Virology Midterm 1 Review: Lecture 1: The Discovery and Nature of Viruses Spontaneous generation of microbial growth disproven upon

n experimentation with closed and open flasks that are sterilized, and possessing a nutrient rich medium. They found that no growth was produced in enclosed flasks whereas open flasks did eventually produce growth. Kochs Postulates, 1. The microbe must be demonstrated in all cases. 2. The microbe must be isolated and propagated in a pure culture medium. 3. The microbe must reproduce symptoms once inoculated into a susceptible individual. Definition of a virus, 1. Submicroscopic-cant see with light microscope. 2. Covers genetic material in a protein coat. 3. Completely intracellular = absolute parasitism. 4. Replicates in the form of their own genetic material, possessing one form of nucleic acid (either RNA or DNA not both) RNA virus will produce a RNA virus and a DNA virus will produce a DNA virus they may be used as intermediates but never will you see a RNA to DNA virus produced from a differing NA. 5. Undergo an infectious phase= phase of replication=hijack hosts machinery. 6. Lack an immune system= no nervous system=cannot respond to external stimuli. 7. Lack a metabolic system- no Lipmanns system = Using enzymes for energy production = cannot produce ATP. 8. Lack motility- exception T4 bateriophage which has a contractile portion for stabilizing and injecting of genetic material into host cell. 9. Alive=replicate their genetic material and can undergo mutation. 10. Infect many organisms= plants, animals, bacteria, etc. Life Cycle 1. Recognition/ Attachment (key and lock theory=why viruses are specific) 2. Penetration-of viral genome into host cell 3. Un-coating- eclipse = just the viral genome and contents no coat=no antigens=cant detect 4. Transcription-to produce translatable portions for protein synthesis 5. Protein Synthesis-proteins that aid in replication and coat production 6. Replication-copying of viral genome 7. Assembly viral genome + protein coat 8. Lysis and release-exits from cell

Lecture 2: Composition of a Virus Part 1 1. 2. 3. 4. 1. Structure briefing, Helical viruses Icosahedral viruses Pleomorphic or amorphic viruses virus encased in a lipid coat known as an envelope. Comes from the membrane of host cell via a process called budding (ex, HIV) -will lead to cell death. Both icosahedral and helical--bacteriophage Viruses consist of, Nucleic acids(RNA or DNA never both)- Note: nucleic acids and proteins are not covalently linked but rather share weak bonds (hydrogen bonds, Vander Waals, ionic bonds, and hydrophobic bonds) thus allowing for un-coating. Increases in bond strength=vaccine. Proteins Lipids, carbohydrates, polyamines Viral Nucleic Acids are composed of -Heterocyclic nitrogenous base, a sugar, and a phosphate. -Glycosidic bond- covalent bond between sugar and base -Phosphodiester bond- covalent bond between sugar and phosphate group -Purines -two ring= A/G -Pyrimidines single ring C/T/U -GtoC= 3 hydrogen bonds -AtoTorU= 2 hydrogen bonds (weaker than GtoC) -RNA contains the nucleotides: A, G, C, and U. -DNA contains the nucleotides: A, G, C, and T. -Bases not a distinguishing factor due to mutation, sugars are, -RNA contains the sugar, ribose, to form ribonucleic acid. -DNA contains the sugar, deoxyribose, to form deoxyribonucleic acid-theorized to have evolved from RNA via the reduction of a hydroxyl group. Nucleic Acid Enzymes, Polymerases (copyases) - synthesizes complementary nucleotide structure. RNA polymerases synthesize RNA as an end product and DNA polymerases synthesize DNA as an end product (codes in the 3 to 5 direction). Does not discriminate between host and viral genetic sequences; machine that can be hijacked. Methylases (add methyl group), ligases (enzymes that can catalyze the joining of molecules by forming a new chemical group, can be RNA or DNA), and terminal transferases (change functional groups of molecules), etc- used in viral modification. Nucleases- are enzymes capable of cleaving phosphodiester bonds between the nucleotide subunits of the sugar phosphate backbone. Two types used to cleave genetic material, -Endonucleases (mid)- cut in the middle of a sequence not at the end- ex, circular NA material. -Exonucleases (end)-cut at the end or edge of NA material-led to the discovery of viroids as they are circular in NA structure and exonucleases are not effective at cleaving or lowering infectivity when put in solution with viroids vice versa for endonucleases.

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Some viruses like that of an mRNA in cells posses a 7-methylguanosine (m7G) cap which is used in ribosomal recognition (as well as AUG methionine codon) and prevents degradation by exonucleases. -All mRNA have a cap -Negative sense RNA viruses do not possess cap because they are originally untranslatable. -Positive sense RNA viruses-majority have cap but not all, some use different mechanisms. -DNA viruses have no cap as they are not translated, must undergo transcription first. Polymerases more detailed, Form complexes with helicase for unwinding of ds NA or jumbled single stranded NA. -Necessary for viral replication, adding nucleotides to 3OH end (3 to 5)of growing strand of DNA or RNA, and sequence is determined by following a template strand. -RNA polymerases do not require a primer. -DNA polymerases do require a primer which is a small segment of DNA/RNA hybridized to a template, allowing for replication. -Most but not all viruses code for their polymerases. Classes of Polymerases, 1. RNA dependent RNA polymerases- convert RNA into RNA, and are encoded by RNA viruses for their replication. 2. DNA dependent RNA polymerases-converts DNA into RNA, production of mRNA from DNA template. 3. RNA dependent DNA polymerases- convert RNA into DNA, example of a reverse transcriptase / retrovirus family. 4. DNA dependent DNA polymerase- converts DNA into DNA, and is encoded by most DNA viruses for their replication.

Lecture 3: Composition of a Virus Part 2 General description of Proteins. -Formed by long chains of amino acids, and are coded by nucleic acids. -Viral proteins can be structural (ex, capsid or envelope proteins) or nonstructural (enzymatic). -Example of a nonstructural enzyme would be an antigen. Antigens contain an epitope/antigenic determinant=part of an antigen that the immune system recognizes via antibodies, beta cells, and T cells. Some viral proteins packaged, majority are not. Levels of protein structure, 1. Primary structure- consists of very strong covalent bonds (structure is stable). Example=peptide bond. 2. Secondary structure- made of many primary structures formed via weak bonds; structures are denoted as alpha helices and beta sheets. 3. Tertiary structure- made of many secondary structures, weak bonds (exception disulfide bonds), begin to form a 3D structure. 4. Quaternary Structure- made by combining tertiary structures, weak bonds; structures are denoted as icosahedral or helical for viruses. Some functions of viral proteins, -Form viral coat. -Used in replication. -Used as receptors to bind to the surface of a host cell. -Viruses are good antigens=elicit the production of antibodies -In plant viruses capsid proteins may also be involved in replication and transportation (movement/transport proteins). Movement proteins (coded by all plant viruses) enlarge plasmodesmata between cells allowing for direct cell-to-cell movement of genetic material. -They may also play a regulatory role in the life cycle and symptoms development in plants. -Other enzymes include polymerases, movement proteins (plant viruses only), integrins, and some will form with sugars to produce glycoprotein. Glycoprotein, -Highly antigenic. -Some involved in protein folding. -Some have enzymatic activity. -Some protect virus against degradation by nucleases, and proteases. -In orthomyxoviruses and paromyxoviruses glycoproteins are used in viral attachment, entry and exit=essential for infectivity. Glycosylation of a viral protein produces a glycoprotein-introduction of a carbohydrate moiety to a protein. Occurs in ER and Golgi apparatus. N-linked glycosylation occurs in AA asparagine and O-linked glycosylation occurs in AAs serine, tyrosine, and threonine. Viruses do not make lipids as lipid synthesis is complicated; therefore, viral lipids originate from host cell. Also, some viruses contain polyamines or metals which neutralize negative charge of NA.

Lecture 4: Isolation and Purification of Viruses Three techniques used in the isolation of viral particles. These include centrifugation, precipitation, and extraction techniques. Viruses are large enough, i.e. larger than proteins, that size can be used to purify them. First obtain a sample containing cells infected with your virus of interest. Then you break the tissue to extract virus (homogenization), ex blender/freeze it. Now separation techniques can be applied to the diffuse material to extract virus. Techniques defined, -Centrifugation- Solution is spun down separating based on weight. Heavier cell debris will sink to the bottom faster than lighter molecules (DNA, viruses, proteins, etc). The supernatant (the upper most layers) will contain the virus and other materials with a similar mass. -Precipitation- Virus has a specific charge. If we know the isoionic charge (pH when charges are neutral) of the virus, it will precipitate out of the solution. -Extraction techniques involve the manipulation of viral properties such as antigens, and how they react to antibodies. Centrifugation, -F=M*g*angular velocity*r also F=W/g*angular velocity*2*r -If you increase mass (molecular weight) or radius you increase force. -Velocity is not relevant -M=RTS/D (1-Vp) -The molecular weight (M) of a particle is a constant, as well as its sedimentation coefficient(S) -S = the coefficient that determines how far the virus will sink which is also proportional to its weight. - The bigger the virus the faster it will sink, ex, virus at 180 S will sink faster than a virus at 80 S. - S depends solely on M not dependent on the speed. Also M of virus is constant. - Ribosomes are similar in size to some viruses. We can dissociate ribosomes using active substance or enzymes (ex, EDTA) that cleave them into 50S and 30S subunits. We then centrifuge again and the virus, now clearly heavier, will spin to the bottom. Removing supernatant will isolate virus. Differential Centrifugation-Alternate between low and high speeds to separate. At low speeds we eliminate denatured proteins and insoluble material. At high speeds (75000g-100000g) we will isolate the virus. Virus can be dissolved in solution to eliminate impurities. Density-Gradient Centrifugation- Two kinds rate zonal and isopycnic (equilibrium) -Virus particles are separated from other components based upon their sedimentation coefficient (rate zonal) or their densities (isopycnic or equilibrium). -Rate-zonal centrifugation=each layer contains decreasing concentration of sucrose (agar). Density is provided by the gradient (separated by weight). Centrifuge for a given time to separate virus. Virus will be located with a layer that corresponds to their S. - Isopycnic or equilibrium centrifugation= density is provided by the virus and other materials. Called a true density gradient; use dissociated cesium chloride (cesium is a heavy element) in solution, which once centrifuged, will form a gradient due to physical properties once in

solution. Denser objects move to areas that have a similar density in solution which is lower in the test tube, and vice versa (separated by density).

Lecture 5: Assaying and Titrating Viruses Infectivity Assay=Depends on ability of virus to multiply in host. Infectious Unit (UI) = Minimum amount of virus capable of producing a reaction/infection. In principle a single virus should be able to infect a host. Also for a virus to be infectious to an organism it must be able to infect that organism. Multiplicity of Infection (MOI) =ratio of input virus to host cell. Two types of symptoms, systemic and necrotic (local lesions). -Systemic means wide spread. -Necrotic means a local lesion/area of dead plant cells. This can be used to quantify infectivity. Local lesion Assay=Can only be done with plants, and involves a monolayer of plant cells that is inoculated with diluted virus. Every lesion will correspond to a single virus. Small # of lesions implies a low infectivity and a large number of lesions imply a high infectivity. 1 virus 1 lesion (low infectivity) 1 virus 10 lesions (high infectivity). Plaque Assay=imitate plant assay by creating a monolayer of animal or bacterial cells. Dilute virus which will aid in finding the minimum number of viruses necessary to produce a response and then inoculate, and cover with a nutrient rich medium (agar) and incubate to allow for virus to attach and replicate. Spread of virus is restricted to neighbouring cells by the gel. More plaques indicate a higher infectivity. End point titration = used because some animal and plant viruses do not produce plaques or foci. The highest dilution of a virus which if delivered to a plant or animal will result in a positive observable effect. A dilution that results in 50% positive and 50% negative results is called the infectivity dose or lethal dose 50 (commonly used for prions). Immunological Assay= Use antigenic response to measure infectivity. Viral lipids are not antigenic viral, viral ssRNA and dsRNA are mildly antigenic, viral proteins are antigenic, and viral glycoproteins are strongly antigenic. Antigen is foreign to organisms body. Antibodies produced by immune system bind to antigens neutralizing the virus by preventing it from binding to host cell, while also acting as a signal for WBCs for digestion. If the body makes enough antibodies immunity results and virus is destroyed or becomes dormant. If there is an insufficient production of antibodies then the organism undergoes a chronic infection=incapable of clearing virus. The Complementation Fixation Test, -If you find an antibody in serum you can deduce that an infection occurred, but not when. No virus implies immunity or vaccination. -Complementation fixation test definitions to note, 1. Antigen=foreign protein body 2. Antibody=a protein that binds to an antigen neutralizing it and signaling immune system for digestion. 3. Complement= protein (globulin) present in serum and is found in the formation of antigen-antibody (Ag-Ab) complex i.e. complement binds to Ag-Ab complex. 4. Serum=(blood) that lacks RBC and clotting factors. Mostly containing proteins, electrolytes, hormones, and most importantly antibodies, and antigens.

-Step by step for complementation fixation test, 1. Serum isolated from patient. 2. Since patient serum already contains complement, therefore to negate effects this may have on the test we heat the serum (56 degrees Celsius)destroying the complement but not the antibodies, and then we must replace them with a known amount of standardized protein complement (which will help us determine antibody concentration and its presence). 3. To test for presence of antibody/immunity we add the antigen of interest to the serum. 4. Now we add sheep RBC which have been pre-bound to antibodies to form a complex that will bind to any free complement in solution. The test is negative if the solution turns pink implying that the added antigen does not have a complimentary antibody and thusly does not form an Ag-Ab-complement complex so the free compliment binds to the sheep RBC-Ab (anti-sRBC) complex causing it to lyse turning the solution pink. On the other hand the test is positive if the solution contains a small pool of RBC implying that the added antigen did form an Ag-Ab-complement complex and all free complement proteins are bound. ELISA=enzyme-linked immunosorbent assay=Use antibodies and colour change to detect the presence of an antibody. -Indirect ELISA=Apply a known antigen of known concentration to a surface (typically a microtiter plate). The antigen will be immobile and fixed in position after which you can wash excess Ag off. Blood sample is added, and AG-Ab complex is formed and remains attached even after a wash. Then an anti-antibody (binds to antibody) is added which are conjugated to a substrate-specific enzyme, which is once again preceded by a washing to remove excess. Finally a substrate is added that allows the conjugate to elicit a chromogenic, fluorogenic, or electrochemical signal which can be seen and measured via a spectrophotometer, spectrofluorometer, or electrochemical device, etc. -Sandwich ELISA opposite to indirect as antibodies are fixated to wells and antigenic level/concentrations are measured. Ab-Ag-primary Ab-secondary Ab with conjugate then visual testing can be done to quantify presence of antibody. Viral Neutralization=Different antibodies are tested against a virus and when the infectivity of a virus is lost upon interaction with a specific body it becomes neutralized. Immunofluoresence=if you want to see the virus during infection you can mark an antibody with an immunofluorescent tag placed in the medium and observe it interaction with the host cell. Radioimmunoassay= radioactively labeled antigen is added to a medium with antibody. Then the serum that may or may not contain a similar antigen is added. Upon mixing the two will compete for the antibody if the labeled wins test is negative for viral antigen presence and vice versa. Radial Immunodiffusion Test= Center well contains the serum with unknown antigen(s) and is surrounded by other wells containing different known antibodies all of which are submerged in an agar medium. Antigen is allowed to diffuse and upon finding a complimentary antibody, a precipitate is formed indicating the composition of viruses in serum.

Haemagglutination (HA Test) = both infectious and damaged viral particles can cause agglutination making the test not one that can detect infectivity; rather it is one that is used to detect viral concentration. Some viruses contain spike like projection proteins that bind to RBC specifically (ex being influenza) causing agglutination. Haemagglutination inhibition (HI Test) = A known antibody is added to the serum prior to addition of RBC. No agglutination implies the existence of complimentary antigen, and agglutination implies that the antigen is still unidentified. RFLP western=protein, northern=DNA, southern=RNA. PCR amplify known sequence to determine nucleotide composition this can be done on RNA sequences by first applying a reverse transcriptase to the medium to create a DNA template and then amplify that sequence using PCR, this is called, RT PCR.

Lecture 6: Virus Architecture and Assembly 1. Helical Viruses The nucleic acid of most viruses is protected from external environment via the use of capsid or coast proteins. Comprised of repeating protein subunits called capsomeres. Virus can have one or many types of capsid proteins, even more complex ones have a lipid coat. Icosahedral= spherical or more elongated (like a sausage). Helical=rod like (strong bonds) or flexible (weak bonds). Subunit Interactions in viruses, -Subunits are stabilized by the maximum number of bonds between them, leading to the lowest free energy state. -Subunit-subunit and subunit-NA bonds are weak bonds. -Not all viruses are fully equivalent in nature (ex icosahedral with T>1) and possess uneven ends this is known as quasi-equivalent or nearly equivalent formation (can also be seen at the ends of helical viruses like TMV). Architecture of TMV, -forms a helical array of identical protein subunits, within which RNA molecules are wound within as a helix. -Comprised of protein subunits linked together to form a disc. Many discs are stacked together to form the helical virus. -17 subunits form a disc. -Height is 4.9 subunits per 3 turns of the helix. -16 1/3 subunits per one turn (remaining 2/3s is placed above the plane of the other subunits). -Approximately 2100 subunits make up the entire virus particle. -RNA is embedded in between subunits. No NA embedded in the center or canal of the virus. -95% protein and 5% NA Assembly of the TMV, -TMV polymerization favoured high temperature. Therefore, the process is endothermic, or entropy driven. Second Law of Thermodynamics all processes must lead in the direction of maximum entropy. Does TMV violate this? Apparently not as the decrease in entropic value during TMV formation is made up by the increase in entropy of the surrounding water molecules which normally structure themselves around individual subunits. -TMV contains the AAs Aspartic and Glutamic acid, normally these two AA forms a dynamic equilibrium at pH 5 whereby we have equal concentrations of COO- and COOH groups. In TMV due to the interactions of other AA this does not happen. Dynamic equilibrium is reached at pH 7 for TMV. At a lower pH like pH5 (COOH=COOH) the protein will aggregate forming an empty helix lacking viral RNA, but at pH 7 disks configure into a lock washer and form a functional TMV in the presence of RNA. At a high pH like pH 9 (COO-=COO-) and low ionic strength, only small aggregates of protein appear, no disks, and no helix.

TMV Assembly, -Each disk has a sedimentation coefficient of 20 S which are formed from aggregation of PS. -Nucleation of the TMV is caused by an interaction with the hairpin loop RNA that travels through the canal placing the 5 end down on the top of the newly forming helix and the 3 end on the bottom of the helix. RNA induces disk to turn into a lock washer configuration. -20 S disks translocate to form a lock washer (can only happen at pH 7) aiding in the formation of overlap between each disk closing the disk and trapping the RNA in between disks. -Remember carboxyl groups prevent the assembly from taking place in the absence of RNA. In disks, carboxyl groups are switched off, and in helices, (lock washer) in presence of RNA, they are switched on (polarity changed in such a way to allow for the formation of a virus). Every subunit has two Asp and Glu AA. -Canal is initially used in the beginning to seed RNA. The 5 end is placed on disks faster than the 3 end of the helix. The 5 end follows elongation of helix away from 3 end. -Elongation of TMV happens distal to the end of the protruding RNA. Assembly of viruses other than TMV, -Main difference lies within the direction of elongation. In those viruses initiation is always at the 5 end of the RNA. Elongation is always polar 5 to 3 direction. TMV is rigid, but some other plant viruses are flexuous rods (consist of weaker bonds).

Lecture 7: Architecture and Assembly of Viruses 2. Icosahedral Viruses Viruses originally thought to be spherical later found to be icosahedral. An icosahedron is a geometric shape made of 20 equilateral triangles. Triangulation Number: Number of sub-triangles found in each of the 20 faces. Triangulation Number (T) gives the number of sub triangles (structural units) in each of the 20 equilateral faces of virion. The structural unit appears under an electron microscope as a cluster of pentamers or hexamers. There are always 12 pentamers in an icosahedron. The number of hexamers is 10(T-1). If T=1 we have 12 pentamers and no hexamers=simplest icosahedron. Icosahedrons have 2/3/5 fold symmetry. A minimum of 60 identical units in equivalent positions are required to construct an icosahedral virus. In its simplest form we have 20 faces and with 3 subunits each making a total of 60 subunits. The structural subunits appear under the electron microscope as either a cluster of pentamers (group of 5) or hexamers (group of 6). Larger subunits can be made by either increasing the number of subunits or by increasing the size of the basic subunits. Since most viral proteins range between 15,000 and 70,000 Daltons, it becomes necessary to increase the number of PS to allow them to exist in equivalent or quasiequivalent positions. T = number of subunit / 60 # of hexamers = # of subunits 60 /6 # of pentamers = 12 ALWAYS Examples, 1. Canine parvovirus (T=1)-12 pentamers and no hexamers. Total number of subunits: 60 20 faces each containing 3 subunits. Tx60=1x60=60. 2. CCMV (Cow Chlorotic Mosaic Virus) (T=3) 12 pentamers + 10(T-1) =20 hexamers. Total number of subunits: Tx60=3x60=180. Concept of Equivalent and Quasi-equivalent, -When the PS forming the virus are in identical positions (surrounded by the same subunit) they are considered equivalent=icosahedron with only pentamers. -In contrast, when they for pentamers and hexamers they are considered quasi-equivalent. -Any icosahedron that does not have T=1 is not equivalent, and therefore is quasi-equivalent. Cannot make a helical virus from icosahedral subunits, due to the maximum number of possible interactions, most stable structure, and lowest free energy. When NA is inside virus it is interacting with the PS, increasing entropy and its stability. When PS interacts with each other they share the maximum # of bonding/ interaction which means this is the most stable structure for PS & lowest free energy state, example polio.

Self-Assembly of Icosahedral Viruses, -The instruction for self-assembly of all simple viruses reside in their protein structure units which aggregate to form minimum free-energy structures (pH 5 icosahedral are typically stable). -Note: TMV not stable at pH 5. -At pH 5 most icosahedral viruses are stable (normal virus) and 88S. When you increase to pH 7 (more COO- groups or like charged groups, therefore more repulsion between PS) the virus becomes swollen and 78S. You can then add NaCl at pH 7 to solution which will dissociate the CP into PS, while also releasing the NA (force holding virus is stabilized by ionic interaction). We can then add Mg2+ which will neutralize uneven charges and stabilize via ionic bonding the structure of the virus now measuring at 85S; this can however jump between 78 and 85 S states as Mg2+ is not stable in itself (both intermediates exist). Now by increasing the acidity of the solution we begin to add more protons into the environment which posses a higher affinity to the viral structure dissociating or precipitating out the magnesium from solution until it once again reaches stability at pH5 and 88S. Models of Self-Assembly of Icosahedral Viruses, Three models, 1. Empty Shell (capsid) model: Capsid is first formed then filled with RNA. Most likely model. 2. Mould Model: RNA might exist in form and shape as it exists inside virus particle, around the RNA the protein forms the capsid. Not as likely as CP and NA are found separately in host cells. 3. Initiation Complex Model: Few protein subunits + RNA initiation complex. Around this complex, the rest of the capsid is formed. Initiation could be very specific. Implies that NA and CP are found together, therefore it is the least likely model. Complex Virus Structure and Assembly, 1. Brick shaped and Pox Viruses-particles are oblong and brick shaped. Examples rhabdoviruses, vesicular stomatitis virus, and vaccinia virus. 2. Large, enveloped, spheroidal, and elongated viruses- Contain membranes, lipids, and glycoproteins. Exit by budding. Nucleocapsid can be helical (e.g. Measles, influenza) or icosahedral (herpes simplex). 3. Encapsulated Viruses: Contain inclusion bodies a crystalline form of protein (viral origin but different from the capsule protein of viral capsid). Insect viruses eg. Autographa californica nucleopolyhedrovirus. Bacteriophage structure and assembly, -Attachment-Entry of phage DNA and degradation of host DNA-Synthesis of viral genome and protein-Assembly-Release. Note some bacteriophages have no tails, and some are filamentous.

Lecture 8: Viruses with Divided Genomes 1.Two Component System: Tobraviruses -Several viruses carry more than one RNA molecule in their genomes (divided genomes). Some viral RNA species are present in two or more separate virus particles; others are encapsidated in one virus particle. -Tobravirus or Tobacco Rattle Virus (TRV) is a bipartite plant virus. Each particle contains a single-stranded plus-sense RNA molecule of positive polarity called RNA1 and RNA2 respectively. -Eukaryotic Ribosomes only translate one gene at a time hence subgenomic formation. In TMV the RNA-1 is monocistronic (mRNA that codes for only one protein) and therefore relies on the production of subgenomic (Sg) RNA. http://www.dias.kvl.dk/Plantvirology/evirusgenes/evirsubgenomic.html Genomic Organization of TRV and subgenomic RNAs, -RNA-1: alone can replicate in plants (in absence of RNA-2) but no virus particle is produced (defective infection: only free viral segments are present). RNA-1 contains genes such as a RNA replicase (RNA dependent RNA polymerase)/methyl transferase/helicase, movement protein, and one responsible for seed transmission. Also contains a termination sequence. - (+)strand RNA is read by incoming ribosome and begins translation, but only the first gene will be translated as the ribosome stops translating once it reaches the stop codon. In order to code for the remaining proteins the RNA virus will form a (-) strand RNA. The (-) strand RNA possessing a new promoter coding for the second gene can allow for the production of a new protein. -RNA-2: codes for the capsule protein, does not replicate on its own. -Note: Polio translates all of its proteins (polycistronic) at once (termination codon only present at end of RNA) making a polyprotein. This polyprotein must be cleaved by proteases to produce functional proteins. TRV stable vs unstable. -Stable (higher plaque density) =both RNAs present, formation of infectious viral particles. -Unstable (lower plaque density) =only naked long strand is present no short strand. Degraded by ribonucleases, but infectivity can be restarted upon the addition of phenol which denatures ribonucleases, allowing only the long naked RNA to become infectious (localized not systemic). 2.Three/Four Component System A.Bromoviruses, -Bromovirus= is tripartite, and displays icosahedral symmetry. -BMV is a + sense single stranded RNA, containing RNA 1(replicase), 2(replicase), 3(movement), 4. RNA 1, 2 and 3 or the three largest RNA components are required for infectivity. RNA 4 is a subsequence of RNA 3. Three icosahedrons containing NA, and RNAs 3 and 4 occur together in an intermediate density particle. RNA 4 is inactive coding for a CP, but RNA 3 has a silent CP gene which is later turned into SgRNA4 which can code for CPs.

-Note: adding RNA4 from one bromovirus, strain to RNAs 1, 2 and 3 of another strain does NOT affect the protein of the strain. This confirms the genetic inactivity of RNA4. -In a rate zonal density gradient test we observe one band (homogeneity). In an isopycnic (true) density gradient test we observe (heterogeneity) three bands. -RNA1to3 are required for infectivity. RNA 3 contains a CP gene that is untranslatable. 3.Three / Four Component System: B. Alfalfa Mosaic Virus - As seen with BMV, the 4 RNAs are encapsidated in one type of capsid. These are called isocapsidic viruses (encapsulated in one type of capsid. In the case of AMV, the four different RNAs are encapsidated in four different capsids heterocapsidic viruses. AMV is a multipartite virus. Capsids are all made up of one type of protein, but are serologically the same. The only difference is in the size and shape of the particles which consists of 3 bacilliform or rod shaped capsids and one spheroidal capsid. The virus consists of 4 different parts. Bottom (B), middle (M), top (Tb), and top (Ta). All capsids of equal width, but varying length. Bottom, Middle, Tb required for replication, and Ta required for production of capsule protein. -Both helical and icosahedral in symmetries. Capsule binding protein (CPB) located on RNA 1 required for infectivity. - RNA 1, 2 and 3 are not infectious, but with the addition of RNA 4 which codes for a CP the virus becomes infectious=CP is necessary for infectivity. A mixture of the 3 larger RNAs becomes infectious after incubation with viral particles. - AMV CP enhances the translational efficiency of viral RNA by binding to the CP binding site (CPB). Multipartite Divided Genomes: Advantages and Disadvantages -Advantage: more recombination events, more heterogeneity and more chances to undergo mutation, and acquire new properties and more opportunities for infection. -Disadvantage: several particles are required for one infection to be successful. This does not apply for single partite genome (e.g. Orthomyxoviruses and Reoviruses). Single Partite (Particle) Divided Genome Viruses, -Divided Genome with several components placed in one capsid (e.g. Reoviruses, Orthomyxoviruses, Bunyaviruses, etc) packaged in a single viral particle. Reovirus, -Double stranded RNA virus with divided genome (10-12 RNAs encapsidated within a single virus particle). -Reoviruses have icosahedral symmetry and a multiple layered capsid (inner and outer capsid). -RdRp located inside core of virus particles. -10 to 12 pieces of double stranded RNA. - + sense RNA is capped and - sense RNA has PPG at the 5 end. -dsRNA is inactive as mRNA; it has to be transcribed first into mRNA (from minus strand). Orthomyxovirus (e.g. Influenza Virus) -Family of RNA viruses with divided genome -Negative Stranded -6-8 (-) RNAs pieces are encapsidated into a single virion.

Lecture 9: Viral Expression Strategies Typical composition of a eukaryotic messenger RNA contains a 5 cap, a start codon, proteincoding segment, stop codon, polyadenylation signal, and a Poly-A tail. While a (+) sense RNAs contain a 5cap, start codon, protein-coding segment, stop codon, and ends with a tRNA cloverleaf. Polioviruses which are also (+) sense RNA viruses have a VPg (viral protein genomelinked) is a protein that is covalently attached to the 5 end of positive strand viral RNA and acts as a primer during RNA synthesis, preceded by a IRES (Internal Ribosomal Entry Site, followed by a stop codon, multiple protein-coding sequences, and a termination codon. Viral RNA-dependent RNA polymerase (RdRp) Replication -Remember all polymerases transcribe in a 3 to 5 direction producing a nascent RNA starting with the 5 end and finishing with the 3 end. Note: Ribosomes translate in the 5 to 3 direction. Protein expression strategies of (+) sense RNA viruses 1. Proteolytic processing of polyprotein- one large polyprotein cleaved by a protease to produce functional proteins 2. Multipartite Genome-separate translatable portions. 3. Subgenomic RNAs- produce an intermediate translatable segment called subgenomic RNA. 4. Translational readthrough- ribosome reads past stop codon. 5. Translational frameshift- ribosome switches nucleotide to prevent the reading of a stop codon. Poliovirus Internal Ribosome Entry Site (IRES)=hairpin strip, detected by ribosome without a cap. Also contains the polyprotein that codes for the capsid along with the protease that cleaves the polyprotein into functional proteins (also destroys 5 cap of host cell mRNA to increase its propensity to be translated by host ribosome), and a RdRp Satellite Viruses, -Some viruses are incapable of performing certain functions essential for their replication so they rely on helper viruses (normal virus) to aid in their replication. These viruses are called satellites. Examples include Tobacco Necrosis virus (TNV) and its satellite (STNV) Adeno-associated virus (AAV), a satellite of adenovirus, Tobacco Mosaic virus and its satellite (STMV), Hepatitis Delta Virus (HDV: satellite to Hepatitis B Virus HBV). General Properties of Tobacco Necrosis Virus and its satellite STNV, -TNV is an icosahedral virus with a satellite virus STNV whose CPs are completely unrelated. STNV can be considered a completely defective virus. TNV is infectious on its own. STNV is not infectious on its own. -STNV codes for its own capsid protein, uses TNV polymerase in replication. -Interactions are highly specific; therefore STNV can only be activated (supported) by TNV machinery. Interference between TNV and STNV -TNV multiplies most efficient alone. -Progressive addition of the STNV results in a decrease in TNV reproduction.

-TNV replicase works for both RNAs one sided complementation. Satellite is parasitic to the helper virus. Satellite Tobacco Mosaic Virus (STMV) -STMV is a small, icosahedral plant virus which worsens the symptoms related to TMV. T=1 and it consists of a single protein and has a single stranded RNA genome which codes for the capsid and one other protein of unknown function. Hepatitis D Virus: A satellite which utilizes Hepatitis B Virus as its helper virus. Definition of a Satellite Virus, -Satellite virus lack the genetic information necessary to code for their own reproduction therefore they rely on the molecular machinery of a helper virus. -Both satellite and helper viruses for the most part code for their own coat proteins, with the exception of hepatitis D satellite which only makes a delta antigen stealing the rest of its required envelope proteins from HBV. - The satellite-helper system is stable. Both viruses multiply in a joint infection. Satellite RNAs and DNAs -Satellite RNAs in contrast to satellite viruses are small RNA molecules which are encapsidated within the coat protein of their helper virus. Example CARNA5, a RNA satellite found in Cucumber Mosaic Virus (CMV). -Virusoids -Satellite DNAs CARNA 5 -found in CMV, contains four RNA molecules, and can have a positive, negative or neutral affect on the infectivity of CMV. Virusoids, -Special type of RNA satellite; they are very small, covalently closed circular (CCC) RNA molecules with a high degree of secondary structure. They are always associated with a helper virus for their replication using the helper virus RdRp. They also get packaged in the helper virus capsid protein and replicate through the rolling circle model. Virusoids also contain RNA self-splicing Ribozymes. Example, Satellite of Lucerne Transient Streak Virus (SLTSV). SLTSV contains mismatched regions that aid in preventing degradation from host cell ribonucleases which recognize double stranded regions and cut them, this is called RNA silencing. - A ribozyme (ribonucleic acid enzyme) is an RNA molecule that is capable of performing specific biochemical reactions (self-cutting), similar to the action of protein enzymes. Also termed catalytic RNA, ribozymes function within the ribosome (as part of the large subunit ribosomal RNA) to link amino acids during protein synthesis, and in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme and the hairpin ribozyme. - The hammerhead ribozyme is a RNA module that catalyzes reversible cleavage and joining reactions at a specific site within an RNA molecule (requires some Mg2+ for catalyzing reactions). Example is the SLTSV which can self splice.

Satellite DNA viruses, -Geminiviruses are plant viruses which have single-stranded circular DNA genomes encoding genes that diverge in both directions from a virion strand origin of replication (i.e. geminivirus genomes are ambisense-containing both positive and negative sense segments). It is the largest known family of single stranded DNA viruses. Begomoviruses (subset of the Gemini family): contain a circular satellite DNA called Beta B satellite Replicate. Some circular sat DNA do not code for proteins.

Lecture 10: RNA virus replication Stages of a Viral Infection -1. Absorption and Entry 2. Eclipse 3. Maturation and Assembly 4. Movement, storage, and release (exit) in some cases. 7. Exit Virus Replication Cycle: -1. Binding to cell receptor (specific). 2. Entry and uncoating (eclipse). 3. Early gene expression. 4. Replication of viral genome. 5. Late gene expression (capsule protein coded late to prevent immediate encapsulation). 6. Assembly of virions. -Note: DNA viruses must enter the nucleus (polymerases located there) in order to replicate themselves and must also send an RNA sequence to the cytoplasm for processing and the production of proteins. RNA viruses can remain in the cytoplasm and are not required to enter the nucleus. -Note: Plant viruses dont have receptors. All animal viruses have receptors. The Eclipse (Lag) Phase, -Following the entry of the viral genome into a host cell, there is a period during which infectious virus cannot be recovered from that cell, known as the eclipse or lag period. In the period, most of the virus components are synthesized de novo (from the beginning). -Similar types of genomes usually replicate in a similar way irrespective of which type of host organism they infect. Examples are that of the ssRNA viruses that replicate similarly in plants, animals, and bacteria. - Eclipse period is preceded by the absorption phase whereby the Virus is added and binds to the host receptor. The eclipse period begins when the virus uncoats and ends when the virus has assembled/exits from the host cell. Absorption and Entry of Plant Viruses, -Due to the fact that plant cells have a thick cell wall, it is necessary to wound the cell in order to allow for infectivity. Plant viruses use insects, physical damage, pinocytosis (cell using endocytosis to absorb extracellular fluid into the cell, virus tags along). Entry into cell after pinocytic stage = eclipse stage. -Coat proteins may play a role in the control of host range as they can be specific. Example is BMV coated with TMV CP did not produce infectious virions. - Plasmodesmata create gaps that connect plant cells directly (cytoplasm to cytoplasm). -Reminder all plant viruses code for a movement protein to allow for the NA to travel from cell to cell. Some experimental evidence for the uncoating of TMV: (a) Virus when uncoated becomes sensitive to UV light. (b) Plant cell with a coated TMV will take longer than a naked RNA molecule to replicate, and mature. Time difference corresponds to the time it takes for TMV to uncoat. (c) RNase sensitive RNA in cell appears after infection. Absortion and Entry of Animal viruses, is dependent recognition of specific cell RECEPTORS. Virus Entry and Receptors, -A variety of surface proteins can serve as specific virus receptors or attachment factors.

Reminder : envelope of animal viruses is not coded by virus. Viral matrix proteins are structural proteins linking the viral envelope with the virus core. They are found in many enveloped viruses including paramyxoviruses, orthomyxoviruses, herpesviruses, retroviruses and other groups. In influenza viruses we have receptor-mediated endocytosis (pH dependent). Proton pump increases H+ concentration causing the environment to acidify and uncoating proceeds. Penetration through cell membranes mediated by fusion proteins that undergo major conformational change leading to fusion. These proteins are activated upon either a low pH or receptor binding. Entry of influenza virus: Influenza virus has envelope proteins (fusion proteins) that bind to the cell membrane of the host cell triggering fusion and entry of the capsid and its contents. Influenza virus being a DNA virus must enter the nucleus of the cell where DdRp are located which can transcribe the viral genome into mRNA sequences. Influenza mRNA segments require 7mg caps which is supplied by the cell. Host cell mRNAs are virtually decapitated and their caps stolen by viral mRNA, eventually leading to cell death. Once viral proteins and genomes are made in a sufficient quantity the virus can now assemble and exit. Replication of TMV (life cycle): TMV enters cell via movement protein/insect/erosion of cell wall. TMV replicates in the cytoplasm (Note: this is not done by all RNA viruses =RT). TMV is a positive sense ssRNA virus and after entry into the cell it begins uncoating and because it is a positive sense RNA virus it is immediately translatable and the first proteins are manufactured (replicase enzymes or RdRp). Intermediate negative sense RNA strands are transcribed; this is known as the replicative form (RF). Because eukaryotic ribosomes are monocistronic (due to the positioning of the start and stop codons only one protein is produced) the RF contains highly active promoters that code for further segments of the positive sense RNA strand but with start codons that can code for the remaining proteins (capsule proteins), this positive sense segment is called a subgenomic RNA segment. This creates intermediates that are both double and single stranded and positive and negative in structure, this is called the replicative intermediate or RI. Once created in sufficient quantities the fully formed viral genomes and the PS bind and form viral progeny, this is finished with the release of the newly formed viruses out of the cell. Note: Ribozyme aids in both the uncoating and translational processing. Note: RNA polymerase in plant and bacterial cells are hijacked by RNA viruses and converted to RdRp for their own use. Note: synthesis of the RF is 3 to 5 but synthesis of the final viral genome is 5 to 3. Replicase has three functions, which include the synthesis of the minus strand over the plus strand (polymerase activity), synthesis of plus strand (progeny) through RF, and finally the synthesis of subgenomic RNAs (mRNAs) from the minus strand. Replicative form (RF) and Replicative Intermediate(RI) - Replicative Form: an intermediate stage in the replication of either DNA or RNA viral genomes that is usually double stranded. -Replicative intermediate: an intermediate in the replication of an RNA virus genome that is partially single stranded and partially double stranded.

-Is genomic RNA transcribed from complementary ssRNA or from dsRNA (composed of parental and complementary RNAs)? If the RNAs are isolated from cells where the viral replication is underway, two types of RNAs have been found: 1. A dsRNA of a specific size is found and called RF or the replicative form. This dsRNA is resistant to RNases. 2. A series of variable size intermediates, ds pieces (resistant to RNases) are called RI or replicative intermediates. The presence of these RI favours the hypothetical mechanism of replication through several intermediary steps. Several polymerase molecules could simultaneously work on the same RNA molecule by displacing each other. Experimental evidence supporting the model of replication of RNA plant viruses, -Actinomycin D prevents transcription, but viral synthesis continues= RNA virus using RdRp. -Infected plant shows polymerase activity=must be producing another strand. -You can isolate RF from AMV, TMV, and TNV= negative strand is being produced during infection. -Polymerase activity and RF presence falls after cessation of viral synthesis. RF is a double stranded segment, RI is a mix between double stranded and single stranded portions. Biochemistry of viral replication, -positive RNA viruses, (ex, TMV/polio) are immediately translated by host ribosome. -negative ssRNA and dsRNA and DNA viruses require a transcriptional step, translation takes place in the host cells cytoplasm, and replication of viral NA may occur in the nucleus, after which viral RNA transcripts travel to the cytoplasm, where translation and virus assembly takes place. Replication of (-) ssRNA viruses -Rhabdo/orthomyxo/paramyxo viruses genome is not infectious. - The viral capsid contains an RdRp necessary for infection. -viral capsid contains an RdRp necessary for infection -is initially untranslatable. -replicase next produces a full sized (+) strand ssRNA that will later be used to produce a full sized (-) strand ssRNA . The polymerase starts at the 3 end of the (+) strand and transcribes all mRNAs, recognizing start and stop codons. These viral mRNAs are capped and a poly-A tail is added. -The mRNA will later code for CP and other enzymes necessary for function and production of new viral progeny. Summary of RNA virus Replication -ss(+)RNA viruses 1. Translation of early gene product from genomic RNA (RdRp). 2. The production of a (-) sense RNA strand from the (+) sense RNA template. Formation of the RF. 3. The production of (+) sense RNA strand produces mRNA, along with subgenomic fragments. 4. Translation of mRNAs, synthesis of late proteins (Cp,MP)

5. Virus Assembly. -ss(-) RNA viruses 1.Synthesis of (+) sense full-length RNA and mRNAs from genomic (-) sense RNA by RdRp derived from viral core. 2. Synthesis of (-) genomic RNAs from (+) sense full length RNA. 4. Translation of mRNAs into viral gene product. 5. Nucleocapsid assembly followed by budding from membrane containing viral envelope proteins. DI Particles -During the replication and assembly of virus some defective interfering particles are produced, called DI particles. They are not infectious on their own and they interfere with normal infectivity. They are not satellites because they share homologous sequences with the virus that has created them. Replication of dsRNA viruses Reoviruses -Note each part of the genome is transcribed into ss mRNA (by a replicase which is a component of the particle core). - Each mRNA is monocistronic (open reading frame which codes for one protein) -The mRNA is also transcribed back into the (-) sense strand by the replicase. -Two kinds of dsRNA virus replication, 1. Conservative Replication - a single stranded (+) sense strand is transcribed from the (-) sense strand of the dsRNA creating an independent single (+) sense strand and the original dsRNA. 2. Semiconservative Replication - the (+) sense strand located on the dsRNA is replaced by a newly formed (+) sense RNA strand using the (-) strand as a template. Replicative strategy of dsRNA viruses. 1. Transcription by viral RdRp within viral core, export of (+) sense RNA to cytoplasm. 2. Translation of (+) sense RNA, accumulation of viral proteins (capped transcripts). 3. Assembly of (+) sense RNA and proteins into immature virions. 4. (-) sense RNA synthesis by viral RdRp to produce dsRNA in virions. 5. Secondary transcription of dsRNA. 6. Finally assembly/maturation of virions.