Virology - Exam 1 - Study Guides
What is particle to PFU ratio?
# of physical particles/# of infectious particles. For example if the particle/pfu ratio is 10,000 that means for every 10,000 particles only 1 is infectious. Semliki forest virus has a particle/PFU ratio of 1-2
Where do viruses replicate?
(+) sense RNA viruses replicate in the cytoplasm of the infected cell. A virus needs to establish an intracellular environment that concentrates the viral proteins and allows productive replication. Virus infections induces rearrangements of host cell membrane and formation of: double membrane vesicles and spherules. Some examples of where viruses replicate: HCV replicates in ER, Rubella replicates in vacuole
When do we observe one-hit and two-hit kinetics of viral infection?
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. For one-hit kinetics, the number of plaques is directly proportional to the first power of the concentration of the virus inoculated. If the concentration of virus is doubled, the number of plaques also doubles. 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). For two-hit kinetics, the number of plaques is directly proportional to the sqaure of the concentration of the virus inoculated.
Describe the process a virus takes to enter a plant cell
1. A viral capsid shell opens to release viral genome, which is translated into proteins that direct the formation of viral factory from membranes of the endoplasmic reticulum and other organelles. 2. Antiviral proteins patrol cells for invading pathogens, but they cannot break into the viral factories. 3. Viral RNA is replicated and exported to the cytoplasm. 4. Viral RNA and newly assembled viral particles move to other cells through plasmodesmata, which can be widened by virus-encoded movement proteins. 5. Some virus particles enter the plant's transport streams.
There are two ways RNA synthesis can be initiated, what are they?
1. De novo initiation 2. Primer-dependent initiation
What type of modification does VPg must undergo to serve as a primer?
It needs to undergo uridylation which means that it needs two U residues to become a primer. To make this happen, 3CD^pol and 3D complex binds to Cre. Next, VPq binds to Cre. There is an A-rich loop on Cre and it templates the uridylation of VPg. VPg-pUpU is going to be the primer for RNA synthesis. Then the genome is circularized for replication (5'-3'). At the 3' end - PABP-poly(A) binding protein binds poly(A). At the 5' end - cloverleaf structure binds cell protein that anchors the RNA to membrane (PCbp-3AB interaction). PABP binds viral 3CD pol and cloverleaf structures. VPg that is uridinylated on Cre makes its way to the 5' end (we don't know how!). VPg serves as a primer for RNA synthesis. RNA polymerase (blue) starts the synthesis
What is RdPd and what is its origin?
It stands for RNA dependent RNA polyermase. It is able to copy viral RNA. RdRps are exclusively viral
What type of viral particle does VZV have? What is the name of the structure it forms?
Its genome is a single copy of linear dsDNA. It is icosahedral (T=16). It has a nucleocapsid. It has the smallest genome of the human herpeviruses. It has a linear genome. It is pleomorphic
What is primer-dependent initiation?
A protein-linked oligonucleotide or capped oligonucleotide serve as a primer for RNA synthesis by RdRp. A terminal protein provides a hydroxyl group (in tyrosine or serine residue) to which the first oligonucleotide can be linked. Ex. of viruses that use this: Picornaviridae, Calciviridae
What is a subunit and how does it differ from structural unit?
A subunit is a single polyprotein chain, for instance in Polio a subunit could be VP1, VP2, VP3. A structural unit is a unit from which capsids or nucleocapsids are build, includes one or more subunits, for instance in polio there are four VPs that build the structural unit
What is the only cell that can take up a virus particle and replicate it?
A susceptible and permissive one
What does it mean that a cell is susceptible?
A susceptible cell has a functional receptor for a given virus - the cell may or may not be able to support viral replication
How do viruses enter the nucleus?
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 nuclear pores, modify it, so that the entire particle enters the nucleus
What is a Baltimore system?
Look at this picture and draw several times...she wants this committed to memory
What are the IE genes and what do they transcribe for? The E genes? the LE genes?
All these genes are stages in transcription. Stage 1: IE genes are immediate early mRNAs. They are proteins that are transported to the nucleus to activate early genes. Afterwards (stage 2), is E gene expression which is early mRNAs. They function in DNA replication and production of substrates for DNA synthesis. 3rd is L gene expression with is late mRNAs. In this case the proteins inserted into rough ER. Some are transported to nucleus for assembly of the nucleocapsid and then DNA cleavage is used to release genomes
What happens during polio virus entry with VP1 and VP4?
Although poliovirus is not pH dependent, it can still enter through the endosome. Interactio of poliovirus with PVR - polio virus receptor causes major conformational change in the virus which leads to the formation of the A particle - physically swollen (less dense). This is how the uncoating process works. 1. receptor binding 2. conformational change 3. Viral peptides: VP4 and VP1 form a channel in the cell membrane. 4. Release of the viral RNA
What specific amino acid residues coordinate two divalent metal ions during two metal ion catalysis mechanism?
Asp
What does assembly mean in the viral life cycle?
Assembly - hundreds to thousands of proteins assemble around the viral nucleic acid to form a protein shell called a capsid
What does attachment mean in the viral life cycle?
Attachment: the process of viral capsid or envelope proteins attaching to the receptors on a target cell
Describe the steps of the viral life cycle: attachment, penetration, uncoating, replication, transcription, translation, assembly, release
Attachment: the process of viral capsid or envelope proteins attaching to the receptors on a target cell; Penetration - the process of entering a host. Some viruses inject their genomes via needle-syringe-mechanism. Other viruses "trick" the cell into engulfing the virus into the cell (endocytosis), enveloped viruses undergo membrane fusion. Uncoating - removal of the viral capsid, unmasking of the genome; Replication - amplification (copying) of the viral genome, virus uses its host machinery. Sometimes occurs in phases (later on that one). Transcription - production of mRNA sgRNA; Translation - production of viral proteins, polyprotein, on protein at the 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 virus progeny following successful reproduction during a host-cell infection.
In the "particle to PFU ratio", "particle" can best be 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 statement is true? A. All viruses make us sick and can be lethal b. Our immune system can mange most viral infections c. humans are usually infected with one virus at a time d. the press is usually correct in their virology reporting e. our immune system cannot handle most viral infections
B. our immune system can manage most viral infections
What type of cells does HPV infect? How?
Basal epithelial cells of skin and mucus membranes through micro-wounds or hair follicles
Why do viruses need metastability?
Because viral particles exist in too states that require them to be stable, but not too stable. In one state, they must protect the genome (stable) then must come apart on infection (unstable)
Why does scratching the blisters cause infection of infected cells?
Because when blisters are scratched, it releases VZV virions from one cell which then attach to the plasma membrane of an adjacent cell and start the replication cycle in a new cell. Thus, scratchung blisters causes fluid to spread and the release of VZV virions which infect uninfected cells
How is Varicella transmitted from person to person?
By droplets in the air from coughing, sneezing, or laughing or by direct contact with fluid from teardrop vesicles. However if you are pregnant and contact chicken pox, it can spread through vertical transmission.
How big is an average virus? A. as big as an animal cell B. as big as a plant cell C. Between a size of ribsome and bacterium D. I don't know
C. Between a size of ribosome and bacterium
A ____ and ___ cell is the only cell that can take up a virus particle and replicate it A. Naive and resistant B. primary and permissive C. susceptible and permissive D. susceptible and naive E. continuous and immortal
C. susceptible and permissive
What are cellular, tissue, and species 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
Who discovered the connection between cervical cancer and HPV?
Dr. Harald zur Hausen
What happens during latent and lytic stages of viral replication?
During latent cycle, a virus is passively replicated along with it host's genome. In this passive cycle, no viruses are produced. When the latent virus is triggered to begin producing viral progeny it will begin to replicate, and we call this process the lytic cyle
When doing a plaque assay, what is the purpose of adding a semi-solid agar overlay on the monolayer of infected cells? A. to stablize 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 two early genes are the main cancer-causing genes in HPV?
E6 and E7
What is the principle behind electron microscopy, x-ray crystallography, and Cryo-EM technologies?
Electron microscopy: produces a shadowing of a viral particle by staining it with an electron-dense material; X-ray crystallography is when you make a crystal of the virus, bombard it w/ monochromatic x-ray beam, then each atom within the virus particle scatters the radiation. Interactions of the scattered rays with one another form a diffraction pattern. (can't use larger viruses); Cryo-electron microscopy: samples are rapidly freezed and examined at very low temp in hydrated, vitrified state that preserve native structure. Increased resolution to atomic level. It creates a reconstruction
What is ELISA?
Enzyme-linked immunosorbent assay detecting viral antigens or antibodies. It is a way to physically measure virus particles. Antibodies used to visualize viral proteins (antigens) in infected cells or tissues. Direct immunostaining-antibody recognizes a viral protein and it is directly couple to a fluorescent dye or enzyme. Indirect immunostaining - primary and secondary antibodies are used (sandwich), provides stronger signal
Besides hemagglutination and ELISA, 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. 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)
What does it mean that HPV has a high host specificity?
It can only bind to a certain host like in this one, only binds to humans
What is the human virome?
It consists of persistant/latent infections (Ex. EBV 100%, VZV 95%, Herpes 80%), transient infections w/ animal cell viruses, endogenous retroviruses (8% of human DNA), bacteriophage predators of bacteria and archaea (10^10-10^11 per gram of stool)
What does it mean that a cell is resistant?
It has no receptor - it may or may not be competent to support viral replication
What are inclusion bodies?
It is a type of cytopathic effect. They are 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 aka they are viral factories of replication. Ex.) negi bodies in rabies, and cowdry bodies in CMV
What is one-step growth analysis and how is it performed?
It is when every single cell is infected at the same time, infection is synchronized. You perform this analysis by applying a virus to a cellular monolayer, letting it absorb, removing unabsorbed inoculum, wash cells, and add new medium. Then at different time point take viral sample and determine titer
What is MOI and how do we calculate it?
MOI stands for multiplicity of infection. It is the number of infectious particles added per cell. It is not the number of infectious particle each cell receive. 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
What are the largest known viruses? What is so special about them?
Mimivirus. They are 400 nm big! Almost as big as a cell. It pretends to be a microbe. It codes for proteins that normal cells do like chaperones or ind. amino acids. However, there is an even bigger virus! A pandoravirus. It is about 1.5 micron long. It codes for 500 proteins. Pithiovirus then e.coli
What types of endocytosis are used by viruses to enter a host cell?
Naked virus - endocytosis: virus attachment to cell surface receptor molecules and sinks into a clathrin coated pit. The pit invaginates and finally closes off creating a clathrin coated vesicle, i.e. Adenovirus. 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 Enveloped Virus - Endocytosis & Membrane fusion: virus enters cell by receptor mediated endocytosis. The cell membrane merges (fuses) with the endosome membrane and so the virus components are released, i.e. influenza virus.
Are viruses alive?
No because they cannot maintain homeostasis, they only replicate inside a host, viruses do not grow. They do not increase their size (no baby virus, mommy virus), Viruses use the cell's sources of energy, but they don't make it. Viruses do not respond to stimuli like a poke. Viruses adapt to their environment, but over a long period of time. Although, they do have different levels of organization. Thus, since they can't pass all the criteria for living things, they are not alive.
Does Varicella infect any animal hosts? Is it able to be zoonotic?
No it can only infect humans
Is chickenpox eradicated like smallpox?
No, 4 million people get chickenpox every year
Can other organisms besides viruses develop RdRps?
No, RdRps are exclusively viral
Can Varicella enter just any cell? What are the criteria of the type of cell it can enter?
No, the cell must be susceptible and permissive
Do viral RNAs have specific structure? What is a purpose of that structure
No...check
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 uridylynated pUpU. The genome is (+) sense and encodes one large polyprotein, which then is cleaved by two proteases 2Apro and 3Cpro into multiple products. VPg (23 aa) 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
What specific host proteins are involved in Polio virus replication?
PABP-Poly(A) - it binds to Poly(A) and PCbp
What is the equation used to calculate the plaque forming units?
PFUcounted/(dilution factor)*(amount plated) = PFU/ML
What does penetration mean in the viral life cycle?
Penetration - the process of entering a host. Some viruses inject their genomes via needle-syringe-mechanism. Other viruses "trick" the cell into engulfing the virus into the cell (endocytosis), enveloped viruses undergo membrane fusion.
Where do the 5' ends of influenza virus RNA come from?
Polyadenylation. It occurs when RdRp is attached to the 5' end of each (-) sense RNA segment and is stationary. The template is pulled through the polymerase. The 5' end of the template is anchored to Pol (red oval), and to the second active site of the enzyme (another red oval) The templated is pulled through until it reached the U track (5-7Us). Pol starts adding As by reiterative copying (non-template additions!)
What is cap-snatching mechanism?
Priming. It is when Cellular mRNAs have a 5' cap structure. Viral RdRp cleaves mRNAs 13 nts downstream and uses this fragment for priming. All eight segments of of influenza RNAs have 5' ends derived from host mRNAs
Describe the stop-start model of VSV mRNA synthesis
RdRp initiates synthesis at the 3' end of N gene (1st gene). After synthesis of N mRNA, RNA synthesis terminates at the intergenic region (ig - reach in Us). This is followed by RdRp reinitiation at the 3' end of the P gene. All 5 mRNAs are synthesized this way: stop-start-stop-start-etc...Once N mRNA is synthesized, the N protein is expressed, coats RNA template and prevents the stopping of RdRp (result: full length RNA synthesis)
How is poly(A) tail added to VSV mRNAs?
RdRp slippage! Poly(A) non-template addition! Poly(A) addition (An) is a result of copying of a sequence of seven Us present in each intergenic region (ig) followed by RdRp slippage. Polymerase slips on Us 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 is VPg?
RdRp. It acts as a primer for RNA synthesis
Why do some viruses need to package their own RdRp and other don't?
RdRps are always packaged into (-) sense RNA virus particles. A. (-) strand RNA genomes: coated with protein, they need to package RdRp within virions because the incoming genome can be neither translated nor copied B. (+) strand RNA genomes: naked (exceptions: retrovirus, coronavirus) don't need to carry RdRp, they encode the polymerase C. dsRNA genomes: carry RdRp because their (-) sense strand cannot be translated. Overall: some viruses need to package their own RdRp because they cannot be translated right off the bat
What is the consequence of genome segmentation?
Reassortment which means that the virus can manipulate itself, so it makes it harder to find a vaccine. One example of this is H7N9 and the influenza
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. Antibody to receptor blocks infection. Receptor gene confers susceptibility (more than one receptor may be involved). Disruption of receptor gene blocks infection
What does release mean in the viral life cycle?
Release - viral shedding refers to the release of virus progeny following successful reproduction during a host-cell infection.
What does replication mean in the viral life cycle?
Replication - amplification (copying) of the viral genome, virus uses its host machinery. Sometimes occurs in phases (later on that one).
What is unusual about dsRNA viruses replication?
Replication occurs in subviral particles. During cell entry, the virion passes through the lysosomal compartment and proteolysis of viral capsid activates the RNA synthetic machinery. The (+) sense RNA strand is used for translation and synthesis of (-) strand. What is unusual though is that genomic RNA never leaves the particle. Replication occurs within the particle. This is because dsRNA is highly immunogenic (produce an immune response) Ex. of this type of virus: Reoviruses, i.e. rotavirus
What types of viruses have helical symmetry?
Sendai virus - paramyxovirus, related to measles; Ebola virus - hemorrhagic virus; Vesicular stomatitis virus (VSV, related to Rabies); Tobacco Mosaic virus
What two human proteins were derived from retroviruses? What is their function?
Syncytin and arc. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. It is a protein produced by certain cells in placenta that directs the formation of the cellular boundary between the placenta and maternal tissue. Because it is similar to a retrovirus envelope protein (env), it causes the virus to fuse with its host cell. Arc is essential for long-lasting information storage in the mammalian brain. It contain homology to the GAG polyprotein that forms the viral capsid and is essential for viral infectivity. Arc self-assembles into virus-like capsids that encapsulate RNA. Arc is released from neurons in extracellular vesicles that mediate the transfer of Arc mRNA into new target cells, where it can undergo activity-dependent translation
What experiment lead to the discovery of RdRp origin?
The baltimore test. In this experiment he monitored the synthesis or viral RNA using radioactive nucleotides. Extracts from virus-infected cells were incubated with 4 ribonucleotides, including one radioactive to measure the incorporation into newly synthesized RNA. Drug inhibiting cellular polymerase was used (actynomycin D). Products appeared and further studies led to purification of active Polio virus RNA-dependent RNA polymerase (RdRp) that was able to copy viral RNA. The first experiments were done in (+) sense RNA virus: Polio virus
What is a viral envelope? Where does the viral envelope come from?
The envelope is a lipid bilayer derived from host cell. It is always derived from the host membrane. This is because the viral genome does not encode lipid synthetic machinery. The envelope acquired by budding of nucleocapsid through a cellular membrane can be any cell membrane, but is virus -specific.
What role does low pH play in influenza virus entry?
The low pH has a second very important role for influenza entry - when virus enters the endosomes 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
What is triangulation number and how is it used to describe viral structures
The number of "initial" triangles per new icosahedral triangle. When you bring the triangles together you create a larger face from same structural subunits
How is ebola virus entry different from other viruses?
The receptor that catalyzes fusion is in endosome. The virus is taken up by pinocytosis and ends up in the endosome. Viral glycoprotein is cleaved by enzyme in endosome and exposes protein NPC1 (cholesterol transporter). Then glycoprotein binds to NPC1 and that triggers the fusion.
How is VSV sgRNA and mRNA synthesis regulated?
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 mechanism is used by RdRp during synthesis?
The two-metal mechanism of polymerase catalysis. It is the universal mechanism for all polymerases. It is when Two Asp residues coordinate 2 Mg2+ ions that remove 2 phosphate groups (nucleophilic attack) leaving behind one that will join nascent T with A and so on...
What is the purpose of viral envelope glycoproteins?
They enable attachment, antigenic sites, fusion
What is the principle criteria for all the Alphaheresviridae viruses?
They have dsDNA genome, there viruses can become latent and reemerge later. They are neurotrophic, infect nerves, but also α herpesviruses, herpes simplex virus types 1 and 2, and varicella-zoster virus, have a short replicative cycle, induce cytopathology in monolayer cell cultures, and have a broad host range
What is a conformation of RdRp?
They resemble a right hand consisting of palm, fingers, and thumb domain, with the active site located at the palm.
How does bacteriophage T4 enter a cell?
This process is also referred to as a "hypodermic syringe" During this process, there is a conformational change in the baseplate (hexagon to extended star-shaped conformation). It 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. Then lysozyme molecules are released which forms a pore through which DNA enters
What two ways can Varicella use to enter the host cell?
Through fusion of viral lipid envelope with the plasma membrane or by endocytosis. During viral entry tegument proteins release and interact with host. Only in susceptible and permissive cells do the icosahedra capsid containing the viral dsDNA genome moving along the microtubules network toward the nucleus form.
What does transcription mean in the viral life cycle?
Transcription - production of mRNA sgRNA
What does translation mean in the viral life cycle?
Translation - production of viral proteins, polyprotein, on protein at the time, numerous strategies.
What does uncoating mean in the viral life cycle?
Uncoating - removal of the viral capsid, unmasking of the genome
What type of envelopes are there?
Unstructured envelope which has a different symmentry then nucleocapsid and it is not interact wit hit. and a Structured envelope that is anchored to the nucleocapsid under the membrane and has the same symmetry as the capsid (interacts with it)
What is the name of the virus causing chickenpox?
Varicella
How abundant are viruses?
Viruses are crazy abundant like we literally eat and breathe billions of virons regularly. There are 10^31 viral particles on earth and 10^13 viruses/mL in sea waters. However, viruses make up approx. 5% of biomass.
How big are viruses if we consider cellular scale (like plant, animal cells? Ribosomes? Proteins? What about in the micrometers or nanometers)
Viruses are very small. There are between a bacteria (10^-6 or 1 micrometer and ribosomes (10^7.5)
What are ambisense RNA genomes? How are they different from dsRNA viruses?
When a virus is made up of part positive (5') and part negative (3') RNA genome. The 5' contains ORFs and can be directly translated, but the 3' needs to be first transcribed into mRNA and then translated. They are different from dsRNA viruses in that one sense strand is positive and the other sense strand is negative. While the dsRNA contains a positive sense strand, it cannot be translated directly. The (-) sense RAN needs to be copied into (+) strand by RdRp, and then used for protein synthesis.
What is de novo initiation of RNA synthesis?
When the RdRp recognizes the end itself and there is no need for primer. A nucleoside triphosphate, sometimes referred to as the one-nucleotide primer, provides the 3' hydroxyl for the addition of the next nucleotide. It may occur at the 3' end of the viral RNA or from an internal base. Some examples of viruses that portray this are HCV and Reoviridae.
What is a provirus?
When the viral genome is integrated into, and replicates along with the host's genome. It is also called a retrovirus
Can chickenpox be prevented by vaccination? And if so what type of vaccine is used to prevent chickenpox?
Yes! It is called Varivax. It is a live atenuated vaccine, so they get part of the virus (weakened form), so their body develops an immunity to it and a long lasting immune response in someone w/o causing pathogenic symptoms
Are these endogenous retroviral sequences of any benefit to humans?
Yes! Some of these old viral 'invaders' in our DNA help us fight off modern infections, researchers found that they produce proteins that block the receptors through which newcomer viruses invade. Some proteins disrupt the replication of attacking viruses, leaving them unable to invade other cells. The vast majority of the viruses that infect us have little or no impact on our health or well being. Most viruses just pass through us. We ingest many non-animal viruses regularly w/ foods. In generally we need viruses because they are important to balance health and disease. They positively and negatively modulate the human microbiome. Viruses access the human body through food and through contact w/ plants, animals, and other humans. Environmental factors. i.e., immunity and genetic greatly influence the composition and dynamics of the human virome.
Do ribosomes and RNA polymerase collide?
Yes, in synthesis in 5' - 3' direction, translation in 3' - 5', but they have different timing, different cellular localization (spherules vs. cytoplasm), RNA structure (on and off switch)
What biophysical property was used to isolate viruses in the past?
a filter sterilization: where a sample from infected tissue is diluted and liquified and sent through a filter w/ pores of 220 nm.
What cellular organelle plays an important role in reovirus entry
a 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 subviral particle i.e. degradation by cellular proteases • The subsequent penetration step is unknown
What is a host range?
a spectrum of host cells the virus can infect. It is determined by the virus' requirements for attachment to host cell (host receptors) and availability within host cell of factors required for replication. You can have a broad host range: rabies virus can infect all mammals. Narrow host range: human cold virus infects only cell lining upper respiratory tract in humans
What is sialic acid?
a sugar moiety that is being used by influenza and parmoxyviruses for entry. Binding has to be terminated for productive infection to occur. Neuramidases cleaves sialic acid in the case of influenza virus. In general, carbohydrate molecules on lipids/proteins allow for viral binding
What is a virome?
a viral component of the human microbiome. The collection of all viruses that are found in or on humans, including viruses causing acute, persistant, or latent infection, and viruses integrated into the human genome, such as endogenous retroviruses
What are bacteriophages?
a virus that infects bacteria
What is affinity and avidity?
affinity is strength of attraction between a receptor and its ligand and avidity is the tightness of bonding due to multiple binding sites
What is the purpose of plaque assay?
allows you to clonally purify a virus population for a single infected cell and isolate mutants
What is a virus?
an infectious, obligate intracellular parasite comprising genetic material (DNA or RNA) surrounded by a protein coat and/or a membrane
During what age range should one get vaccinated for HPV?
b/t 9 and 26
Other than cervical cancer, what other cancers can HPV cause?
cancer of vulva, vagina, penis, anus, and oropharynx
What form is the VZV genome in when it leaves the nucleus (circular or linear)
circular
Describe the protein priming mechanisms during Polio virus replication.
circularization of the genome, role of PABP, PCbp, Vpg, dis acting motifs, protein priming
Influenza virus pleiomorphy characterized by
cryoelectron tomography
Where does translation happen in the cell?
cytoplasm. Translated proteins go back to cytoplasm to be packaged into newly made capsids to leve the nucleus in vesicles
What does it mean that virions are spring loaded with energy?
during assembly energy is put into the structure. That energy is used for diassembly if cell provides proper signal. Virus particles go through energy transitions. Unfavorable energy barrier must be overcome i.e. through interactions w/ receptor, pH, cleavage of their coat proteins
what is the first event of virus life cycle?
entry into host cell. It often limits infection to the "correct" cell. Viruses are too large to simply cross the membrane without involvement of other complex molecular interactions (too large to diffuse)
What are the other components of virions?
enzymes, polymerases, integrases, accessory proteins, proteases, poly(a) polymerase, capping enzymes, topoisomerase, cellular components, tRNAs, histones, lipids
How is the one-step growth curve different for viruses with high particle-to-PFU ratio vs viruses with low particle-to-PFU ratio?
for one you get this high ratio because of defective particles.
What viruses do not use receptors as a point of entry to the host cell?
fungi because they spread during cellular division and plants (insects, mechanical damage)
Where does fusion occur and how?
fusion occurs at the plasma membrane on the cell surface. For measles/mumps virus: viral HN glycoprotein recognizes the cellular receptor that triggers conformational change in viral fusion peptide, that inserts its hydrophobic domain into cellular membrane. Another example is HIV: HIV SU protein is right next to TM (transmembrane protein that includes fusion peptide), binding to CD4 first, and then CCR co-receptor triggers the rearrangement of the fusion peptide
What is the shape of the HPV capsid?
icosahedral capsid (T=7)
What is a provirus?
integrated cDNA of retrovirus into host genome
What does it mean that virus is asymptomatic?
it doesn't produce or show symptoms. Polyomavius is an example of this. However, polymavirus can cause life-threatening pathologies in immunocompromised hosts.
What is the significance of the polymavirus?
it has been with humans forever. Everyone has it. Because of this, it evolved w/ humans and because of this, you can use it to track human migration by looking at their genomes and how they changed.
What is the sputnik virus?
it infects mimivirus, affects the normal morphogenesis and production of mimivirus
What is transfection? How is it used to study viruses?
it is an infectious DNA clone. transfection is the production of infectious virus after transformation of cells by viral DNA , first done with bacteriophage lambda. It is used to engineer mutations into viral genomes through deletion, insertion, substitution, nonsense, missense, and viral vectors. Transformation-infection
What is end-point dilution assay? And when is it used?
it is for viruses that do not form plaques or obvious CPE. Serial dilutions of a virus stock are inoculated into replicates (multi-well plate). The number of test units that have become infected is then determined for each dilution (cytopathic effect). At high dilutions, no cells are infected (10^_7), too dilute. 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 at 50% infectious does (TCID50) per mililiter. Ex.) 10^5 ID50 per mL
What role does differentiation of the host cell play in the expression of HPV genes?
it is linked to the replication cycle
What is a virus receptor?
it is where the virus binds on the cell surface. It can be ubiquitous/specific, with variable density. A virus is a host cell surface component recognized by the virus as a gateway to entry into the cell
What is plaque assay?
it provides a measure of the number of particles capable of forming plaque (infectious particles) per unit volume (PFU/ML). To do this, you have a viral stock. Then perform a 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. In the end if you see a plate will a circular clear zone this is because of an infectious viral particle. Thus an 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 stain). Only plates with 10-100 plaques are statistically valid. Plaque forming units (PFU/ML) can be calculated as follows: PFU counted/(dilution factor)*(amount plated) = PFU/ML
What does a virus' existence depend on?
its host
What type of countries have a higher incident rate of HPV infection?
less developed countries
Viral genomes must make _______ that can be read by host ribosomes
mRNA
RdRps are always packaged into ___ sense RNA virus particles?
negative
Are all viral RNA genomes (+)-sense?
no
What information is never encoded in viral genomes?
no genes encoding the complete protein synthesis machinery (AARS, elF, TRNAs), no genes encoding proteins involved in energy production or membrane biosynthesis, no classical centromeres or telomere found in standard host chromosomes
Is there a cure for the virus?
no, but there are vaccines to prevent infection
Are all viruses dangerous to humans?
no, some actually help fight off infections.
Do all viruses bind to the same receptors?
no, they are specific to each viruses. Some may overlap, but not all
Where does transcription take place in the cell?
nucleus
When an infectious viral particle creates a circular clear zone of infected cells, this is called a
plaque
What are syncytia?
rounding of the infected cell, fusion with adjacent cells (e.g. paramyxoviruses: measles). Viral glycoprotein mediates fusion of an infected cell with neighboring cells leading to the formation of multi-nucleate enlarged cells called syncytia (mutlinuclear cells). Usually these syncytia are the result of expression of a viral fusion protein at the host cell membrane during viral replication
What is a subgenomic RNA?
shorter than genome RNA templates.
What was the first known viral receptor?
sialic acid
What type of genome does HPV have?
small non-enveloped double stranded dNA
What disease was chickenpox mistaken for?
smallpox
What is the cytopathic effect?
structural changes in host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to reproduce. This helps you know that the virus infected your cell. During this affect you may have morphological alterations like nuclear shrinking, proliferation of membrane, vacuoles in cytoplasm, synctium formation (cell fusion), or you could have inclusion bodies (virions in nucleus, clumps of ribosomes in virions or clumps of chromatin in nucleus)
What is the function of the late genes?
structure
What is the name for the characteristic blisters that form from having a Varicella infection?
teardrop vesicles that form from maculopapular rash
What did the Hershey-Chase experiment prove?
that DNA is the transforming material. They conducted this experiment by growing bacteriophages and placing radioactive isotopes P32 and S35 inside. Then centrifuged the bacteria to separate it. The bacterial pellet showed high P32 which proved that most of the phage DNA was in the bacteria. Thus proving that the genes of this phage are made of DNA
Once HPV enters the host cell, where does it go?
the circular dsDNA goes to the nucleus
What triggers the production of late genes for HPV
the differentiation of a cell to the granular epithelial layer
What is a virion?
the inanimate phase of a virus. Its like a crystal. Its there. They are not replicating.
What is hemaggulatination?
the physical measurement of virus particles or according to google, the clumping together of red blood cells. In a two-fold dilution of samples of differnt 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 dectectable virus (button present). Sample D causes hemaggluation up to the 1:1024 dilution, therefore the HA titer of this virus stock is 1024. It is the clumping of RBC b/c of virus is causing them to clump b/c virus is attaching to RBC if there is an antibody, the virus will not attack the RBCs
What does it mean that viral genomes are structurally diverse?
they are a lot of ways the genomes can be structured i.e. linear, gapped, double-stranded
How do histones affect gene regulation, transcription, and/or translation of HPV genes?
they function to regulate the expression of early and late genes
What is remarkable about retroviral genome replication?
they have a provirus
How are influenza virus and VSV RNA synthesis similar?
they have sgRNA production, polyadenylation, and replication regulated by proteins
What is the universal mechanism for all polymerases?
two-metal mechanism of polymerase catalysis
What is the function of the early genes?
viral gene expression, replication, and survival
What is the correlation between viral genome size and mutation rate?
viruses with smaller genomes tend to mutate faster. Higher mutation rates, coupled with rapid replication, are also the basis for high rates of nucleotide substitution (mutations)
What is quasi-equivalence and what is the reason behind it?
when the positioning of the triangles are bond by interacting tail-to-tail and head-to-head. In this design, you end up with pentagons and hexagons. You get whereall A proteins are equivalent, all B are the same, and all C are the same. The reason for quasi-equivalence is there is evolutionary pressure to make a larger capsid using larger subunits helps very little, using more smaller subunits helps a lot . It is not possible to form icosahedral shell (of identical units in identical environments) with more than 60 subunits
You perform a serial dilution and count 17 plaques. Are you able to use this culture?
yes, it is between 10 and 100
How is capsid different from nucleocapsid?
A capsid is a protein shell surround genome. A nucleocapsid is nucleic acid/protein assembly within particle, subassembly with a clear substructure, i.e. in HIV
What does it mean that a cell is permissive?
A cell has the capacity to replicate virus - it may or may not be susceptible
What does HPV stand for?
Human Papillomavirus (HPV) it is a type of cervical cancer
Where does the active site reside in RdRp?
If you look at the palm conformation, it resides in the palm
You perform a serial dilution and count 17 plaques per 0.1 mL from 10^-6 dilution. How many PFU/mL is there?
1.7x10^8 PFU/ML -> 17/0.1x10^-6 = 17/10^-7 = 1.7x10^8 PFU/ML
What percentage of all cancer is attributed to HPV?
4.5%
What is the typical MOI we use for experiments? Why is this number usually higher than 1?
5-10. This number is usually higher than one because a low MOI means you have multiple bursts.
How many structural subunits build an icosahedral particle with T=1
60
How many types of viral genomes do we distinguish?
7
What percentage of human genome is derived from retroviruses?
8%
Where did viruses come from? There are three theories.
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. One reason for this could be that mimvirus does not differ appreciably from parasitic bacteria. Overall: cell that lost some things in replication 3. The virus-first hypothesis: the progressive and regressive hypothesis both assume that cells existed before viruses. Koonin and Martin postulated that viruses existed in a precellular world as self-replicating units. Over time these units, they argue became more organized and more complex. Eventually, enzymes for the synthesis of membranes and cell walls evolved, resulting in the formation of cells. Overall: people believe viruses were first and slowly evolved as they need more complex processes and assembled into a beautiful creation and then there was a cell
What are the functions of structural proteins?
1. Protection of the genome (assembly of a stable protective protein shell, specific recognition and packaging of the nucleic acid genome (high specificity), interaction w/ host cell membranes to form the envelope); 2. Delivery of the genome (bind host cell receptors, uncoating of the genome, fusion w/ cell membranes, transport of genome to the appropriate site)
What are the universal rules for RNA-directed synthesis?
1. RNA synthesis initiates and terminates at specific sites on the template. 2. RdRp may initiate synthesis de novo (like cellular DdRp) or require a primer. 3. Other viral and cell proteins may be required 4. RNA is synthesized by template-directed stepwise incorporation of NTPs, elongated in 5'-3' direction. 5. Some non-templated synthesis, i.e., polyadenylation
How do plant viruses enter a host cell?
1. 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. 2. Other viruses infect plants through a wound site created by a leaf-munching insect such as a beetle
How is metastability achieved?
1. Stable structure (subunits are very stable, repeating subunit is the key, created by symmetrical arrangement of many identical proteins to provide maximal contact. 2. 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)
Why is it difficult to study the origin and age of viruses?
1. The origin of viruses remains mysterious because of their patchy molecular and functional makeup - the fast evolution and high mutation rates. 2. Viruses 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 (they evolve and mutate. Thus it is difficult to track their phylogenetic origin). 3. No single gene has been identified that is shared by all viruses - there are common protein motifs in viral capsids, but these have likely come about through convergent evolution or horizontal gene transfer. 4. Viruses steal genes from cells - it has been suggested that viruses influence evolution of cells by donating new genes. 5. Just because viruses are simple doesn't mean that they are old
What viral properties do we use to classify viruses?
1. nature and sequence of nucleic acid in virion. 2. Symmetry of protein shell (capsid) 3. Presence or absence of lipid membrane (envelope) 4. Dimensions of virion and capsid.
What is a fusion protein? How is its conformation change triggered?
Certain viral fusion proteins induce cell-cell fusion when expressed on the cell surface as a consequence of infection, and cell-cell fusion can contribute to viral spread, virulence, persistence. There are two types of fusion proteins: type 1 and type 2. Type 1 fusion protein is the most common and understood fusion protein, trimer, i.e. influenza, VSV, retrovirus. Type II fusion proteins are not proteolytically (breakdown of peptides into smaller amino acids) activated, they have internal fusion peptides and no "coiled-coil" form; they are principally Beta-sheet, i.e. Dengue virus, Zika virus. The fusion of class II fusion proteins is triggered by low pH. For measles/mumps virus: viral HN glycoprotein recognizes the cellular receptor that triggers conformational change in viral fusion peptide, that inserts its hydrophobic domain into cellular membrane. Another example is HIV: HIV SU protein is right next to TM (transmembrane protein that includes fusion peptide), binding to CD4 first, and then CCR co-receptor triggers the rearrangement of the fusion peptide
What are the differences between the vaccines available for HPV?
Cervarix is a two-valent vaccine targeting HPV 16 and 18. Gardasil is a four-valent vaccine targeting HPV 16/18 and low risk types HPV 6 and 11. Gardasil 9 is a nine-valent vaccine that targets HPV 6/11/16/18 and the next five most carcinogenic types (HPV 31/33/45/52/58)
How does HPV transcribe and translate its DNA in the host cell?
Circular dsDNA reaches the nucleus and associates to the host cell DNA. In high-risk HPV types, the E2 gene is often lost leading to dysregulation of expression of oncoproteins E6 and E7. Transcription is polycistronic with overlapping open reading frames. Post-transcriptional events include polyadenylation and alternative splicing. Translation of viral mRNA is performed by host cell machinery
Which of the following methods for studying virion structure provides the best resolution?
Cryo-electron microscopy. During this process samples are rapidly frozen and examined at very low temp in hydrated, vitrified state that perserve native structure. It increases resolution to atomic level. It creates a 3D structure by looking at different angles and piecing them together
What systems are used to culture and store viruses?
Currently, a lot of viruses are being store in an embryonated chicken egg (fertilized). Different egg elements inoculated with different viruses depends on the suceptibility and permissively of the virus. Most influenza vaccines are grown in eggs. In the egg, there are four sites of inoculaton. The chorioallantoic membrane inoculation which is where herpes simplex virus, poxvirus, and rous sacroma virus are inserted; amniotic inoculation which are where influenza virus and mumps virus are injected; Yolk sac inoculation which is where the herpes simplex virus is inoculated. The nthe allantoic inoculation which is where the influenza virus, mumps virus, newcastle disease virus, and avian adenovirus are injected. Viruses can also be cultivated in tissue like primary human foreskin fibroblasts (have a limited amount of cycles), mouse fibroblast cell line (3T3), and human epithelial cell line (HeLa) (3T3 and HeLa cells are immortal they replicate continuously)
36 plaque formed from 1 mL of virus solution diluted to 10^-5. What is the PFU/ML? A. 36 B. 360 C. 36 x 10^6 D. 3.6 x 10^6 E. i don't know
D. 3.6 x 10^6
What is the function of genome diversity?
DNA and RNA based (RNA genomes appeared first in evolution, switch to DNA genomes, only RNA genomes on planet today are viral, viroids (relics of RNA world?), linear, circular, segmented, ds, ss, (+), (-). *Accommodation of various replication, transcription and translation strategies. Expansion of their coding capacity, recombination, reassortment, protection from mutations, evolution in general, expansion of host, vector range, general tropism
What first experiments lead to virus discovery in plants and humans?
First thought of viruses go back to ancient times to like 700 B.C. there are artifacts from egypt where their ruler was drawn as having polio. The greek poet homer mentioned rabies. Antonie van Leeuwenhoek created the first drawings of microorganisms. He was trying to prove that microorganism are everywhere. Pasteur disproved the spontaneous generation hypothesis by boiling broth and proving that it would stay sterile in a bottle neck swan flask. Then Koch developed the set of criteria that characterized a pathogen including viruses. The first idea that viruses existed was in plants. Ivanovsky filtered juice from crushed tobacco leaves and rubbed it on healthy plants and they got the disease. Then beijerinck filtered the juice and noticed tiny particles in juice and was like this must be the poison. The first animal disease discovered was foot and mouth disease discovered by loefler and frosch. They noticed that the tiny particles were passing through tiny filters (0.2 micrometer filters) and spread it on animals. Key concept: agents not only small, but replicate only in the host, not in broth.
How is fusion regulated and why?
Fusion must not occur in the wrong location. In neutral pH (plasma membrane), there is a second protein receptor interaction. In low pH, fusion occurs. Proteolytic cleavage activates the fusion protein for cleavage (class I). Cleavage of a second protein (class II) activates the fusion protein. Endosome fusion receptor.
All retroviruses contain at least 3 genes. What are they?
Gag (encoding structural proteins), pol (viral enzymes), env (surface envelope proteins). As well as long terminal repeats (LTRs) called retrotransposons
What information is encoded in viral genomes?
Gene products and regulatory signal 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 specific cellular receptors are bound by HIV, influenza viruses, adenovirus, and coxackievirus?
HIV: CD4 and CCR5/CXCR4 Influenza: Adenovirus and Coxsackievirus: use receptor CAR
How does HPV attach and enter the host cell?
HPV binds to the cell surface via interactions b/t L1 and HSPG (Heparan sulfate proteoglycans). A conformational change to expose L2 and interact with secondary receptor. The nHPV internalization (entry) occurs by clathrin-dependent endocytosis very slowly over several hours. Then it is possible that HPV could make use of multiple internalization pathways
What receptor(s) is(are) involved in attachment of HPV to the host cell?
HSPG (Heparan sulfate proteoglycans)
What are the main types of viral particles?
Helical, Icosahedral, Complex
