Virology Exam 1 Study Guides

What is VPg?

A protein that acts as a primer for RNA synthesis. It provides a 3' OH group for the replication.

What does it mean that cell is: susceptible, permissive or resistant?

A susceptible cell has a functional receptor for a given virus - the cell may or may not be able to support viral replication. A permissive cell has the capacity to replicate a virus - it may or may not be susceptible. A resistant cell has no receptor - it may or may not be competent to support viral replication. A susceptible AND permissive cell is the only cell the can take up a virus particle and replicate it.

What is the typical structural arrangement of all polymerases? A. Fingers-Palm-Thumb B. Fingers and Beta-sheets C. Helical arrangement D. Tubular arrangement

A. Fingers-Palm-Thumb

Which is a universal rule about RNA directed RNA synthesis? A. RdRp may initiate de novo or require primer B. RNA synthesis initiates randomly on the RNA template C. RNA is synthesized in a 3'-5' direction D. RNA synthesis is always template-directed

A. RdRp may initiate de novo or require primer

Which DNA genome, on entry into the cell, can be immediately copied into mRNA? A. dsDNA B. Gapped dsDNA C. Circular ssDNA D. Linear ssDNA E. All of the above

A. dsDNA

What is the function of genome diversity?

Adapt to change in different environments. Why did viruses create DNA and RNA genomes and have such diverse structural arrangements? We cannot say, but have a hypothesis: The life started based upon RNA, and RNA is the first molecule of primitive viruses. We know this because of remnants: pathogenic RNA molecules that can cause disease called viroids. How do we know that RNA was the first molecule? Because, it would have to be a molecule that could perform two functions: code and have catalytic activity.

What is affinity avidity, and the difference between them?

Binding in viral entry is characterized by affinity and avidity. Affinity is low, which means the strength of the attraction between a receptor and a ligand (glycoprotein of the virus) is low. Avidity is high, which means that the interaction/binding is very tight because there are oftentimes multiple receptors involved in that reaction -Sometimes there is a main receptor, followed by co-receptors that stabilize and dock a viral particle.

36 plaques formed from 1 mL of virus solution diluted 10^-5. What is the PFU/mL? A. 36 B. 360 C. 36 x 10^-5 D. 3.6 x 10^6 E. Idk

D. 3.6 x 10^6

Why is mRNA placed at the center of the Baltimore scheme? A. Because all virus particles contain mRNA B. There is no specific reason C. Because all viral genomes are mRNAs D. Because all viral genomes must make mRNA E. Because Baltimore studied mRNA

D. Because all viral genomes must make mRNA They all actually contain DNA, not mRNA They all must MAKE mRNA

Viral fusion peptides are exposed for insertion into the host cell membrane when: A. The virus particle is near a cell B. The virus particle is in the cytoplasm C. Trimers of the fusion peptides form D. The endosome becomes acidified E. The virus is docked on the nuclear pore

D. The endosome becomes acidified What triggers the conformational change in fusion peptides? Being near the cell is not enough, it needs to bind to receptor. Cytoplasm? Too late, already entered Trimers are already there! They form when the virus is formed (when its released from the cell) Virus docked? Too late, already inside cell

Viral capsids are metastable because: A. They must protect the viral genome outside of the cell B. They must come apart and release the genome into a cell C. They have not obtained a minimum fee energy conformation D. They are spring-loaded E. All of the above

E. All of the above

Viral receptor on the cell surface: A. Can bind directly to icosahedral virus capsid proteins B. Interact with glycoproteins of enveloped viruses C. Can be carbohydrate, protein molecules, or lipids D. Have cellular functions E. All of the above

E. All of the above

Which of the following are characteristics of icosahedral symmetry in viral capsids? A. Produces a solid with 20 faces, each an equilateral triangle B. Allows formation of a closed shell with 60 identical subunits C. Fivefold, threefold, and twofold axes of symmetry D. The T number describes the number of facets per icosahedral face E. All of the above

E. All of the above

What information may be encoded in a viral genome? A. Gene products that catalyze membrane biosynthesis B. Gene products that catalyze energy production C. Complete protein synthesis systems D. Centromeres or telomeres E. Enzymes to replicate the viral genome

E. Enzymes to replicate the viral genome Energy production depends on the host cell Not complete protein synthesis because its still missing ribosomes

When doing a plaque assay, what is the purpose of adding a semi-solid agar overlay of the monolayer of infected cells? A. To stabilize progeny virions B. To ensure that cells remain susceptible and permissive C. To act as a pH indicator D. To keep cells adherent to the plate during incubation E. To restrict viral diffusion after lysis of infected cells

E. To restrict viral diffusion after lysis of infected cells

What is a host range?

Host range is defined as spectrum of host cells the virus can infect. It is determined by: -virus requirements for attachment to host cell (host receptors) -availability within host cell of factors required for replication Broad host range: rabies virus can infect all mammals Narrow host range: human cold virus infects only cells lining upper respiratory tract in humans.

What types of viruses have helical symmetry?

Identical subunits are distributed with helical symmetry for rod-shaped viruses. (Icosahedral symmetry for round viruses.) Animal/human virus with helical nucleocapsid: -Sendai virus - paramyxovirus, related to measles, responsible for a highly transmissible respiratory tract infection in mice, hamsters, guinea pigs, rats, and occasionally pigs and marmosets -Ebola virus - hemorrhagic virus All animal/human viruses with helical nucleocapsid are enveloped Animal virus with helical nucleocapsid: -Vesicular stomatitis virus (VSV, related to rabies) TMV?

What other assays can we use to measure viral infectivity?

Immunochromatographic assay: The sample is placed on an absorbent pad at one end and is drawn across by capillary forces. The antigen in the sample reacts with a specific antibody, which is conjugated to a detector. The antigen-antibody complexes move across the membrane until they are captured by a secondary antibody (visible line). Polymerase Chain Reaction-Molecular Assays: You have specific primers that amplify a specific viral antigen. If the viral infection is there, it generates a product that you can then analyze by gel electrophoresis. If you are looking for another stimulation of a specific oncogene, you would have primers that would prime for that region to see expression/presence of RNA from that oncogene. Deep, high-throughput sequencing: Metagenomics, identification of new viruses in environmental samples, identification of new pathogens, human genome

How can we reconstruct recombinant Influenza virus? Why is it different from Polio reconstruction?

In order to recreate infection of influenza, we use transfection, but we have 8 plasmid construct. Plasmid construct in poliovirus was just a simple one DNA plasmid. We need 8 because influenza has 8 pieces of negative sense RNA, so each plasmid needs to copy or carry information about one of these segments. Those plasmids need to have 2 promoters: 1. Gives rise to negative sense RNA because that is the influenza genome, and 2. Gives rise to positive sense mRNA to produce viral proteins. 8 plasmids because there are 8 segments of viral RNA, and each should have 2 promoters; one for negative sense viral RNA production, and one for positive sense RNA production. Negative sense is just the genome that will be packed into progeny virions, and positive mRNA is used for translation of proteins, which will come together with RNA and build viral particles.

Which version of influenza affects only humans?

Influenza B

What was the first known viral receptor? What kind of molecule is it?

Sialic acid for influenza virus entry Surface carbohydrate

How do plant viruses enter a host cell?

Some viruses can infect plants when aphids and other insects tap into the phloem to feed. Such insect vectors can also pick up virus particles and carry them to new plant hosts. Other viruses infect plant cells through a would site created by leaf-munching insects such as beetles.

What specific RNA structures regulate Polio virus genome replication?

Specific cis-acting motifs are recognized by Polio virus RdRp, and this is how viral polymerases "know" what RNA it should replicate. 1. Cloverleaf: VPg attachment 2. Cre: Cis-acting Replication Element 3. Pseudoknot: Involved in replication, motif bound by RdRp

How is the metastability achieved?

Stable structure: subunits are very stable Repeating subunits is the key Created by symmetrical arrangement of many identical proteins to provide maximal contact Unstable structure: structure is not usually permanently bonded together (non-covalently bonded) Can be taken apart or loosened on infection to release or expose genome

What is cytopathic effect?

Structural changes in host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell, or the cell dies without lysis due to an inability to reproduce. You have symptoms, or effects, and this basically indicates that you are infected. The infectious virus gets inside the cell and does something to its molecular makeup, causing it to react by lysing.

What type of modification VPg must undergo to serve as primer?

Uridynylation pUpU

How is VSV sgRNA and mRNA synthesis regulated?

VSV RNA synthesis is regulated by N concentration. Viral negative sense strand genome is a template for the production of either sgRNAs or full length positive sense strand RNAs. Polymerase is associated with the 3' end of the viral genome. Poly (A) addition (An) is a result of copying a sequence of seven Us present in each intergenic region. The switch from sgRNA synthesis to mRNA synthesis is mediated by the concentration of N protein. -Low N: sgRNA synthesis -High N: mRNA synthesis for packaging of the genome and release

WHAT IS THE MAIN WAY TO PREVENT CHICKENPOX? (HINT, SLIDE 22 SAYS IT FOUR TIMES)

Vaccination

NAME OF THE VIRUS THAT CAUSES CHICKENPOX? • WHICH CAUSES CHICKENPOX AND WHICH CAUSES SHINGLES?

Varicella-zoster virus Shingles: Varicella-zoster virus reactivation

What does it mean that viral genomes are structurally diverse?

Viral DNA or RNA genomes are structurally diverse. -They can have linear, circular, segmented, or gapped structures. -They can be single-stranded positive or negative sense strands, single stranded ambisense, or double-stranded. -They can have covalently attached proteins, cross-linked ends of double stranded DNA, or DNA with covalently attached RNA.

What is a viral envelope? Where does the viral envelope come from?

Viral Envelope: always derived form the host membrane Envelope is a lipid bilayer derived from host cell; viral genome does not encode lipid synthetic machinery Envelope acquired by budding of nucleocapsid through a cellular membrane; can be any cell membrane, but is virus specific -Takes it to additionally protect its genome/content, and trick another cell into thinking that it is just a vesicle Nucleocapsids inside the envelope may have helical or icosahedral symmetry

What is a conformation of RdRp?

Viral RdRps resemble a right hand consisting of palm, fingers, and thumb domains, with the active site located in the palm. It is right-handed.

What is a Baltimore system?

Viral genomes must make mRNA that can be read by host ribosomes. All viruses on the planet follow the rule. mRNA has been put in the center of this classification system. This system classifies viral genomes into 7 groups: 1. dsDNA 2. gapped dsDNA 3. ssDNA 4. dsRNA 5. ss (+) RNA 6. ss (-) RNA 7. ss (+) RNA with DNA intermediate Depending on a combination of their nucleic acid (RNA/DNA), strandedness (single or double), sense, and method of replication.

What specific pathway is crucial for exporting viral nucleoproteins through nuclear pores?

Viral ribonucleic proteins have NLS's that bind to cellular nuclear import machinery to enter the nucleus.

What are cellular, tissue and species tropisms?

Virus entry can be primary determinant of tropism. -Cellular tropism: the virus replicates in one cell type, but not another -Tissue tropism: the virus replicates in a particular tissue or organ, but not another -Species tropism: the virus replicates in one host species but not another

Why viruses need metastability?

Virus particles are metastable - stable but not too stable Two states of viral particles: -Must protect the genome (stable) -Must come apart on infection (unstable)

What is a receptor?

Virus receptor: a host cell surface molecule recognized by the virus as a gateway to entry into the host cell. Receptors are molecules on the cell surface that are there just because the cell uses them for whatever processes it requires -They are not here to support virus entry, they are there for the cell, and the virus just so happens to abuse/use the opportunity of the receptor to enter the cell -The interactions are very specific, and that initial binding to the receptor is followed by penetration and uncoating of the viral particle

What is de novo initiation of RNA synthesis?

de novo: without a primer (from scratch) De novo initiation is a mechanism of RNA synthesis where RdRp recognizes the end itself and there is no need for a primer. A nucleoside triphosphate, referred to as the one-nucleotide primer (NTP), provides the 3'-hydroxyl for the addition of the next nucleotide. Two types: -The 3'-terminal initiation occurs at the 3' end of the viral RNA, and -internal initiation occurs from an internal base. Examples of viruses include HCV and reoviridae.

What viral genome after the cellular entry can be directly transcribed?

dsDNA Only genome that after entering the cell can immediately transcribe from dsDNA to mRNA, and from mRNA it can express proteins. Ex: adenovirus, herpesvirus, papillomavirus, polyomavirus, poxvirus

DESCRIBE THE STRUCTURE OF THE VZ VIRUS.

dsDNA genome Nucleocapsid Tegument Lipid envelope Glycoprotein spikes

What are bacteriophages?

viruses that infect bacteria

Where do the 5' ends of influenza virus RNA come from?

Host cellular mRNA 5' cap structure. Host mRNAs.

What information is never encoded in viral genomes?

-No genes encoding the complete protein synthesis machinery (AARA, elFs, tRNAs) --None encode for ribosomes; without ribosomes, no translation -No genes encoding proteins involved in energy production or membrane biosynthesis -No classical centromeres or telomeres found in standard host chromosomes -Probably we haven't found them yet-90% of giant virus genes are novel

Describe the protein priming mechanisms during Polio virus replication.

-One long ORF, RNA is polyadenylated (3' end) -VPg (RdRp) is covalently attached to the 5' end of the genome -VPg acts as a primer for RNA synthesis after it is uridynylated pUpU -The genome is (+) sense and encodes one large poly protein, which is then cleaved by two proteases not multiple products -VPg is covalently linked to poliovirus genomic RNA via phosphodiester bond -The linkage is cleaved by a cellular enzyme as soon as the genome is released to the cytoplasm -VPg needs two U residues to become a primer -3CD and 3D complex binds to Cre -VPg binds to Cre; loop of Cre is A-rish and templates the uridynylation of VPg -VPg-pUpU is going to be the primer for RNA synthesis Poliovirus uses a protein for priming, called VPg protein. It is ALWAYS attached to its genome at the 5' end. VPg is NOT RdRp, it is just a protein primer that happened to have that 3'OH group that acts as a primer. VPg is a positive sense RNA virus, so it does not package its RdRp. It is attached at the 5' end of the genome, and it is used for its replication. The genome is positive sense so it could be translated into proteins, and those proteins are cleaved into small pieces by proteases. One of them is a polymerase because it is the first protein that needs to be cleaved, and it is needed fro positive sense to go into negative sense and so on. VPg has to be uridynylated in order to be called a primer.

What are the universal rule for RNA-directed RNA synthesis?

-RNA synthesis initiates and terminates at specific sites on the template. -RdRp may initiate synthesis de novo (like cellular DdRp) or require a primer. -Other viral and cell proteins may be required. (spherules) -RNA is synthesized by template-directed stepwise incorporation of NTPs, elongated in 5'-3' direction. -Some non-templated synthesis , i.e. polyadenylation.

Where did viruses come from? (know three hypothesis)

1. Escape Hypothesis: Viruses originated through a progressive process. Mobile, genetic elements, pieces of genetic material capable of moving within a genome, gained the ability to exit one cell and enter another, similar to retrotransposons. 2. Regressive Hypothesis: Existing viruses may have evolved from more complex, possibly free-living organisms that lost genetic information over time, as they adopted a parasitic approach to replication. Mimivirus being similar to parasitic bacteria supports this hypothesis. 3. The Virus-First Hypothesis: The progressive and regressive hypotheses both assume that cells existed before viruses, but what is viruses existed first? Koonin and Martin postulated that viruses existed in a precellular world as self-replicating units. Over time, these units became more organized and more complex. Eventually, enzymes for the synthesis of membranes and cells walls evolved, resulting in the formation of cells

What first experiments lead to virus discovery in plants and humans?

1. Ivanovsky: Filtered juice from crushed tobacco leaves and killed healthy plants. He named it tobacco mosaic disease and said that "poison" caused the disease. 2. Beijerinck: Said that tiny particles in the juice cause the disease, the agent multiplied only in cells that were dividing, and viruses are liquid in nature

What happens during polio virus entry with VP1 and VP4?

1. Receptor binding 2. Conformational change in the particle 3. Viral peptides: VP4 and VP1 form a channel in the cell membrane --VP1 goes outside and fuses itself with the membrane --VP4 is inserted into the membrane forming a channel, facilitating uncoating and release of viral RNA 4. Release of the viral RNA

WHEN WAS THE VIRUS MADE READILY AVAILABLE IN THE U.S.?

1995

What Baltimore Classification does influenza fall under and why?

5 It is a single stranded RNA genome that is negative sense

What is the total number of subunits that build the virus with a T number of 5?

5 x 60 = 300

What percentage of human genome is derived from retroviruses?

5-8% 8% of the genome is derived from sequences with similarity to infectious retroviruses

How many subunits do you need to build the smallest icosahedral structure?

60

How many structural subunits build an icosahedral particle with T=1

60 Icosahedral symmetry: smallest viruses are made of 60 identical subunits -1 type of subunit involved 60 of them arranged in closed shell -Consists of 20 facets (equilateral triangles) -Each subunit is in exactly the same environment as any other -Symmetry: 5-, 3-, and 2-fold rotational symmetry axes allow placement of 60 identical units with equivalent contacts between each of them

How many types of viral genomes do we distinguish?

7

What are ambisense RNA genomes? How are they different from dsRNA viruses?

Ambisense viruses contain partly positive (5') and partly negative (3') RNA genome. The 5' end is positive sense, and the 3' end is negative sense. dsRNA viruses have two RNA strands: one (+) sense strand and one (-) sense strand. -While dsRNA contains (+) sense strand, it cannot be translated directly. -The (-) sense RNA needs to be copied into (+) strand by RdRp, then used for protein synthesis. -Only the positive strand is taken for translation of the protein because the negative sense RNA is blocking positive sense, and they need to come apart. Only positive strand can be used for translation; negative sense would have to be transcribed into positive.

What is a virus?

An infectious, obligate, intracellular parasite comprising genetic material (DNA or RNA surrounded by a protein coat and/or a membrane

What systems are used to culture and store viruses?

Animal viruses at first could not be routinely propagated in cultured cells. Most viruses were grown in laboratory animals. 1. Embryonated chicken egg (fertilized): Different egg elements inoculated with different viruses, depends on susceptibility and permissiveness of the virus. Most influenza vaccines are grown in eggs 2. Cell systems: Enders, Weller, an Robbins propagate poliovirus in human cell culture - primary cultures of embryonic tissues. We have different human cell lines that can be used for propagation of a virus. There are primary human cell lines where you take a piece of tissue, digest it with specific enzymes, and grow the cells. The majority of primary human tissues are human foreskin fibroblasts. We also have cultures that are derived from model systems like mouse fibroblasts and human epithelial cell line (HeLa Cells).

What is plaque assay?

Assays ask: How many viruses are in a sample? How much do we need to use to infect our cells? How do we know that cells actually get infected? (not all viruses cause CPE) Plaque Assay: provides a measure of the number of particles capable of forming plaques (infectious particles!) per unit volume (PFU/mL). Perform 10-fold serial dilutions of viral stock, apply each dilution to a plate with monolayer of cultured cells, incubate to allow for virus absorption, overlay the cells with nutrient medium to restrict the spread of viruses to neighboring cells. Result: infectious viral particle creates a circular zone of infected cells, so called plaque. If the infected cells are damaged, the plaque can be distinguished from the surrounding monolayer after staining (crystal violet). Only plates with 10-100 plaques are statistically valid.

I have 5 x 10^3 (5000 viral particles/ul) of lentivirus stock. How much do I have to take (ul) to get MOI of 1 if I plate 1 x 10^5 (10000) cells in a 12 well plate? A. 1 ul B. 2 ul C. 5 ul D. 10 ul E. I don't know

B. 2 ul

In the "particle to PFU ratio", "particle" can be best described as: A. One of the proteins which makes up the virion B. A virus which may or may not be infectious C. A virus which is infectious D. A virus which is not infectious

B. A virus which may or may not be infectious

Which of the following statements are true? A. All viruses make us sick and can be lethal B. Our immune system can manage most viral infections C. Humans are usually infected with one virus at a time D. Our immune system cannot handle most viral infections

B. Our immune system can manage most viral infections

Which of the following describe virus symmetry and self-assembly? A. The bonding contacts of subunits are usually covalent B. The bonding contacts of subunits are usually non-covalent C. Each subunit has different bonding contacts with its neighbors D. Self-assembly of virus particles does not occur E. None of the above

B. The bonding contacts of subunits are usually non-covalent Non-covalent means they can come apart. If they were covalent, it would be very difficult to break them apart. Each subunit has almost identical contacts with their neighbors, not different.

Which of the following methods for studying virion structure provides the best resolution? A. Electron microscopy B. X-ray crystallography C. Cryo-electron microscopy (CryoEM) and cryo-electron tomography D. Nuclear magnetic resonance spectroscopy (NMR)

B. X-ray crystallography -Make a crystal of the virus, find the conditions, very laborious process -Virus crystal is bombarded with a monochromatic x-ray beam -Each atom within the virus particle scatters the radiation -Interactions of the scattered rays with one another from a diffraction pattern -Larger viruses lie beyond the power of this method -2-3 angstroms for viruses C. Cryo-electron microscopy -Samples rapidly frozen and examined at very low temp in hydrated, vitrified state that preserves native structure -Increased resolution to atomic level -Reconstruction of 3D structure

What role does a low pH play in influenza virus entry?

Binding is through sialic acid, but after initial binding, virus ends up in endosome where there are specific pumps that pump H ions inside and lower the pH of the endosome, resulting in conformational change of HA protein that allows it to release a fusion peptide that enters itself into a cellular membrane of the endosome, and that brings the two membrane layers to be within close proximity, allowing release of genome content. Low pH role in influenza entry: when the virus enters the endosome, its interior gets acidified. That causes a conformational change in HA protein and exposition of fusion peptide and insert into a cell membrane. Pulling on the membrane releases the viral genome.

How big is an average virus? A. As big as an animal cell B. As big as a plant cell C. Between the size of a ribosome and bacterium D. I don't know

C. Between the size of a ribosome and bacterium

Which is a part of the poliovirus replication strategy? A. The production of subgenomic mRNAs B. De novo (without primer) initiation of RNA synthesis C. Circularization of template for initiation of RNA synthesis D. All of the above

C. Circularization of template for initiation of RNA synthesis

Which of the following does not play a role in virus entry? A. Clathrin-mediated endocytosis B. Fusion of viral and plasma membranes C. Diffusion of virus particles in the cytoplasm D. Microtubule-mediated transport E. Sialic acid

C. Diffusion of virus particles in the cytoplasm

A ___ and ___ cell is the only cell that can take up a virus particle and replicate it. A. Naïve and resistant B. Primary and permissive C. Susceptible and permissive D. Susceptible and naïve E. Continuous and immortal

C. Susceptible and permissive

What is cap-snatching mechanism?

Cap-snatching mechanisms (priming). Cellular mRNAs have a 5' cap structure. Viral RdRp cleaves off the 5' capped segment of mRNAs and use this fragment for priming. All 8 segments of influenza RNAs have 5' ends derived from host mRNAs.

How is capsid different from nucleocapsid?

Capsid (capsa=latin, box): protein shell surrounding genome. Nucleic acid genome. Coat. Nucleocapsid: (core) nucleic acid/protein assembly within particle, subassembly with a clear substructure, i.e., in HIV Within the virion; used when this assembly is a discrete substructure of a particle Core

What specific host proteins are involved in Polio virus replication?

Cell membranes allow replication to occur in a condensed and protected environment. The genome is circularized for replication (5'-3'). At the 3' end, which is the poly A tail, there is a poly A binding protein (PAbp) that binds to it. At the 5' end, there is a VPg protein and a cloverleaf structure which is bound by PCbp protein that attaches the genome to the 3AB protein in the membrane Just remember that polio replication occurs through circularization, which is mediated by tons of proteins, and during that entire process, VPg finds its way to the 3' end of the template and starts synthesis.

What is sialic acid?

Cell surface carbohydrate, Receptor for influenza Influenza binds to the cell surface carbohydrate Sialic acid, present in oligosaccharide chains that are covalently attached to cell surface. The interaction is strong due to multiple interactions among Hemagglutinin (HA) molecules on the surface of the virus. Neuraminidase (NA) cleaves the receptors and it is essential for viral release. Sialic acid is bound by hemagglutinin that is subsequently cleaved by neuraminidase

What are syncytia? Inclusion bodies?

Common examples of CPE include: Syncytia - rounding of the infected cell, fusion with adjacent cells (e.g. paramyxovirus: measles). Viral glycoprotein mediates fusion of an infected cell with neighboring cells leading to the formation of multi-nucleate enlarged cells called syncytia (multinuclear cells). Usually, these syncytia are the result of expression of a viral fusion protein at the host cell membrane during viral replication. Inclusion bodies - another CPE: elementary bodies , are nuclear or cytoplasmic aggregates of stable substances, usually proteins. They typically represent sites of viral multiplication in cell and usually consist of viral capsid proteins (e.g. Negri bodies in rabies, Cowdry bodies in CMV).

What types of polymerase can we distinguish?

Drug inhibiting cellular polymerase actinomycin D was used in the determination of RdRp being viral or cellular.

What is the principle behind electron microscopy, x-ray crystallography and cryo-EM technologies?

Electron microscopy: most widely used method, biological material have little inherent contrast; need to be stained with an electron-dense material (negative staining to obtain contrast, i.e. uranyl acetate), limited resolution 50-75 angstroms (protein alpha helix ~10 angstroms), detailed structural interpretation impossible, only a shadowing of the viral particle X-ray crystallography: make a crystal of the virus, find the conditions, very laborious process, virus crystal is bombarded with a monochromatic X-ray beam, each atom within the virus particle scatters the radiation, interactions of the scattered rays with one another form a diffraction pattern, larger viruses lie beyond the power of this method, 2-3 angstroms for viruses Cryo-EM: samples rapidly frozen and examined at very low temp in hydrated, vitrified state that preserve native structure, increased resolution to atomic level, reconstruction of 3D structure

What is transfection? How is it used to study viruses?

Engineering mutations or changes into a genome by use of viral vectors that carry genetic information. -Production of infectious virus after transformation of cells by viral DNA, first done with bacteriophage lambda

Where does the fusion occurs and how?

Enveloped virus - Membrane fusion Virus enters the cell when its outer membrane fuses with the plasma membrane at the cell surface. The viral contents are then spilled into the cytoplasm of the cell (i.e. HIV)

What are the other components of virions?

Enzymes: -polymerases, integrases, accessory proteins -proteases (for cleaving) -poly (A) polymerase -capping enzymes -topoisomerase Cellular Components: -tRNAs (HIV: tRNA serves as a primer for its replication) -histones -lipids

What information is encoded in viral genomes?

Everything that is important for the virus to reproduce, infect the cell, leave the cell, and continue infection. Gene products and regulatory signals for: -Replication of the viral genome -Assembly and packaging of the genome -Regulation and timing of the replication cycle -Modulation of host defenses -Spread to other cells and hosts

What is end-point dilution assay? And when is it used?

For viruses that do not form plaques, or obvious CPE, serial dilutions of a virus stock are inoculated into replicates (multi-well plate). You basically perform serial dilutions of viral stock and put those dilutions on a 96 well plate that has a monolayer of cells in each, then you visually inspect which of the dilutions caused 50% death of cells. The number of test units that have become infected is then determined for each dilution (cytopathic effect). Shown: At high dilutions, no cells are infected (10^-7), too diluted. At low dilutions, every culture is infected (10^-2). The end point is the dilution of virus that affects 50% of the tested units (in between). The results are expressed as 50% infectious dose (TCID50) per milliliter. RESULTS: 105 ID50 per ml

How do viruses enter nucleus?

Four modes of nuclear entry: A. Influenza virus: genome released from endosomes, RNP enters the nucleus as a complex B. Herpesvirus: pores in the coat protein docks on the nuclear pore C. Adenovirus: partial disassembly at the nuclear pore D. Parvoviruses: bind to the nuclear pores, modify it, so that the entire particle enters the nucleus

What viruses do not use receptors as a point of entry to the host cell?

Fungi: spread during cellular division Plants: insects and mechanical damage

What two human proteins were derived from retroviruses? What is their function?

Gag: Encodes structural proteins Pol: Surface envelope proteins (encodes polymerase and encodes envelope) -envelope is lipid bilayer surrounding virus

What is a fusion protein? What is its relation to the viral glycoprotein?

Glycoproteins binding to the cellular receptors triggers conformational change in the fusion peptide and inserts its hydrophobic domain in the membrane. That insertion pulls the membrane together in close proximity, and because both membranes are hydrophobic, they have a high affinity for themselves and start fusing. Fusion peptide going outside and inserting itself through the hydrophobic domain to the cell membrane is what brings two cells together.

What specific cellular receptors are bound by HIV, influenza virus, adenovirus, coxackievirus? (remember these!)

HIV: Receptor is CD4 -Coreceptors: CCR5 and CXCR4 -HIV entry, one receptor for docking and one for endocytosis Adenovirus and Coxsackievirus: Receptor is CAR -Coxsackievirus and Adenovirus Receptor

What are the main types of viral particles?

Helical Icosahedral Complex

What types of viral enveloped are there?

Helical nucleocapsids: Unstructured envelopes-has a different symmetry than nucleocapsid and does not interact with it -Loosely surrounds the capsid protein -Ex: influenza and Ebola Icosahedral nucleocapsids: Structured envelopes-is anchored to the nucleocapsid under the membrane and has the same symmetry as the capsid (interacts with it) -Goes into interactions with the coat protein; has to be symmetrical to interact with it -This is what defines its shape

What 3 transmembrane proteins make up the lipid bilayer of the influenza virus?

Hemagglutinin Neurominidase Matrix 2

How does bacteriophage T4 enters a cell?

Initial binding is reversible and electrostatic: the outer most part of the long tail fiber binds to surface lipopolysaccharides (LPS) of the bacterium. Binding is "additive" until all six tail fibers are bound, binding of 3 fibers is needed to initiate infection. The virus may "browse" the surface, looking for a suitable site for penetration. (LPS=weak spot). Conformation change in baseplate. Initiates sheath contraction (to 37% of its original length). Sheath of the helical tail slips and forms a shorter helix. The tube is pushed down and contacts the membrane - note, the tail does not directly punch through. Lysozyme molecules are released, which forms a pore through which DNA enters.

What is the purpose of viral envelope glycoproteins?

Integral membrane glycoproteins Ectodomain: attachment, antigen sites, fusion Internal domain: assembly Oligomeric spikes Before a virus leaves and takes a part of the lipid bilayer for its own protection, it modifies it -It has some specific proteins that attach to it: some face exterior, some face interior. They are often called spikes/spike proteins, and they serve and important function in docking the virus on a specific cell receptor Ex: HA trimer for docking influenza Ex: Flavivirus dimer for docking Zika virus

When do we observe one-hit and two-hit kinetics of viral infection?

Kinetics of viral infection are the dose-response curve of the plaque assay. How many viral particles are needed to initiate infection? Perform serial dilutions, count plaques for each, plot the relationship. For majority of viruses there is a linear relationship between the number of infectious particles and the plaque count: 1 viral particle = 1 plaque. One infectious particle is sufficient to initiate infection, and the virus is said to infect cells with one-hit kinetics. Some viruses require more than one particle to initiate infection. If two different types of virus particles must infect a cell to ensure replication, we are talking about two-hit kinetics (parabolic dose-response curve).

What biophysical property was used to isolate viruses in the past?

Loeffler and Frosch said that the agent of foot and mouth disease is filterable. They filtered an infected sample through a 0.2 um (220 nm) pore, and since they knew the size of a bacterium, they knew that the filter would allow them to deduce what new entity was present. This filter size, for a long time, was what defined a virus as a virus.

What cellular organelle plays important role in reovirus entry?

Lysosome Reoviruses have a complex double capsid, which is very stable to low pH (gastrointestinal viruses, rotavirus). The lysosomal proteases degrade the outer capsid to form a sub viral particle i.e. degradation by cellular proteases. The subsequent penetration step is unknown.

What are the largest known viruses? What is so special about them?

Mimivirus and Pithovirus. Mimivirus is 400 nm and is known as the mimicking microbe. It infects amoebae and its genome size is 1.2 Mb (million bases). It codes for 1260 proteins involved in DNA repair and chaperones. Pithovirus is even bigger at 1.5 um. Its genome is 650k base pairs and it codes for 500 proteins. It also infects amoebae. These viruses can be seen under a regular microscope.

What viral properties do we use to classify them?

Morphology: The easiest. way to classify something is to basically look at it and assume morphology is a way to classify it. People also classified viruses by the presence or absence of a viral envelope, which is another morphological feature. Genetic Make-Up: This is the most important classification we will follow. This scheme is called the Baltimore system. It tells you that, in general, there are seven groups of viruses that we can divide/classify according to their genetic makeup (nucleic acid). As long as you know what kind of nucleic acid a virus contains, you will know everything. The information embodied in this classification provides immediate insight into the steps that must take place to initiate replication and expression of the viral genome.

Explain the stop-start model for VSV mRNA synthesis

RdRp initiates synthesis at the 3' end of the N gene (first gene). After synthesis of N mRNA, RNA synthesis terminates at the intergenic region (ig-reach in Us). This is followed by RdRp reinitiating at the 3' end of the P gene. All 5 mRNAs are synthesized this way: start, stop, start, stop.. Once N mRNA is synthesized, the N protein is expressed, coats RNA template, and prevents the stopping of RdRp. Result: full length RNA synthesis.

What is MOI and how do we calculate it?

Multiplicity of infection (MOI)-number of infectious particles ADDED per cell. Not the number of infectious particles each cell receives! Infection depends on the random collision of virions and cells. When susceptible cells are mixed with virus, some cells are uninfected, some receive one, two, three, or more particles. To calculate, take the number of viral particles used per well then divide by the number of cells originally seeded in the well. An MOI of 5 indicates that there are five transducing units (virions) for every cell in the well. Different cell types may require different MOIs for successful transduction and knockdown of the target gene. Typically used MOI is 5-10 to achieve one-step growth cycle. MOI of 1 means that each 1 infectious particle added per cell. 10000 cells = 2 x 5000 viral particles/ul = 2 ul.

How is fusion regulated and why?

Must not occur in the wrong location, highly regulated. Induce cell-to-cell fusion by undergoing conformational change that can be triggered by: 1. Low pH - influenza 2. Viral glycoprotein binding to the receptor - HIV 3. Binding to the second cellular receptor (co-receptor) interaction 4. Proteolytic cleavage Very important for interacting with specific cell receptor, they determine that diffusion does not occur in a wrong location (highly regulated/controlled)

How are influenza virus and VSV RNA synthesis similar?

NP protein expression is similar to N protein in VSV. As soon as NP (nucleoprotein) protein is expressed from mRNA, it coats nascent transcript and prevents that termination. NP protein coats the whole thing, and by doing this, supports more efficient replication/production of positive sense.

What are the three antiviral drugs currently used to combat the flu and how do they function?

Neuraminidase inhibitors -block neuraminidase enzyme on influenza CAP-dependent endonuclease inhibitors -impede viral RNA transcription and block viral replication Adamantases -target the M2 proton channel, which plays a big role in viral RNA into the cytoplasm, only fluA

Are all viruses dangerous to humans?

No, 95% of the time, we have them under control and they do what they are supposed to do: control and regulate the microbiome. Stressful moments trigger viral over replication and this is what tips the balance towards disease.

Does corona virus undergo reassortment?

No, because it has only one positive sense RNA genome; does not have segments

Are all viral RNA genomes (+)-sense?

No, there are negative senses: ssRNA (-) sense. The (-) strand RNA genome must be copied into (+) strand to make proteins. There are two configurations: segmented and non-segmented.

Do ribosomes and RNA polymerase collide?

No. Synthesis is 5'-3' direction Translation is 3'-5' direction Different timing, different cellular localization (spherules vs. cytoplasm), RNA structure (on and off switch)

Are viruses alive?

No. They cannot maintain homeostasis, grow, or use energy. It is possible for them to reproduce, but it is called replication. It is unknown if they respond to stimuli. They can have different levels of organization and adapt to their environment.

Do all viruses bind the same receptors?

No. There is a variety of molecules used by viruses to enter cells: protein (glycoprotein), carbohydrate (sugar), lipid (glycolipid). -Their receptor basically determines the virus tropism and pathogenesis; this is what makes the cell susceptible, the presence of a proper receptor. Some receptors can be used by multiple viruses: Sialic acid is used not only by influenza, but also measles and respiratory syncytial virus; so, many viruses can use the same receptor.

What is Particle-to-PFU ratio?

Number of physical particles divided by the number of infectious particles. A single particle can initiate infection. Not all viruses are successful: damaged particles, mutations, complexity of infectious cycle.

What is virion?

Obligate, molecular parasites. A virus "exists" in two phases: a virion, which is an inanimate phase, and an infected cell, which is a multiplying phase. Visions are infectious particles outside of a living organism or cell.

DESCRIBE THE VIRAL REPLICATION, KEEPING IN MIND THE TWO PROMINENT PROTEINS INVOLVED.

Occurs in a cascade that leads to the synthesis of viral proteins that are classified as IE, E, and late. IE62 is predicted to be an important immediate-early protein because it functions as the most potent. VSV GE is the most abundant glycoprotein in infected cells and is a prominent envelope glycoprotein.

How it the one-step growth curve different for viruses with high Particle-to-Pfu ratio vs viruses with low Particle-to-PFU ratio?

One-step growth cycle: every single cell is infected at the same time, infection is synchronized. Information about the kinetics of viral replication: eclipse period where virus finds the cell and attaches to the receptor to penetrate it and uncoat, burst of replication, and plateau. If you dilute viral stock too much, all cells will be infected synchronously; you will have an eclipse period, first burst of replication, and a plateau followed by a second burst. The second burst is needed to infect them all (too much dilution or high particle to PFU ratio).

How to we calculate PFU/ML?

PFU counted/(dilution factor) *(amount plated) = PFU/ML Ex: 17 plaques per 0.1 mL from 10^-6 dilution = 17/10^-7 = 1.7x10^8 PFU/mL

What experiment lead to the discovery of RdRp origin?

PhD experiment by Baltimore. Monitoring the synthesis of viral RNA using radioactive nucleotides showed that the increase in RNA concentration correlated with viral PFU. The conclusion was that there must be an enzyme responsible for viral RNA synthesis. Extracts from virus-infected cells were incubated with 4 ribonucleotides, including one radioactive to measure the incorporation into newly synthesized RNA. The drug inhibiting cellular polymerase actinomycin D was used, but products still appeared. Further studies led to purification of active Polio virus RNA-dependent RNA polymerase (RdRp) that was able to copy viral RNA. It is exclusively viral and it is the first protein expressed after (+) sense RNA virus entry. Proteins cannot be expressed from (-) sense strand, and must be converted to (+) sense strand, but the conversion requires RdRp. The solution is that RdRps are always packaged into (-) sense RNA virus particles. Polymerase was discovered in (-) strand virus particles.

What is ELISA?

Physical measurements of virus particles: Enzyme-linked immunosorbent assay (ELISA) detecting viral antigens or antibodies. Antibodies used to visualize viral proteins (antigens) in infected cells or tissues. Direct immunostaining - antibody recognizes a viral protein and it is directly coupled to a fluorescent dye or enzyme. Indirected immunostaining - primary or secondary antibodies are used (sandwich), provides stronger signal. You basically have a paper strip, and you have an antibody that is specific for, or that detects, the viral antigen (some sort of specific protein).

What is hemagglutination?

Physical measurements of virus particles: Hemagglutination. Two-fold dilutions of samples of different influenza viruses were prepared, mixed with chicken red blood cells, and added to the wells of a plate. After 30 minutes the wells were photographed. The sample in row C contains no detectable virus (button present). Sample D causes hemagglutination up to the 1:1024 dilution; therefore, the HA titer of this virus stock is 1024. Some viruses like hemagglutination because the virus makes the blood sticky. You place the dilution, and check for buttons. Dots mean that the blood got heavy and clumped. By knowing what dilution you plated there, you will know what the concentration of the virus was.

How is poly(A) tail added to VSV mRNAs?

Poly (A) is added by RdRp slippage, Poly(A) non-template addition. Poly (A) addition (An) is a result of copying of a sequence of seven U's present in each intergenic region (ig) followed by RdRp slippage. Polymerase slips on U's and starts adding more A's. When concentration of N proteins is high, it coats template, no access to U's, no slippage, and full length mRNA is produced.

What mechanism is used by RdRp during synthesis? Describe it!

Polymerase catalysis involves a two-metal mechanism. This mechanism is universal for all polymerases. Two Asp residues coordinations 2 Mg2+ ions that remove 2 phosphate groups through a nucleophilic attack, leaving behind one phosphate, that will join nascent T with A and so on. Polymerase needs the 3'OH group, and sits on the 3' OH group of adenosine. The two aspartic residues coordinate to the magnesium ions, and the magnesium ions attack the previous nucleotide adenosine which has three phosphate groups (nucleophilic attack on the triphosphate). They remove two phosphates and, providing energy, and leave one behind that will connect to oncoming T residue (connects one nucleotide to another). Synthesis occurs from the 5' to 3' direction.

What is primer-dependent initiation?

Primer-dependent initiation is a mechanism of initiation of RNA synthesis that includes a protein-linked oligonucleotide or capped oligonucleotide serving as a a primer for RNA synthesis by RdRp. A protein provides a hydroxyl group from tyrosine or serine residue to which the first oligonucleotide can be linked. An oligonucleotide proves 3' OH group. Examples of viruses include picornaviridae and calciviridae.

What are the functions of structural proteins?

Protection and delivery of the genome. -Protection: assembly of a stable, protective protein shell, specific recognition and packaging of the nucleic acid genome (high specificity), interaction with host cell membranes to form the envelope. -Delivery: bind host cell receptors (binding to external receptors of the host cell), uncoating the genome (transmission of signals that induce uncoating of the genome), fusion with cell membranes (induction of fusion with host cell membranes), transport of genome to the appropriate site (interaction with internal components of the infected cell to direct transport of the genome to the appropriate site).

What is RdRp and what is its origin?

RNA-dependent RNA Polymerase. Exclusively viral and it is the first protein expressed after (+) sense RNA virus entry. Viral origin. We don't have it, if viruses need it they have to package and code it into RNA themselves.

Why some viruses need to package their own RdRp and some don't? What are they?

RdRps are always packaged into negative sense RNA virus particles because their genome is not prepared to associate with ribosomes, so they package their own polymerase inside of their genome. Positive sense RNA viruses templates can associate with ribosomes immediately after entry into the cell, and then translate the protein. Negative strand viruses have to go from negative to positive to make a polymerase. In order to go from negative to positive, you need an enzyme, which is why they package. -Negative sense strand RNA genomes have RdRp and RNA is coated with protein nucleocapsid. -Positive strand RNA genomes do not have RdRP and are naked, exceptions being retrovirus and corona virus. -dsRNA genomes have RdRp and naked RNA, but even though they contain positive sense RNA, it is blocked by negative sense RNA, so it needs to be transcribed so we need RdRp

What is a consequence of genome segmentation?

Reassortment: -happens when two parental viruses infect cells (influenza) -they have segmented negative sense RNA genomes -when they infect the cell (2 different viruses), they release their genome -some genomes come from L parent, some come from M parent -they get mixed up, and as a result, their progeny could be identical to one parent, or could have reassortment -reassortment has reasserted genes -reason why we need new flu vaccine every year Reassortment is only possible if the virus has a segmented gene

What are the criteria to distinguish a cell protein as a viral receptor? What methodologies can we use to help that process?

Receptor binds virus particle -Strip virus of spike protein, incubate them with suspected molecule, if they bind, that cellular molecule is responsible for binding of the viral antigen Antibody to receptor blocks infection -Putting an antibody against that receptor, receptor binds to it, and blocks infection. Receptor gene confers susceptibility; more than one receptor may be involved -Switch off gene that codes for that protein by CRISPR Cas system Disruption of receptor gene blocks infection -Block it by RNAi pathways using siRNAs to block a given expression of a gene or mRNA Cloning and Transfections

What types of endocytosis are used by viruses to enter a host cell?

Receptor-mediated endocytosis. Macropinocytosis Naked Virus-Endocytosis: virus attaches to cell surface receptor molecules and sinks into a clathrin put which invaginate and closes off, creating a clathrin coated vesicle (i.e. Adenovirus) After initial entry, all viruses move within the cellular milieu in association with motor protein (no diffusion!)

What is unusual about dsRNA viruses replication?

Replication occurs in sub viral particles. During cell entry, the vision passes through the lysosomal compartment and proteolysis of viral capsid activates the RNA synthetic machinery. Positive sense RNA strand is used for translation and synthesis of negative strand. Positive and negative dsRNA come apart for positive to act as a template for replication. Genomic RNA never leaves the particle; replication occurs within particle, why? dsRNA is highly immunogenic. Thought to occur inside particle, kind of like a spherule.

What is remarkable about retroviral genome replication?

Retroviruses with positive sense RNA genomes enter the cell and do not get translated, but go through a DNA intermediate. There are two strands of RNA that get reverse transcribed into DNA, and that DNA gets integrated into a host DNA that is later on transcribed into mRNA. There are two pieces of that positive sense DNA that gets reverse transcribed into negative sense DNA, that gets fixed to dsDNA, and that creates a virus. A virus is a piece of DNA that reflects the genetic material of the retroviruses that get integrated into host DNA. One of the reasons why we can't cure HIV is because their genetic material is integrated into human DNA. After DNA is integrated, it gets transcribed into RNA and translated into proteins that build viral particles.

Why some viruses use complex structures with mixed symmetry? Do you know examples of these viruses? What these modifications serve for?

Structurally complex capsids: -Largest viruses; distinct components with different symmetries -Presence of proteins devoted to specialized roles Large complex capsids: -Distinct components with different symmetries -Presence of proteins devoted to specialized roles -Ex: Adenovirus - structures sticking out of the virus are called fibers built with different proteins which follow different rules of assembly; these fibers are proteins that dock adenovirus with the receptor on cellular surface Complex capsids with two icosahedral protein layers: -Ex: Retrovirus - has an outer layer with a T number of 13 and underneath that is another layer with protein protection with a T number of 2, so it has two capsids to protect its genetic material from low pH of stomach acid Complex structure: Tailed Bacteriophages -Icosahedral structure at the top with 5-fold symmetry -Helical body -Base plate at the bottom with unique structure: spike - three subunits repeating that form a spike, allowing it to pierce the cell membrane of bacteria Herpes simplex virus capsid: -Holes for entry and exit of DNA -Repeating subunits with slight distortion that forms a hole in its structure which is what releases the genome of the virus into the nucleus of the cell

What is one-step growth analysis and how is it performed?

Studying the viral infectious cycle, how different steps of viral infection proceed. One-step growth cycle: every single cell is infected at the same time, infection is synchronized. Information about the kinetics of viral replication: eclipse period where virus finds the cell and attaches to the receptor to penetrate it and uncoat, burst of replication, and plateau. Apply virus to cellular monolayer, let it absorb, remove unabsorbed inoculum, wash cells, add new medium, and at different time point take viral sample and determine titer (by plaque assays). Once we have our viral stock, we know its concentration, and we know the growth kinetics, we now need to know how to block its replication (infectivity) cycle. It is done in a similar manner to plaque assay. We have a monolayer of cells, apply addition of virus, let it absorb, dilute that culture, and assay. If you dilute viral stock too much, all cells will be infected synchronously; you will have an eclipse period, first burst of replication, and a plateau followed by a second burst. The second burst is needed to infect them all (too much dilution or high particle to PFU ratio).

What is a subgenomic RNA?

Subgenomic RNA - shorter than genome RNA templates. From negative strand RNA, there is a site called an internal promoter, which allows for internal initiation of the synthesis. That is how a subgenomic (not exactly full length) RNA is synthesized. That subgenomic RNA is used as a template for translation of other proteins that are used later during replication. Subgenomic RNAs are made in order to regulate the timing of translation. They are needed later when there are copies of the genome, when there are more viruses produced, so that allows regulation of the timing.

What is a subunit and how does it differ from structural unit?

Subunit: single polyprotein chain, for Polio: VP1, VP2, etc. Single, folded, polypeptide chain. Structural unit: unit from which capsizes or nucleocapsids are built, includes one or more subunits, for polio: 4 VPs bull structural unit One protein subunit or multiple, different protein subunits

How do the symptoms between influenza and the common cold differ?

Symptoms of the flu come on sooner than those of the cold.

WHAT ARE THE MAIN SYMPTOMS, THE TWO TYPES OF SEVERE CAUSES OF THE VIRUS, AND WHO IS MOST SUSCEPTIBLE?

Symptoms: Rash with itchy, fluid-filled lesions that scab when healing Fatigue Fever Headache Loss of appetite Severe causes: Unvaccinated children Immunocompromised people Susceptible: Adults aged 45+

Are these endogenous retroviral sequences of any benefit to humans?

Syncytin is nearly identical to a retroviral envelope protein (Env). It is a protein produced by placental cells that directs formation of the cellular boundary between the placenta and maternal tissue. Arc protein, derived from retroviral gag protein, participates in memory formation. Arc protein is homologous to retroviral Gag protein. Since 8% of our DNA is viral, endogenous retroviruses have been repurposed to fight modern viruses. They produce proteins that block the receptors through which newcomer viruses invade. Some proteins distribute the replication of attacking viruses, leaving them unable to invade other cells.

How is Ebola virus entry different from other viruses?

The receptor that catalyzes fusion is in the endosome! Virus is taken up by pinocytosis and ends up in the endosome. Viral glycoprotein is cleaved by enzyme in endosome and exposes protein NPC-1 cholesterol transporter. Glycoprotein binds to NPC1, and that triggers the fusion.

What is a quasi-equivalence and what is the reason behind it?

T=1, 60 subunits forming pentamers, all subunits in identical environment. With high T number, even if all proteins are identical, some will be in an environment of 5 neighbors (pentagons) and others will be in a 6 neighbors environment (hexagons). Hence the positioning of each protein is not equivalent between each protein and any other, but only quasiequivalent: bonding interactions tail-to-tail and head-to-head. Most T=3 viruses have 180 proteins (3 proteins x 60 triangles). Their positions are often referred to as A, B, C quasi-equivalent positions. All A proteins are equivalent to one another, and similarly for B and C proteins. "Quasi-equivalence" proposed in 1962 by Caspar and Klug: -"Identical" subunits in slightly different environments. -Not all protein subunits are equivalent. -Only certain numbers of subunits can be packed into closed regular lattice. Reason for quasi equivalence: -Clear evolutionary pressure to make larger capsid: using larger subunits helps very little and using more subunits helps a lot -Not possible to form icosahedral shell (of identical units in identical environments) with more than 60 subunits

Where does the active site resides in RdRp?

The "palm" of the polymerase.

Can (-) sense RNA genome be transcribed?

The (-) sense RNA needs to be copied into (+) sense strand by RdRp, and then used for protein synthesis. The virus needs to carry its own RdRp, which is packaged into virions.

How big are viruses if we consider cellular scale (like plant, animal cells? Ribosomes? Proteins? What about in the uM or nM scale?

The average size of a virion ranges between 10^-6 and 10^-8 meters (10nm-150nm). Most viruses end up on a meter scale somewhere between bacteria and ribosomes. Viruses on a logarithmic scale are larger than proteins and smaller than plant and animal cells. Proteins are less than 10 micrometers, ribosomes are about 150 nm, and plant and animal cells are about 10-100 um. Viruses exist between 10 nm and less than 1 um.

What happens to gapped dsDNA genome when it enters the cell?

The cell must fix that gapped dsDNA: Viruses use cellular enzymes to fix their genome, including dsDNA. The gapped dsDNA genome needs to be fixed before it can be used as a template for the synthesis of RNA which is to be translated to protein.

What did Hershey-Chase experiment prove?

The genetic code of viruses is nucleic acids Nucleic acid is what is passed on through generations, carrier of genetic material

Why is it difficult to study the origin and age of viruses?

The origin of viruses is mysterious because of their patchy, molecular makeup. They cannot be included in the tree of life because they do not share characteristics with cells. While cellular life has a single, common origin, viruses are polyphyletic - they have many evolutionary origins. No single gene has been identified that is shared by all viruses. Viruses steal genes from cells, and it has been suggested that viruses influence the evolution of cells by donating new genes. Cross-species jumps, mutations, recombination, and reassortment all play a big role in virus evolution.

What is human virome?

The viral component of the human microbiome The collection of all viruses that are found in or on human, including viruses that cause acute, persistent, or latent infection, and viruses integrated into the human genome, such as endogenous retroviruses (viral DNA in our DNA)

Know consecutive steps of viral life cycle: attachment, penetration, uncoating, replication, transcription, translation, assembly, release.

The virus attaches to a receptor, which mediates its entry and release of genomic material. Then, there is a translation, genome replication, assembly, and release. Attachment: viral capsid/envelope proteins attaching to receptors on a target cell Penetration: process of entering a host Uncoating: removal of the viral capsid, unmasking of the genome Replication: amplification (copying) of the viral genome, virus uses its hosts machinery, sometimes occurs in phases Transcription: production of mRNA, sgRNA Translation: production of viral proteins, polyprotein, one protein at a time, numerous strategies Assembly: hundreds to thousands of proteins assemble around the viral nucleic acid to form a protein shell called a capsid Release: viral shedding refers to the release of progeny following successful reproduction during a host-cell infection

What does it mean that virus is asymptomatic?

They usually do not affect us unless we are immunocompromised. In that case, they tend to cause pathologies.

What is triangulation number and how is it used to describe viral structures?

Triangulation number, T. The number of "initial" triangles per new icosahedral triangle. Combining several triangular facets allows assembly of larger face from same structural subunit. T=1, 60 subunits forming pentamers, all subunits in identical environment. With high T number, even if all proteins are identical, some will be in an environment of 5 neighbors (pentagons) and others will be in a 6 neighbors environment (hexagons).

What specific amino acid residues coordinate two divalent metal ions during two metal ion catalysis mechanism?

Two Asp (aspartate) residues coordinate two Mg2+ (magnesium) ions.

What is the typical MOI we use for experiments? Why this number is usually higher than 1?

Typically used MOI is 5-10 to achieve one-step growth cycle. To achieve synchronous infection (key to one-step growth cycle) we need to infect all the cells. Different cell types may require different MOIs for successful transduction and knockdown of the target gene.

How abundant are viruses?

Viruses are the most abundant biological entities in the oceans, comprising 94% of DNA/RNA containing particles The comprise ~5% of the biomass because of their size 90% of us have herpes simplex 1 98% of us have Epstein-Barr (mono) The human virile is part of the human microbiome

Where do viruses replicate and why?

Viruses need to establish intracellular environments that concentrate the viral proteins and allows productive replication. -They need to arrange the cell environment in a way that concentrates all viral components needed for replication. The components are the genome, polymerase (RNA dependent RNA polymerase, RdRp), and all necessary accessor proteins that dictate replication. Virus infections (viral proteins) induce rearrangements of host cell membranes and formation of double membrane vesicles called spherules. Viruses create replication factories that are frequently located within rearranged membranes. Depending on the virus, this could be cellular, mitochondrial, vacuole, or ER membranes. The rearrangement forms spherules. These spherules contain everything that is needed for replication of the virus. They function by concentrating the compounds and catalyzing reactions, which is what the virus wants: fast replication followed by reinfection. The purpose is to concentrate all the compounds, and the second purpose is to protect these components from degradation. Viral factories, or double-leaflet vesicles, could be created from different membranes: Some viruses ER, others use Golgi, others use endosomes from mitochondria peroxisomes. Remember that different membranes can be arranged by an infection of the virus, and the purpose of that is to create replication factories (spherules).

What is the correlation between viral genome size and mutation rate?

Viruses with small genomes mutate faster: -High mutation rate, coupled with rapid replication, are also the basis for the high rates of nucleotide substitution

What does it mean that virions are spring loaded with energy?

Visions are not inert structures: virus particles go through energy transitions Unfavorable energy barrier must be overcome Visions are spring loaded during assembly: energy is put into the structure That energy is used for disassembly if cell provides proper signal

How can we reconstruct recombinant Polio virus?

We cannot clone the RNA into a vector, so we have to reverse transcribe that viral RNA into cDNA (copied DNA). Next, put cDNA into a plasmid by transfecting it into culture cells by electroporation. dsDNA goes through and is immediately used as a template for the synthesis of mRNA; one is poliovirus mRNA expressed into proteins that build particles, and the other is recombinant poliovirus that we designed.

How do we know that these sequences are derived from retroviruses?

We know we have these because the human genome was sequenced The majority of things in our genomes are actually noncoding sequences Endogenous retrotransposons/retroelements are frequently not coding anything because if they code it would result in presence of an infectious virus

What happens during latent and lytic stages of viral replication?

When a virus is passively replicated along with its host's genome, we term it the latent cycle. In this passive cycle, no viruses are produced (herpesviruses). -The latent cycle is when the herpes virus genome gets attached (not integrated) to your chromosomes and stays "quiet" to avoid immune stimulation and recognition. It is coated with a protein that keeps it quiet. When the latent virus is triggered to begin producing viral progeny, it will begin to replicate, and we call this process the lytic cycle. -When there is a trigger (stress), the virus receives the signal that the immune response is down and it can begin replicating: starts making proteins and more expressing RNAs. The lytic cycle is triggered by different environmental conditions, and it leads to active replication of the viral genome, expression of viral proteins, and production of progeny viruses and release

What is provirus?

When the viral genome is integrated into, and replicates along with, the host's genome, we call this viral genome a provirus (retroviruses). We cannot cure HIV because its genome is forever integrated into your DNA. Because it becomes part of the host genome, it gets replicated, transcribed, and translated over and over again.

Can (-) sense DNA genome be transcribed?

Whether it is positive or negative sense, it needs to be converted into into dsDNA for transcription. Only dsDNA can be used as a template for the synthesis of RNA.

Do viral RNAs have specific structure? What is a purpose of that structure?

Yes. RNA falls into these complex structures to assert the binding of other molecules: proteins, DNA, RNA. By forming these complex structures, it forms a docking/binding site for these molecules. Because RNA is highly flexible, it can form and dissolve these structures easily. On the level of genetic material, viruses have a lot of control over what happens in the timing of these processes. We care about RNA structure because it leads to better drug targeting.