BIOC20 Lec 1-6 (Lecture Notes) PDF

Lecture 1: Key concepts 1. The nature of viruses 2. Viral morphology 3. Viral genomes 4. Viral classifica6on 5. RNA viruses 6. Why study viruses? 7. History of virology Sec6on 1: - Viruses need to get into the host cell to survive due to the fact that they don’t have itself metabolism machines, which...

Lecture 1: Key concepts 1. The nature of viruses 2. Viral morphology 3. Viral genomes 4. Viral classifica6on 5. RNA viruses 6. Why study viruses? 7. History of virology Sec6on 1: - Viruses need to get into the host cell to survive due to the fact that they don’t have itself metabolism machines, which means they cannot survive without relying on other species’ metabolic machines Ø Synthesis of basic biological building blocks, e.g. nucleo6des, amino acids, carbohydrates, lipids, etc. Ø Genera6on of usable chemical energy (ATP) Ø Protein synthesis machinery - Viruses infect all forms of life - Gene6c material is either DNA or RNA, which encode for proteins (e.g. replica6on, expression, packaging genome) - Virion: A complete virus par6cle outside of the host cell Ø Consists of a nucleic acid genome, a protec6ve coat (capsid) and some contain a lipid envelope - Viruses are consider the simplest and smallest forms of life - One virus can infect mul6ple hosts but not mul6ple species - Once the virus inside the host cell, virus par6cles disintegrate/break down (uncoa<ng)> releases gene6c material inside the cell> synthesis of mRNAs and viral proteins which then direct replica6on of viral genome using host machinery. Ø Viral structural proteins encapsidate the newly replicated genome? New progeny virus par6cles Sec6on 2: - Viruses come in different shapes and sizes Sec6on 3: - Viral genome can be either RNA or DNA (not both) and can be: Ø Single stranded, double stranded, ad circular Sec6on 4: - Chrema6s6cs used to determine viruses: types of genome, symmetry of capsid, presence or absence of envelope, and dimension - Genome data is used to understand evolu6onary rela6onships and assign viruses to specific family. - “+” strand> encodes for a protein. - “-“ strand> complementary strand Sec6on 5: - Only known form of life that can have RNA genomes Two main issues of having a RNA genome: Ø 1) mRNA must be synthesized from a RNA template Ø 2) RNA genome must be replicated - Most RNA viruses encode their own RNA dependent RNA polymerase - Retroviruses have RNA genome which gets converted to DNA by the host cell using reverse transcriptase (cDNA copy of RNA) encoded by viral genome Sec6on 6: - Viruses can infect all forms of life and are important disease-causing agents in humans - Viruses are the most abundant biological en66es. Since it is so small, we cannot see them with naked eyes but there are around 200 millions - Study viruses helped us discovered that Ø DNA carries inherited gene6c informa6on Ø Iden6fica6on of promoters for eukaryo6c RNA polymerase Ø Enzymes involved in cellular DNA replica6on Ø RNA splicing in eukaryo6c cells Ø Isola6on of numerous cellular oncogenes and the understanding that cancer is caused by their muta6on or unregulated expression - Viruses play a cri6cal role in various ecosystems, disease, and are an excellent tool for understanding the biology of cellular processes. - Hershey and Chase experiment: Discovered that non-radioac6ve DNA came from replica6on and 2) DNA is the carrier of inheritance informa6on. Sec6on 7 - Viruses were dis6nguished from other microorganisms by filtra6on Ø Scien6st found that agent causing mosaic disease could pass through filters made of find earth or porcelain (bacteria did not pass through)> a fillable infec6ous agent >> Tobacco mosaic disease caused by Tobacco Mosaic Virus> first studies to show that there were infec6ous agents smaller than bacterial. - Ques6on: Are viruses alive? Inanimate when genomes are packaged in virions, ability to mutate and evolve, ability to reproduce once inside the host cell - Bacterriphages are viruses that infect bacteria - Study of bacteriophages by scien6sts lead to an understanding of the basic processes of life: Ø Bacteriophages show heritable traits> must contain and express genes like other organisms Ø Small, simple and easy to grow> can be used as a model system >> Isola6on and analysis of phage genomes >>gene6c mapping of phage and bacterial genes using gene6c crosses >>Developing of plaque assays Lecture 2 Key concepts 1. Detec6on and measurement of viruses 2. Viral replica6on cycle 3. Bal6more classifica6on of viruses 4. Virus structures Sec6on 1: - Plaque assay allows for the quan6ta6on of viruses Ø Bacterial growth can be measured with a spectrophotometer> intact bacteria diffract visible light> dense bacterial cultures look cloudy Ø Bacteriophages lyse their host cell> lysis causes a loss of diffrac6on, leading to a clearing of the bacterial culture Ø Can also be done on solid bacterial culture - Upon infec6on, phage binds to bacterial cells, replicate and releases progeny phage par6cles> taken up by surrounding cells and the cycle repeats Ø Repeated cycles lead to lysing of cells in area surrounding the ini6al infec6on> observed as a clear area (plaque) against uninfected cells> reported as plaque forming units (allows to count the number of infec6ous virus par6cles in a suspension) - Plaques are generally visualized by staining the cells (dead cells do not stain well)> plaques appear clear - We doing dilu6ons because it is easier for us to count the number of viruses. - Red blood cells are commony used for assaying iruses Ø Visible due to colour Ø Can be isolated and stored easily Ø Have carbohydrate containing receptors on their surface> a number of animal viruses bind to such molecules Ø Hemagglu<na<on Assay: binding of excess of virus with red blood cells result in ‘agglu6na6on’ (clumping) >> Sensi6ve to condi6ons (pH, temperature, buffer composi6on, etc.), some viruses only cause agglu6na6on in par6cular mammalian or avian species - Not all par<cles are infec<ous. There is a lot more par<cles than infec<ous par<cles Ø Disrupted or defec6ve virions (disrupted envelopes), empty capsids (no gene6c materials), defec6ve genomes (muta6ons, dele6ons), cellular an6-viral defenses (can interfere with virus replica6on), latent infec6ons (not all viruses undergo reproduc6ve cycles, gene6c material stays in host un6l condi6ons are right) - Electron microscopy can be use to see virus par6cles. Ø Viruses do not take up the stain> observed as light image against a dark background (nega6ve staining) - MOI: Mul6plicity of Infec6on: Virus replica6on cycles are studied by infec6ng 10^3-10^6 cells to get enough amount of viral gene6c material and proteins. Ø All cells must be infected at the same 6me to synchronize the events of replica6on >>Infect with excess of virus to ensure that each cell receives at least one infec6ous par6cle cell. However, steps in replica6on cycle can overlap> difficult to study Sec6on 2: - Ini6al drop in infec6ous 6ter due to: 1) defec6ve par6cles, 2) Uncoa6ng inside the host cell If the DNA goes up, the RNA goes up There is a lag because when the virus uncoated, they are not infec6ous un6l the genome get replicate. - Analysis of viral macromolecules can reveal the detailed pathways of virus replica6on - Virus replica6on cycles vary greatly depending on host cell, type of viral genome, complexity of virus, etc. 1) AIachment of Viral Adsorp<on: Recogni6on and binding to a host cell Ø Some viruses bind to surface proteins only present on certain cell types> limit virus tropism Ø Non-specific primary receptor, followed by a more specifc secondary receptor 2) Entry into the host cell: Either the virus par6cle (virion) or its gene6c material Ø Bacteriophages ‘drill’ holes in cell walls and membranes to inject their gene6c material Ø Enveloped viruses fuse their lipid envelope with the host plasma membrane> releases capsid or gene6c material inside the cell Ø Endocytosis? Releases the virion or genome in the cytoplasm 3) Genome replica<on and gene expression: Viral genome is copied, genes expressed> make viral proteins Ø Replica6on can be in nucleus or cytoplasm, depending on the virus/genome type Ø Proteins can be structural or non-structural Ø ‘Early’ proteins enable viral genome replica6on Ø All RNA viruses must encode RNA-dependent RNA polymerase (except retroviruses)> not present in host cells. Early proteins combined with host proteins form RNA replica6on complexes Ø Early proteins of DNA viruses induce synthesis of cellular enzymes involved in DNA replica6on> generally by ac6va6ng a signalling cascade Ø ‘Late’ mRNAs are made from newly replicated genomes. ‘Late’ viral proteins package viral genomes and assemble virions >> Structural proteins are usually the most abundant (can be single or mul6ple, depending on complexity of the virus >> Some viruses produce scaffolding proteins (involved in assembly but not part of the mature virion) 4) Assembly and morphogenesis: Genome copies+ viral proteins> genome enclosed by capsid proteins (for enveloped viruses, host membrane is used for envelopes) 5) Release/Exit: Complete virus par6cle exis6ng the cell and infec6ng other cells Sec6on 3: - Developed by David Bal6more> all viruses can be divided into seven groups Sec6on 4: - Structures of virions have evolved such that RNA or DNA genome can be transmiked efficiently - Virion must: assemble, exit the cell, survive/withstand extra-cellular environment, akach and enter another cell, and release its genome into the host - Capsid: a rigid, symmetrical container for viral genome> generally made with viral proteins - Nucleocapsid: capsid with enclosed viral genome - - - - Envelope: Lipid bilayer membrane surrounding capsids/nucleocapsids Viruses composed of many copies of iden6cal subunits (gene<c economy). Iden6cal subunits give the capsid symmetry. And they can assemble spontaneously (selfassembly) Ø Gene<c economy: Principle that organism with a small/limited size genome use their protein-coding capacity economically Ø Avoid exhaus6ng the coding capacity of a small viral genome Iden6cal subunits can have one of three kinds of symmetry: 1) Tetrahedral 2) Cubic 3) Icosahedra: the most common one Capsids with helical symmetry are organized as helical tubes composed of iden6cal, repea6ng subunits> extends along a single helix axis Ø Helical nucleocapsids can accommodate a number of genome lengths> the longer the genome, the longer the helical nucleocapsid. Especially advantageous for viruses with variable genome lengths Ø The symmetry of helical capsids is defined by two parameters: 1) the number of subunits per turn, 2) the displacement along the helical axis between one capsid subunit and the next (p) For nega<ve strand RNA viruses, the genome winds along a groove that follows a helical path of protein subunits Ø Each protein subunit binds a fixed number of nucleo6de Lecture 3 Key concepts 1. Virus envelopes and proteins 2. Packaging of genomes 3. Virus envelopes and budding 4. Virion Disassembly 5. Virus classifica6on 6. Major virus groups 7. Satellite viruses, nucleic acids and viriods Sec6on 1: - Viral envelopes composed of: Ø Lipid bilayers that have a similar protein composi6on as the cellular membrane from which they were derived Ø Viral glycoproteins embedded in the lipid bilayer Ø Envelopes can adpt a variety of shapes> not always symmetrical - Budding: the process of developing viral envelopes at the cell membrane Ø Can be internal membranes (e.g. endoplasmic re6culum) or surface membrane (plasma membrane) Ø Viral glycoproteins are inserted into the cell membrane - - - Ø Nucleocapsid is wrapped in a membrane into which viral glycoproteins have been inserted >> Some nucleocapsids interact directly with cytoplasmic tails of envelope proteins (viral spike) during budding >> Some viruses use a Matrix (M) protein to interact with both viral spikes (HA and NA) and nucleocapsids (helical RNP-ribonucleoprotein) The lipid content of the lipid bilayer reflects the composi6on of the membrane it was derived from Ø E.g. influenza virus buds from cell surface and has cholesterol and phospholipid in propor6ons that are similar to the plasma membrane Ø By analyzing the lipids, we can figure out where it budded from. Aside from lipid analyses, we can also do electron analysis. Glycoproteins (spikes) are inserted into the lipid membrane to form the nature viral envelope. Ø Characteris6cs of envelope glycoproteins: 1) large glycosylated external domain (ectodomain), 2) hydrophobic transmembrane anchor domain, 3) short internal (cytoplasmic) tail Envelopes proteins are synthesized on ribosomes in the ER> inserted in plasma membrane via standard export pathways for cell surface proteins. 1) Type I integral membrane proteins: N-terminus facing lumen of ER or extracellular space 2) Type 2 integral membrane protein: Opposite orienta6on to type I- C-terminal facing lumen or extracellular space, N-terminal near the anchor, faces cytosol 3) Signal sequence one he envelop protein directs membrane inser6on. >> Type I: Cleaved N-terminal signal sequence by a pep6dase when inserted in the ER during synthesis >>Type II: use the transmembrane anchor as the signal sequence >> if the proteins going through post-transla6onal modifica6on (e.g. glycosyla6on), it would occurs in ER/Golgi - HA (hemagglu<nin from influenza virus: Ø 1) Ectodomain> binds to cell receptors and mediates fusion between viral envelope and cell membrane Ø Transmembrane anchor domain> alpha helices that spans the 3nm thick hydrophobic part of lipid membrane Ø Tail faces the cytoplasm before a virus buds off - Glycosyla6on process prevents dehydra6on of the external surfaces of virus par6cles and reduces protein-protein interac6ons to prevent viral aggrega6on Sec6on 2: - Mul6ple modes of capsid assembly exist depending on, 1) size, shape, and complexity of capsid, 2) genomic composi6on Ø Some viruses, capsids assemble around genome and in some, genome is inserted into a preformed shell Ø May require scaffolding proteins >> Assist with forma<on of procapsid: forerunner par6cle that co-assembles with capsid proteins >> does not included in final, mature virion Concatemer: repeated sequences of DNA form together to form a single strand. Purpose is to fill in the empty space in the genome - Enzymes know when to cut of DNA when they encounter certain sequences and when the amount of DNA is enough in the genome. - - Packaging signals direct the specificity for incorpora6on of viral genomes into virions Ø Nucleo6de sequence of DNA or RNA viral genome that gets recognized by packaging proteins Ø Packaging sequence on viral genome interact with capsid proteins - Core proteins: pro6ens associated with the DNA viral genome inside the capsid (nucleocapsid complex) Ø Neutralize the “-ve” charges on DNA (phosphate groups) Ø Involved in condensing the viral DNA for op6mal packaging> can use cellular and/or viral histones> resembles chroma6n Sec6on 3: - Interac6ons between viral proteins drives the forma6on of viral envelopes by budding - Viral envelope proteins inserted into the cell membrane form localized aggregates> most cellular proteins are excluded Ø Proteins can move around in fluid membrane Ø Envelope proteins can have side-by-side interac6ons or can interact throough their cytoplasmic rails to form clusters. But it doesn’t mean it cannot interact with other proteins - Budding involves membrane curvature> wraps around the nucleocapsid Ø Budding can be driven by different mechanisms (but generally is driven by interac6ons between envelope glycoproteins, matrix or nucleocapsids drives budding) >> nucleocapsids can assemble in cytoplasm and approach membrane via bidning to cytoplasmic tails of envelope proteins> progressive forma6on of contacts between envelope protein and capsid. - Budding can be driven by different mechanisms: Ø Many use internal Matrix proteins: encoded by viruses and present just underneath the envelope> forms connec6ng bridges between envelope and nucleocapsid Ø Retroviruses, assembly of nucleocapsids directly at the membrane during budding> precursor proteins (gag protein) is cleaved to generate matrix and nucleocapsid proteins >> Some retroviruses can me ‘bald’ par6cles. Layer of ‘gag’ proteins (group-specific an6gen) on inner plasma membrane surface interact strongly with lipid bilayer> drives budding Ø Some viruses can generate empty envelopes (without nucleocapsid)> budding driven by envelope glycoproteins Ø Many viruses require cell proteins for the final pinch off. E.g. ESCRT protein Sec6on 4: - Virions are primed to enter cells and release their genomes. The release of the viral genome can occur by mul6ple mechanisms: Ø Proteoly6c cleavage of capsid proteins (mul6ple triggers/mechanisms)(e.g. selfcleavage, pH dependent cleavage, membrane fusion)> most common Ø Unspooling of genome into the cell Ø Interac6on of genome with cytoplasmic components (structural transi6ons) - Virions are energe6cally metastable (intermediate state of stability)> can easily dissociate with the right trigger e.g. binding to a receptor/ protein on cell surface or cytoplasm Ø Assembly and disassembly are not a simple reversal of the processes. Sec6on 5: - Taxonomy: The science of classifica6on Ø Virus classifica6on is based on 1) molecular architecture, 2) gene6c relatedness, 3) host organism Ø Viruses are grouped into species, genera, and families - Molecular criteria used to classify viruses: 1) type of nucleic acid genome (DNA or RNA), 2) Strandedness of nucleic acids (single or double stranded), 3) Topology of nucleic acids (linear, circular, fragmented), 4) Capsid symmetry (icosahedral, helical, none), 4) Presence or absence of an envelope - Comparing genomic sequences and amino acid sequences for viral proteins can help determine gene6c relatedness Ø Other features: order of genes, mechanisms of encoding mRNA, use of reverse transcriptase, etc. - Virus adapt to their host organisms and evolve with them Ø Viruses that are gene6cally and evolu6onarily related tend to infect related organisms (excep6ons exist) >> virus taxonomy takes into account viruses infec6ng different categories of organisms - Viruses hosts can be divided into 6 categories of organisms: 1) bacteria, archaea, lower eukaryotes (fungi, protozoa, algae), plants, invertebrates, vertebrates (including humans) - Virus species: share a high degree of nucleic acid homology, similar amino acid sequence and an6genemic proper6es. Ø Infect limited organisms, or specific target cells/6ssues (trophism) Ø Common gene6c lineage - Species are grouped into genera using share characteris6cs such as: 1) Genome organiza6on and size 2) Structure of the virion 3) Replica6on strategies 4) Related by evolu6on byt may have divergences in nucleo6de and amino acid sequences - - 5) Infect different organisms or cells/6ssues within an organism Genera are grouped into virus families: share overall.general genome organiza6on, virions structure and replica6on mechanisms (presumed to the evolu6onarily related) Ø Can vary greatly in virion size and genome length Ø May have unique genes not present in other family members. Ø Could have evolved separately, with limited homology of nucleo6de or amino acid sequences Ø ICTV recognizes at number of virus families Bacteriophages: include name of the host bacterial genus and an arbitrary number e.g. Bacillus phage SP01 Plant viruses: named aner host plant species which the virus was first isolated and disease symptoms caused by virus e.g. tobacco mosaic virus Insect viruses: include La6n name of the insect species from which the virus was isolated followed by virus genus name Vertebrate viruses: a number of different conven6ons: 1) host species of origin, 2) loca6on of isola6on, 3) disease caused Sec6on 6: - Major groups of viruses leads to understanding of shared characterisitcs and replica6on pathway Ø Virus with ssDNA genomes tend to be small and have few genes with ssDNA being suscep6ble to degrada6on >> they don’t have an envelope Ø Virus with dsDNA genomes include some of the largest know viruses. Most viruses with dsDNA genomes have segmented genome and capsids with icosahedral symmetry (mostly infect fungi) >> larger have envelopes Ø Viruses with a reverse transcriptase (RT) step in their replica6on cycle can have RNA or DNA genomes >> RT enzyme is packaged in the virion>> these enzymes are group separately because they have an RT enzyme to make a DNA copy of RNA in their replica6on cycle >>’-‘ RNA and dsRNA also package RNA-dependent RNA polymerase in their virion Ø Most “=” RNA genomes that less than 10kb does not have envelopes, except for COVID. Ø All ‘-‘ strand viruses that infect vertebrates are enveloped. May have a segmented or unsegmented genome Ø dsRNA have fragmented genomes and capsids with icosahedral symmetry >> unique mechanism of transcrip6on where capsids act as 6ny intracellular machines> capsid/subviral par6cle remains intact intracellularly while mRNAs are transcribed from each individual segment> using viral RNA polymerase packaged in the virus par6cle along with the genome> newly synthesized mRNAs extruded into cytoplasm >>>Capsid provides a structure to posi6on RNA polymerase with each segment and allows for direc6onal transfer of mRNAs to cytoplasm >>dsRNA tends to have broad host range because they have their own RNA replica6on machinery> less dependent on specific cell environment Sec6on 7: - Satellite viruses and satellite acids require a helper virus to replicate> need simultaneous infec6ons Ø Satellite viruses only encode their own capsid proteins - Satellite nucleic acids replicate only in the presence of a helper virus and either encode no proteins, or encode only non-capsid proteins. Ø Their genomes are encapsidated by the helper virus capsid - Viriods do not encode for proteins, but replicate independent of other viruses> reprogram cellular RNA polymerase to copy its genome RNA> virus-like single circular RNAs Ø Can replicate and can cause disease Ø Do not code for proteins, not encapsidated Lecture 4 Key concetps 1. Evolu6onary origin of viruses 2. Virus entry 2.1. General steps 2.2. Enveloped vs Non-enveloped 2.3. Cell-to-cell transmission 2.4. Akachment 2.5. Internaliza6on Sec6on 1: - Viruses must have evolved alongside host cells> need host to replicate - - Viroids and RNA viruses may have originated in the RNA world 1) catalyze some steps in replica6on without the host protein, 2) depend on RNA polymerase> either from host or helper viruses The transi<on to the DNA-based world: self-replica6ng RNA molecules> produc6on of deoxyribonucleo6des from ribonucleo6de> make DNA copies of RNA (first reverse transcriptase) Ø DNA is much more stable than RNA Ø Small and medium-sized DNA viruses could have arisen as independently replica6ng gene6c elements in cells >> Limited number of viral genes that encode for: 1) structural proteins, 2) proteins that s6mulate DNA replica6on enzymes in host, 3) proteins that recognize and bind to viral DNA and assemble cellular replica6on machinery Ø Large DNA viruses could have evolved from cellular forms that become obligatory intracellular parasites >> likely, genes that overlapped with cellular genes were lost due to similar func6on(s) Sec6on 2: - Mission of virus are to transport the viral genome from an infected cell to uninfected cell, organism to organism Ø Have to ‘evade’ host cell defence barriers Ø Evade host immune system - Viruses of bacteria, acrchae, algaea and plants use different entry mechanisms than animal viruses - Bacteriophages may puncture through cell wall - Plant viruses may enter through mechanical means then spread through plasmodesmata Seciton 2.1: Four major steps: 1) akachment, 2) entry, 3) transport, 4) uncoa6ng. Sec6on 2.2.: - Enveloped and non-enveloped viruses have dis6nct penetra6on strategies - Enveloped viruses can enter by: 1) fusion and fusion of the envelope with the plasma membrane; 2) receptor-mediated endocytosis followed by fusion/fission with an endosome Non-enveloped viruses do not use fusion for entry> form channels or rupture membrane Ø Passage of genome through a channel in membranes (e.g. surface or endosomal membrane)> confirma6on change aner receptor binding or internaliza6on> form membrane channels to release the genome Ø Rupture/lysis endosomal membrane Sec6on 2.3 - Entry of enveloped viruses Ø Fusion: Fusion can occur at two main lova6ons (cargo=nucleocapsid, envelope= transport vehicle) 1) Fusion of envelope with cell membrane, followed by delivery of nucleocapsid into the cytosol 2) Fusion of envelope with membranes of early or late endosomes aner receptor mediated endocytosis - Entry of non-enveloped viruses Ø Non-enveloped viruses do not have a lipid membrane> need to use other entry or exit mechanisms 1) Receptor mediated endocytosis followed by rupture of endosomal membrane 2) Passage of viral genome through a channel in the membrane - Cell-to-cell Transmission: Not all viruses need the release of virions from the infected cell to infect neighbouring cells. Many viruses can be transmiked cell to cell using a number of mechanisms: 1) cell-cell contact via ac6n based extensions called filopodia; 2) Virus assembly and transmission at virological synapses- small region of close contact between neighbouring cells that allows passage of enveloped viruses from one cell to another> no exposure to extracellular environment; 3) forma6on of syncy<a (singular syncy6um)- large mul6nucleated cells resul6ng from fusion of infected and uninfected cells; 4) Intracellular channels such as plasmodesmata>> forma6on of tubules> intact virion (b) OR modifica6on of plasmodesmata> non-encapsidated form (c) 1. 2. 3. 4. Sec6on 2.4 - Akachment of virus par6cle to the host cell is the first step for virus entry. - A variety of cell surface molecules can serve as specific virus akachment factors and/or receptors e.g. proteins, lipids, carbohydrates Ø Serve a normal func6on in cell> usually high conserved, unlikely to mutate dras6cally Ø Presence or absence of such molecules determines host tropism - - - AIachment factor(s)/Adhesion receptors: cell surface component(s) involved in binding of virion to cell but not uptake (primary receptors) Ø E.g. carbohydrates moie6es on glycoproteins Entry receptors: play an ac6ve role in conforma6onal changes, cell signalling endocytosis, etc. (secondary or co-receptor) Host cell-receptors interact with surface components of a virion: viral glycoproteins, surface protrusions, surface depression (“canyons”) Ø Recepetors and virus interac<on leads to changes in the virus and cell which facilitates entry via variety of mechanisms: 1) exposing fusion proteins on virus; 2) New binding sites revealed on virus and receptors; 3) Ini6a6ng endocytosis (signaling cascades in cells) Specific cellular receptors can akach to mul6ple features on virion surface e.g. primary and secondary receptors Ø Affinity of individual interac6ons can be fairly low but akachment can be excep6onal 6ght due to mul6ple interac6on with the receptors. Receptor-mediated endocytosis: viruses enter the cell Ø Carries nucleocapsid into cytosolic vacuoles/vesicles and then enter the cytosol Ø May occur at clathrin coated pits, caveolae or lipid rabs> traps virus at plasma membrane, invagina6on Ø Virus then delivered to early endosomes> late endosomes> cytoplasm through fusion, lysis, or permeabiliza6on - Virus entry for non-enveloped: Non-enveloped viruses transfer their gene6c material form endosome to cytoplasm through permeabiliza6on of endosomal membrane or lysis> rupture of endosomal membrane Ø Nucleocapsid can escape directly or only the genome is released into the cytoplasm - Macropinocytosis: transient ruffling of plasma membrane and internaliza6on fluid, solutes, membranes and small par6cles akached to plasma membrane> generally nonspecific Ø Macropinosomes: large vacoules Ø Viruses can trigger macropinosytosis by exploi6ng cell signalling. - Passage from endosomes to the cytosol triggered by a low pH Ø Low pH signals to the virus it has reached intracellular compartments> leads to conforma6on changes Lecture 5 Key concepts 1. Membrane fusion 2. Membrane lysis and pore forma6on (non-enveloped) 3. Intracellular transport 4. Nuclear import 5. Preven6ng virus entry 6. “+” sense RNA viruses Sec6on 1: - Membrane fusion is mediated by specific viral fusion proteins, fusogens. It is generally type I transmembrane proteins. Ø Due to the conforma6on changes, energy were presence and fusion occurred, which mean it does not need ATP/metabolic energy. Ø Fusion proteins are synthesized, folded and assembled in ER> must undergo matura6on >>1) Cleaved while transi6ng ER-Golgi to plasma membrane by cellular proteases >>2) Cleaved by host cell proteases once bound to target cell >>3) Conforma6onal changes that reveal a fusion pep<de- short hydrophobic region within a viral envelope protein> gets inserted into target cellular membrane during virus induced membrane fusion. >>Fusion proteins play a cri<cal role in viral infec<on> can be therapeu<c targets - - Class I fusion proteins: mostly alpha-helical, tend to form trimers. Have two dis6nct conforma6ons: 1) perpendicular to the envelope surface; 2) hairpin conforma6on aner fusion Class II fusion proteins: Mostly beta-sheets, tend to form dimers. Fusion involves rearrangement of mul6mers of protein subunits but only minor conforma6onal changes at ter6ary level. Rearrangement aner a triggering event (e.g. pH change) before final trimer Is formed - Class III fusion proteins: Combina6on of alpha-helices and beta-sheets. Generally consist of two subunits: fusion subunit and receptor binding subunit. Func6onality is similar to Class I and II fusogens Sec6on 2 - Non-enveloped viruses penetrate by membrane lysis or pore forma6on Ø Major conforma6onal changes in response to receptor binding or low pH in endosomes Ø Viral genomes may pass through the pores in host membranes Ø Non-enveloped viruses can also penetrate without any lysis or rupture of host membranes Ø Can be pH independent but s6ll dependent on endocytosis Ø Binding to receptor triggers a conforma6onal change> loosens up the capsid wall Ø Hydrophobic proteins in capsid can also create membrane channels followed by genome egress Sec6on 3 - For many viruses, transpor6ng to cytosol is not enough, it needs to find a specific site for replica6on 1) Viruses take advantage of cellular transport systems normally used for moving membrane vesicles and other large macromolecules> efficient transport 2) Some virions/capsids are transported along microtubules using molecular motors (e.g. dynein) (microtubule motor) and dynac6n (adapter complex) - Sec6on 4: - Many viruses (par6cullary DNA viruses) use the nucleus as site of replica6on because it is where the cellular machinery for transcrip6on is (RNA processing, RNA export, DNA replica6on) Ø Viruses can also establish latency in the nucleus, or integrate their genome into the host genome, however, it needs to pass through the nuclear membrane, which is very hard. Piggyback on machinery (cell u6lizes for protein and nucleic acid trafficking) is a strategy for them to use to pass through the membrane >>E.g. interact with nuclear targe6ng receptors such as impor6ns (aka karyopherins)> interact with nuclear localiza6on signals in proteins> imported through nuclear pore complex (NPC) - There are other ways to enter the nucleus: 1. Dissocia6on of the nuclear membrane during mitosis- must wait un6l cell divides (most retroviruses)> infec6on only possible in cells that undergo mitosis (usually non-dividing cells are not the target) 2. Par6al disassembly in cytoplasm and entry through nuclear pore via impor6ns (a.b) 3. Disassembly at the nuclear pore for largest capsids (c,d) 4. Transport of intact virions through the nuclear pore (small capsids,e,f,g) Sec6on 5: - Every step in the virus replica6on cycle can be viewed as a target for an6viral drugs> can be specific (narrow spectrum) or generic (broad spectrum) - - Intercept virus before it reaches cell with neutralizing an6bodies. However, there are other ways to prevent virus entry 1) Flood extracellular space with targets for virions to bind> prevents binding of the virion to receptors on cell surface 2) Use molecules (e.g. proteins) that bind to the receptor> occupy receptor sites or induce receptor internaliza6on 3) Interfere with cellular processes need for internaliza6on/penetra6on (e.g. preven6ng endosome acidifica6on by using lysosomotropic agents (weakly basic compounds), carboxylic ionophores (bind to monovalent ions and increase their membrane permeability) or specific inhibitors 4) Inhibit membrane fusion using pep6dase designed to bind conforma6onal intermediates of viral fusion proteins 5) Inhibit uncoa6ng of capsids: >>Using amantadine> inhibits ac6vity of proton channel (M2) of influenza A virus> no acidifica6on of virus interior >>WIN compounds> stabilize capsid by binding to a pocket under receptor-binding canyon in human rhinoviruses Advantages and disadvantages of narrow and broad spectrum an<virals Sec6on 6: - Coronaviruses: Spherical enveloped par6cles studded with trimeric spikes. Cause humans and veterinary diseases. Ø Linear, single-stranded posi6ve sense RNA genome. Ø The order of genes is highly conserved among all coronaviruses Ø Genome ends have a 5’ terminal cap (methylguanosine) and a 3’ poly (A) tail>>>very similar to RNAs. Ø First 2/3rd of the en6re genome> translated into polyprotein(mul<ple proteins in a single transla<on)- cleaved to form mature func6onal protein Ø Final 1/3rd generally encoded for virion structure and non-structural proteins (nsp) - Family: Coronaviridae. Three genera based on genome homologies: Alpha-,beta-, and gammacoronaviruses Lecture 7 Key Points 1. Coronaviruses (+ sense RNA viruses) 1.1. History of infec6on in humans 1.2. COVID-19 6meline and impact 1.3. Genes and proteins 1.4. Entry by fusion 1.5. Synthesis of viral proteins 1.6. Genome replica6on 1.7. Assembly and release Sec6on 1 - A newly emerged coronavirus caused a worldwide epidemic of severe acute respiratory syndrome (SARS) Ø Caused fever and progressive pneumonia which led to hypoxemia, and death Ø SARS coronavirus might have came from bats Sec6on 1.1 - SARS found in bats because they are the most common natural reservoir of CoV and don’t fall ill - Zoonosis transfer from one animal to another Sec6on 1.2 - Coronaviruses have enveloped virions containing helical nucleocapsids (which binds to ‘+’ sense RNA genome) - Spikes important for how the virus infect the cells, how it enter the cells. - Nucleocapsids formed from viral N-protein bound to viral RNA in a helical fashion Ø Most other ‘+’ strand RNA viruses have nucleocapsids with icosahedral symmetry Ø Covid resemble ‘-‘ strand RNA viruses with helical nucleocapsid - Some covid may have a spherical core structure (shell) formed via the M-protein> spherical core with internal helical nucleocapsid - Corona virions contain mul6ple envelope proteins (E) - Corona virions contain mul6ple envelope proteins; spike (S), membrane (M), envelope (E), nucleocapsid (N) and in some case HE (hemagglu6nin esterase) protein Ø Spike protein: Major surface transmembrane glycoprotein protruding from the surface; Responsible for viral entry and tropism; Targeted by neutralizing an6bodies and T-Cells in infec6on> possible vaccine/therapeu6c targe; Generally forms trimers >>Protein synthesized as a single polypep6de chain> cleaved by a cellular proteinase to yield an N-terminal S1 domain (or subunit) and a C-terminal S2 domain (subunit) S1 recognizes specific cellular receptors and ini6ates akachment (Receptor Binding Domain (RBD) is present within S1 for most corona); S2 forms the stalk with a short C-terminal tail, a hydrophobic transmembrane domain and exterior domain of interac6ng alpha-helices >> Spike proteins bind to a variety of cellular receptors: 1) Alphacoronaviruses bind to aminopep<dase-N, a family of zinc binding metalloproteinases (use metal ions in cataly<c mechanism present on cell surface): 1.1 Broadly distributed on epithelial and fibroblast cells in small intes6ne, kidneys, etc. 1.2 Sepcies specific 2) Covs that have HE can bind to sialic acid (9 carbon sugar), found on a variety of glycoproteins and glycolipids 3) Betacoronaviruses use a variety of receptors: 3.1 SARS-CoV binds to metalloproteinase Angiotensin-conver<ng enzyme (ACE2) and an co-receptor, L-SIGN (a Lec6n-carbohydrate binding proteins that recognize specific sugar mo6fs) 3.2 Transmembrane serine protease 2 (TMPRSS2), a cellular serine protease for host-cell entry> cleaves at S2 to ac6vate viral fusion >>In Sar2, spike protein cleavage by TMPRSS2 or cathepsin L (endosomal route) is required 3.3 SARS CoV-2 also binds ACE2 and possible other receptors (this is s6ll unknown) Sec6on 1.4 - Spike protein generally mediates entry via fusion Ø External S1 subunit mediates akachment Ø Stalk subunit S2 (a class I fusion protein) facilitates fusion Ø Series of conforma6onal changes> inser6on of S2 into target cell membrane> brings cell membrane and viral envelope into close contact Ø Some CoV S proteins can also cause forma6on of syncy6a (individual cells fused together) >>In some cases, fusion can be pH dependent Ø Fusion at plasma membrane is also required for egress(departure) from cells Sec6on 1.5 - Aner fusion, inside the cell, synthesis of viral proteins that organize and catalyze viral RNA synthesis - The replicase gene (gene 1) is translated from genomic RNA into a polyprotein that is processed by viral proteinases Ø Gene 1 is composed of ORF1a and ORF1b par6ally overlapping reading frames >>ORF1a translated by ribosomes paused at pseudoknot (secondary structures formed in RNA via hydrogen bonds) and framshin (ribosomal frameshib; ribosome ‘slipping’ by one nucleo<de in 5’(-1 nt) or 3’(+1nt)) aallowing transla6on of ORF1b> ieylds two polyproteins, pp1a and pp1ab (aner frameshin) (pseudoknots) (ribosomal frameshin) Ø Transla6on starts at the start codon (AUG)> progresses 5’-3’ - it tells me that the gene is extremely important RNA helicase and nucleoside triphosphatase ac6vates assist with replica6on and packaging of the viral genome (ORF1b, nsp13) RNA exonuclease (ORF1b, nsp14) has proofreading ac6vity> rare for RNA viruses Various nsps may be involved in various roles/ac6vi6es Sec6on 1.6 - Membrane associa6on of viral RNA synthesis is common among ‘+’ strand RNA viruses of eukaryotes - Replica<on complexes are the site of viral RNA synthesis (RNA factories)> consists of viral and cellular proteins associated with membrane in the cytoplasm of the infected cell Ø Rearrangement of subcellular architecture (cytoplasmic membranes) induced upon CoV infec6on Ø Replica6on complexes commonly observed on double membrane vesicles by EM - Re<culovesicular network- the virus induced membrane altera6ons. DMW= double membrane vesicles, CMs= convoluted membranes; DMSs= double membrane spherules Ø Networks are formed by a combina6on of cell s6muli to produce new membrane and modifica6on of exis6ng membranes by virus proteins Ø Nucleocapsid protein can also be found in abundance at these sites> encapsida6on of newly synthesized genome RNA can also occur at these sites - Genome replica6on proceeds via a full-length nega6ve-strand intermediate (an<genome)> used to direct synthesis of full-lengths ‘+’ strand genome Nega6ve-strand RNAs account for 1-2% of total viral RNA. Mostly are ‘+’ strand. These nega6ve strand are the determina6on of ‘+’ RNA strand in a replica<ve intermediate. Replica<ve intermediate: RNA molecule on which - - one or several growing RNA strands are being synthesized. The growing strand typically forms base-pairing to template RNA only near their growing 3’ end. Aner replicated genes, all the gene downstream on the replicase gene occurs from a series of subgenomic mRNAs (all have iden6cal “untranslated leader” (UTR) sequences at the 5’ end, poly (A) tails, and represent a nested set of mRNAs and all contain overlapping sequences on their 3’ end) Ø UTR is akached to a unique mRNA ‘body’ sequence with one or more ORFs Subgenomic (sg) mRNAs are transcribed from subgenomic nega6ve strand mRNA templates Ø Nega6ve strand mRNA templates are made by discon<nuous transcrip<on 1. Polyermase begins at 3’ end (because that’s where the template is being read) and stops at the end of a transcrip<on-regula<ng sequence (TRS) 2. Polymerase then pauses and disassociates the nascent RNA chain from TRS> jumps to TRS located at the end of the leader sequence (template switching) - - Dissociated nascent RNA chain forms RNA-RNA hybrids of complementary sequences at 5’ TRS Ø RNA pol can pause and dissociate at any of the TRSs Ø Each of the subgenomic ‘-‘ strands are used to make a ‘+’ strand mRNA, then transcript it into proteins This discon6ous model explain recombina6on between viral genoms: 1. Viral RNa polymerase can switch between two different posi6ve-strand genome RNAs if they are both in the same cell 2. Could be two different virus strains infec6ng the same cell, or muta6ons in the virus genome during replica6on 3. Template switching may help with genome repair and/or generate new viral strains/variants >>Aner dissocia6on, polymerase has to find another template or transcrip6on will abort> can lead to template switch Sec6on 1.7 - Assembly of virions takes place at intracellular membrane structures- ERGIC (endoplasmic re<culum Golgi intermediate compartment); involves in transport, processing and modifica6on of proteins. Generally located in the perinuclear (around or near the nucleus) region of the cell 1. Helical nucleocapsids (curved blue lines) containing genome RNA are delievered from site of synthesis to these membranes packaging 2. Virus par6cles are formed by budding into the lumen of these membranes (virions acquire donut-shaped cores) 3. Progress to smaller and more uniformly dense cores as transit through Golgi membrane, envelope proteins also undergo glycosyla6on 4. Secretory vesicles transport virions to cell surface, for fusion with plasma membrane and release. - - M and E proteins play important roles in the forma6on of the virus envelops by budding Ø Enveloped virus-like par6cles can be formed in ERGIC when only M and E are expressed> which indicates that M and E proteins are sufficient in forming the par6cles C-terminal cytoplasmic tail of M is though to interact with packaging signals in N> ensures only full-length viral RNA gets packaged into virons HE (if present) and S are incorporated into the membrane through interac6ons with the M protein As all these proteins transverse through Golgi, envelope proteins are glycosylated, mature virions are packaged into vesicles> targeted to plasma membrane for release