ML=Maximum likelihood MP=Maximum parsimony CPE=cytopathic effects MOI=multiplicity of infection PM=plasma membrane TMD=transmembrane domain GPI=Glycosyl phosphatidylinositol ORF=open reading frame RNP=ribonucleoprotein RdRp=RNA dependent RNA polymerase EM=electron microscopy VRC=viral replication complexes VIGS= virus-induced gene silencing IRES=internal ribosome entry site (for translation) RVR=rapid response EVR=early response eRVR=extended rapid response DAA=direct acting antiviral drugs SOF=sofosbuvir INF=pegylated interferon alpha RBV=ribavirin