virology test 1

what is ELISA

A direct viral detection technique that uses virus‐specific antibodies to detect virus particles or secreted viral proteins in fluid.

what is plaque assay

A virological assay developed to count and measure viruses. We count these up to determine how many viruses are present, as each virus copy infects one cell. # of plaques (holes) = # of viruses in a sample

how do viruses enter the nucleus?

A. Influenza virus: genome released from endosomes, RNP enters the nucleus as a complex B. Herpes virus: pores encoat? protein docks on nuclear pore C. adenovirus: partial disassembly at nuclear pore D. parvovirus: bind nuclear pores, modify it, so that entire particle enters the nucleus

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

1. clathrin dependent endocytosis -- virus attaches to cell surface receptor molecules and sinks into clathrin coated pit (form a bubble around the virus) 2. receptor mediated endocytosis -- cell membrane merges with endoscope membrane and virus components release 3. membrane fusion -- when virus enters cell when its outer membrane fused with plasma membrane at cell surface

what is the function of genome diversity

DNA and RNA bases ???

What two early genes are the main cancer causing genes in HPV?

E6 and E7

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

SYNCYTIN (produced in placenta and directs the formation of cellular boundary between placenta and maternal tissue), and ARC (essential for long lasting information storage in the mammalian brain and also essential for viral infectivity)

what is siallic acid

Siallic acid is a sugar moiety that's being used by influenza for entry

what happens during polio virus entry with VP1 and VP4

VP1 AND VP4 form a channel in the cell membrane to allow the release of viral DNA

describe the protein priming mechanisms during polio virus replication

VPg is covalently attached to the 5' end of the genome (acts as a primer for RNA synthesis after it is uridylynated)

how are influenza virus and VSV RNA synthesis similar

as soon as NP (nucleoprotein) iw expressed from mRNA it coats nascent transcript and prevents that termination

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

aspartic acid residues

Why does scratching the blisters cause infection of infected cells?

because it releases the fluid -- releases the VZV virions

why do viruses need metastability

because they need to be stable enough to protect the genome but they also need to be able to come apart on infection in order to release the genome

Can Varicella enter just any cell? What are the criteria of the type of cell it can enter?

can ONLY enter susceptible and permissive cells (the viral dsDNA genome moves along the microtubule network toward the nucleus)

Other than cervical cancer, what other cancers can HPV cause?

cancer of the vulva, vagina, penis, anus, and oropharynx -- infected individuals can often be asymptomatic and the virus can remain dormant for years

what is cap-snatching mechanism

cellular mRNA have 5' cap -- viral RdRp cleaves mRNA 13nt downstream and uses the fragment for priming *it is a priming mechanism*

what is hemagglutination

clumping together of red blood cells

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

criteria: - receptor binds virus particle - antibody to receptor blocks infection - receptor gene confers susceptibility (more than one receptor can be involved) - disruption of receptor gene blocks infection methodologies = ?

what types of viral enveloped are there

ectodomain (attachment, antigenic site, fusion), internal domain (assembly), oligomeric (spikes)

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

electron microscopy (most widely used method, limited resolution -- detailed structural interpretation impossible because4 it is only shadowing of viral particle), X-ray crystallography (viral crystals are bombarded with monochromatic x ray beams and each atom within the virus particle scatters the radiation and the scattering forms a diffraction pattern), cryo-EM (uses frozen samples that preserve the native structure and increases resolution to atomic level)

what does it mean that irons are spring loaded with energy

energy is put into their structure -- when closed/stable there are energy stores and when you want to release, it goes through different energy stages to allow open and release. the energy is used for disassembly if cell provides proper signal

what is the first event of virus life cycle

entry (often limits infection to the "correct" cell ; permissive and susceptible cell)

how abundant are viruses?

extremely abundant -- viruses outnumber hosts by 10 fold in sea water. 95% abundance but only 5% of biomass bc of small size

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

fungi (they spread during cellular division) and plants (insect, mechanical damage)

how is fusion regulated and why

fusion is regulated based on location, neutral pH (low pH evokes a change to fusion peptide)

what information is encoded in viral genomes

gene products and regulatory signals for replication of viral genome, assembly and packaging of genome, regulation and timing of replication cycle, modulation of host defenses, spread to other cells and hosts

what information is NEVER encoded in viral genomes

genes encoding: complete protein synthesis machinery, proteins involved in energy production or membrane biosynthesis, classical centromeres or telomeres found in standard host chromosomes

what are the main types of viral particles

helical, icosahedral, complex

what disease was chicken pox mistaken for

herpes

how is the one step growth curve different for viruses with high particle-to-PFU ratio vs viruses with low particle-to-PFU ratio

high particle to PFU ratios has a larger "burst" and ones with low particle to pFU have smaller burst

What does HPV stand for?

human papilloma virus

What is the shape of HPV capsid?

icosahedral capsid (T=7) -- composed of L1 and L2 capsid proteins

what is one step growth analysis and how is it performed

if a virus has one step growth cycle means that every single cell is infected at the same time (synchronized infection)

what other assays can we use to measure viral infectivity

immunochromatographic assay, PCR

what is a virus

an infectious, obligate intracellular parasite compromising genetic material (DNA or RNA) surrounded by a protein coat and/or membrane (obligate = require host, parasite = not beneficial to host usually)

what systems are used to culture and store viruses

animals are helpful (ex. embyonated chicken egg) -- different egg elements inoculated with different viruses, depends on susceptibility and permissiveness of the virus . cell systems are used to study the infectious cycle

why some viruses use complex structures with mixed symmetry? do you know examples of these viruses? what these modifications serve for?

the presence of proteins is devoted to specialized roles. ex. adenovirus, reovirus (two layers to survive the gut), bacteriophages (tails allow for more movement)

what is a host range

the spectrum of host cells a virus can infect

how is the metastability achieved

the subunits are very stable (repeating subunits is the key -- symmetrical arrangement of many identical proteins to provide max contact) but the structure is not usually permanently bonded together (no covalent bonding -- can be taken apart or loosened on infection to release genome)

what is human virome

the viral component of the human micro biome. the collection of viruses that are found in or on humans

do viral RNAs have specific structure? what is the purpose of that structure

there are many loops that are different landing sites for the proteins that regulate transcription/translation and can turn on/off things for replication

what is a consequence of genome segmentation

there are multiple strains of the same virus -- mixed segments/progeny mixed

what is the principle criteria for all the Alphaherpesviridae viruses?

they are all three neurotropic human viruses (invade nerve cells), broad host range, short rep cycle

where do the 5' ends of influenza virus RNA come from

they are derived from host mRNA

what is the purpose of viral envelope glycoproteins

they are important for docking the virus to the cell membrane (ex. ectodomain, internal domain, oligomeric)

why is it difficult to study the origin and age of viruses

they have patchy molecular and functional makeup (they have *fast evolution* and *high mutation rates*) -- they also have *many evolutionary origins* (no single gene has been identifies that is shared by all viruses)

how do we know that these sequences are derived from retroviruses

they have similar homology to certain poly proteins

what is cytopathic effect (CPE)

this is how you know that the virus infected you cell -- structural changes in host cells that are caused by viral invasion. Also, infecting virus causes lysis of host cells or when the cell dies without lysis due to inability to reproduce

Where does transcription take place in the cell? Where does translation take place in the cell?

transcription in nucleus??? and translation happens in the cytoplasm

How does HPV transcribe and translate its DNA in the host cell?

transcription is polycistronic with overlapping reading frames and translation of viral mRNA is performed by host cell machinery

what is transfection? how is it used to study viruses

transfection is the production of infectious virus after transformation of cells by viral DNA. it is used to study the function of genes or gene products, by enhancing or inhibiting specific gene expression in cells

what is the typical MOI we use for experiments? why is this number usually higher than 1

typical MOI is 5-10 to achieve one step growth cycle -- why is it larger than 1??????

what type of modification VPg must undergo to serve as primer

uridylynation

what is the name of the virus causing chicken pox

varicella

how big are viruses if we consider cellular scale (like plant, animal cells? ribosomes? proteins? what about the uM or nM scale?)

very small -- between bacteria and ribosomes.

What is the function of the early genes?

viral gene expression, replication, and survival

what is a Baltimore system

virus classification systemthat groups viruses into families, depending on their type of genome and their method of replication.

what is the correlation between viral genome size and mutation rate

viruses with small genome tend to mutate faster. largest known viral genome = pandoravirus and smallest is viroid

what is provirus

when the viral genome is integrated into and replicates with the host genome (also known as provirus)

what role does a Low pH play in influenza virus entry

when virus enters endosomes its interior gets acidified

what is de novo initiation of RNA synthesis

where the primer is 1 nucleotide

do all viruses bind to the same receptors

- Viruses can bidn multiple distinct receptors, and individual cellular proteins may be receptors for multiple viruses

what is affinity and avidity

- affinity = strength of attraction between a receptor and its ligand - avidity = binding due to number of binding sites

why some viruses need to package their own RdRp and some don't

- genomes of all RNA viruses *except retroviruses* encode RNA-dependent RNA polymerase (RdRp) 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

What are the IE genes and what do they transcribe for? The E genes? The LE genes?

- IE gene = immediate early mRNAs (proteins transported to nucleus to activate early genes) - E gene = early mRNAs (function in DNA replication and in the production of substrates for DNA synthesis) - LE gene = late mRNAs (they are proteins inserted into rough ER and some are transported to nucleus for assembly of nucleocapsid -- DNA cleavage to release genome)

how does bacteriophage T4 enter a cell

- Initial binding is reversible and electrostatic - the outer most part of long tail fiber binds to surface Lipopolysaccharides of the bacterium - Binding is "additive" until all 6 tail fibers are bound, binding 3 fibers is needed to initiate infection - The virus may "browse" the surface looking for suitable site for penetration

what is primer dependent RNA initiation

- Protein-linked oligonucleotide or capped oligonucleotide serve as a primer for RNA synthesis by RdRp - Terminal protein provides a hydroxyl group (in tyrosine or serine residue) to which the first oligonucleotide can be linked

explain the stop-start model for VSV mRNA synthesis

- RNA polymerase binds at the 3' end of N gene - initiation of mRNA synthesis at 3' end of N gene - synthesize N mRNA and terminate at intergenic region - reinitiate at 3' end of P gene

what are the universal rules for RNA directed RNA synthesis

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

how is Ebola virus entry different from other viruses

- Receptor that catalyzes fusion in endosomes - Virus taken up by pinocytosis and ends up in endosome

what is the particle-to-PFU ratio

(# of physical particles/ # infectious particles) it can determine how many viral particles are actually infectious

what mechanism is used by RdRp during synthesis

*two metal ion catalysis*: two conserved aspartic acid residues coordinate two divalent metal ions one metal ion promotes deprotonation of the 3' OH group of the nascent strand. the other ion stabilizes the transition state at the alpha phosphate of NTP

where do viruses replicate

+ sense RNA viruses replicate in cytoplasm of infected cells. viruses need to establish an intracellular environment that concentrates the viral proteins and allows productive replication

what is RdRp and what is its origin

RdRp are enzymes (responsible for RNA synthesis) and are essential for all viruses with RNA genomes. their origin = viral

what specific RNA structures regulate polio virus genome replication

1. Cloverleaf - VPg attachement 2. Cre-cis acting replication element 3. Pseudoknot- involved in replication ?? *genome is circularized for replication 5'-3'*

how do plant viruses enter a host cell

1. Viral capsid shell opens to release the viral genome, which is translated into proteins that direct the formation of viral factory from membranes of the ER and other organelles 2. Antiviral proteins patrol cells for invading pathogens, but 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 plants transport streams

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

1. attachment (process of viral capsid or envelope proteins attaching to the receptors on a target cell), 2. penetration (process of entering a host: some inject their genome via needle syringe mechanism and some "trick" the cell into engulfing the virus into the cell by endocytosis), 3. uncoating (removal of viral capsid, unmasking of the genome), 4. replication (amplification, copying, of the viral genome and virus uses host machinery), 5. transcription ( production of mRNA, sgRNA), 6. translation (production of viral proteins, polyprotein, one protein at the time, numerous strategies), 7. assembly (hundreds to thousands of proteins assemble around the viral nucleic acid to form a protein shell called capsid), 8. release (viral shedding refers to the release of virus progeny following successful reproduction during a host cell infection)

how is varicella transmitted from person to person

1. by droplets in the air from coughing, sneezing, or laughing 2. by direct contact with fluid from teardrop vesicles (and if you are pregnant with chicken pox, it can spread through vertical transmission)

what are cellular, tissue, and species tropisms

1. cellular tropism = virus replicates in one cell type but not another 2. tissue tropism = virus replicates in one particular tissue or organ but not another 3. species tropism - virus replicates in one host species but not another

where did viruses come from (know three hypothesis)

1. escape hypothesis (virus originated through progressive process, mobile genetic elements capable of moving within a genome gained the ability to exit one cell and enter another0 2. regressive hypothesis (existing viruses may have evolved from more complex organisms that lost genetic info over time 3. virus first hypothesis (the theory that viruses existed first -- koonin and Martin thought that viruses existed in a pre cellular world as a self replicating units and overtime they became more organized and complex forming cells

What two ways can Varicella use to enter the host cell?

1. fusion of viral lipid envelope 2. endocytosis

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

1. susceptible = has a functional receptor for a given virus -- the cell may or may not be able to support viral replication. virus can bind a receptor, 2. resistant = has no receptor -- may or may not be competent to support viral replication, 3. permissive = capacity to replicate virus -- may or may not be susceptible **susceptible AND permissive cell is the only kind of cell that can take up a virus particle and replicate it**

What percentage of all cancer is attributed to HPV?

4.5%

how many types of viral genomes do we distinguish

7 (dsDNA, gapped dsDNA, ssDNA, dsRNA, ss+ sense, ss- sense, ss+ RNA with DNA intermediate)

what percentage of human genome is derived from retroviruses

8%

During what age range should one get vaccinated for HPV?

9-26

are all viral RNA genomes +/- sense

?

what is remarkable about retroviral genome replication

?

what does it mean that viral genomes are structurally diverse

??

what types of polymerase can we distinguish

??

what is a fusion protein? how is its conformation change triggered

Certain viral fusion proteins induce cell-cell fusion when expressed on cell surface as consequence of infection, and cell-cell fusion can contribute to viral spread, virulence, persistence

Who discovered the connection between cervical cancer and HPV?

Dr. Harold Zur Hausen

what specific cellular receptors are bound by HIV, influenza, adenovirus, coxackiecirus

HIV, utilizes CD4 and CCR5 or CXCR4 = one receptor for docking and one for endocytosis, Siallic acid is a sugar moiety that's being used by influenza, Adenovirus and coxsackievirus B3 have common primary receptor, CAR= cozackie virus and adenovirus receptor

How does HPV attach and enter the host cell?

HPV binds to the cell surface via interactions between L1 and HSPG -- conformational change to expose L2 and interact with secondary receptor. HPV entry occurs by clathrin-dependent endocytosis slowly

How do histones affect gene regulation, transcription, and/or translation of HPV genes?

Histones can function to regulate the expression of the early and late genes

what did Hershey-chase experiment prove

It proved that DNA is the hereditary material, not protein.

What receptor(s) is(are) involved in attachment of HPV to the host cell?

L1, HSPG, and L2

what is MOI and how do we calculate it

MOI is the multiplicity of infection (number of infectious particles ADDED per cell) -- to calculate take the number of viral particles used per well then divide number of cells originally seeded in the well

are viruses alive

NO!! VIRUSES ARE NOT ALIVE -- do not grow and do not maintain homeostasis

what is VPg

RdRp, orange (a protein)

what specific host proteins are involved in polio virus replication

PABP-polyA binding protein binds polyA and pcbs is a cell protein that anchors the RNA to the membrane

how do we calculate PFU/ML

PFU/mL = PFU counted/((dilution factor)x(amt plated))

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

T=1, 60 subunits forming pentamers (all in identical environment)

what types of viruses have helical symmetry

sendai virus (paramyxovirus, related to measles, responsible for highly transmissible respiratory tract infection in mice), Ebola virus, VSV **all animal/human viruses with helical nucleocapsid are enveloped**

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

The extent of similarity between these structurally unique environments occupied by the chemically identical subunits in the virus capsid. The reason behind it: using larger subunits helps to make larger capsid, using more smaller subunits helps a lot??

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)

can chickenpox be prevented by vaccination? and if so, what type of vaccine is used to prevent it

YES -- varivax is a live attenuated vaccine (weakened forms of the virus) and it is able to create a long lasting immune response

what are ambisense RNA genomes? how are they different from dsRNA viruses?

a genome which both nucleic acid strands encode for proteins. viruses with dsRNA, −ssRNA, or ambisense genomes must carry their own RdRp protein into the cell in order for transcription to occur. ??

what is a virus receptor

a host cell surface component recognized by virus as a gateway to entry into the cell (essential for all viruses except fungi and plants)

what are bacteriophases

a virus that parasitizes a bacterium by infecting it and reproducing inside it.

what is the purpose of plaque assay

allows you to clonally purify a virus population for a single infected cell. also an isolation of mutants

Once HPV enters the host cell, where does it go?

it goes to the nucleus to associate to the host cell DNA

What type of cells does HPV infect? How?

it infects basal epithelial cells through micro wounds or hair follicles

what is unusual about dsRNA virus replication

it is located in diff place

what is the end point dilution assay and when is it used

it is used for viruses that don't form plaques or obvious CPE. the end point is the dilution of virus that affects 50% of the tested units (in between)

What form is the VZV genome in when it leaves the nucleus (circular or linear)?

it starts as linear but becomes circular after leaving the nucleus

what happens during latent and lytic stages of viral replication

latent stage = when a virus is passively replicated along with the host genome (no viruses produced in this passive cycle), lytic cycle = when the latent virus is triggered to begin producing viral progeny and begins to replicate

What type of countries have a higher incident rate of HPV infection?

less developed countries

what first experiments lead to virus discovery in plants and humans

loeffler and fresh did an experiment -- agent foot and mouth disease is filterable (key: agent not only small, but replicate only in the host not it broth)

what cellular organelle plays important role in reovirus entry

lysosome -- - Reovirus have complex double capsid, which is very stable to low pH (gastrointestinal viruses; rotavirus) - Lysosomal proteases degrade the outer capsid to form a subviral particle i.e. degradation of cellular proteases

What does it mean that HPV has a high host specificity?

means that they are highly specific in which hosts they infect. HPV is specific for humans (with no known vectors)

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

mimivirus (mimicking microbe/pretends to be a microbe), pandoravirus, sputnik virus (can infect other viruses) -- they infect amoeba

is chickenpox eradicated like smallpox

no

are all viruses dangerous to humans

no most of then have little or no impact on our health -- help with balance between heath and disease

do ribosomes and RNA polymerase collide

no they do not -- different timing, different cellular localization (spherules vs cytoplasm) and RNA structure (on/off switch)

is there a cure for the virus?

no treatment for virus but there are treatments for the symptoms and vaccinations to prevent infection

Does Varicella infect any animal hosts? Is it able to be zoonotic?

no-- only infects humans

what is triangulation number and how is it used to describe viral structures

number of initial triangles per new icosahedral triangle

where does the fusion occur and how does it happen?

occurs at the plasma membrane and it happens when the binding of specific receptor triggers conformational change

What role does differentiation of the host cell play in the expression of HPV genes?

once the cell differentiates to the granular epithelial layer, L1 and L2 proteins are expressed to encapsulate the viral genomes to prepare for release

when do we observe one hit and two hit kinetics of viral infections

one-hit kinetics is when the virus is said to initiate infection with only one infectious particle. two-hit kinetics ow when two different types of virus particles must infect a cell to ensure replication

how is polyA tail added to VSV mRNAs

poly A addition is a result of copying a sequence of 7 Us present in each intergenic region followed by RdRp slippage -- polymerase slips on Us and starts adding more As

what are the functions of structural proteins

protection of the genome (assembly of stable protective protein shell), delivery of the genome 9makes sure the virus goes to cell thats susceptible and permissive-- bind host cell receptors, uncoating of genome, fusion of cell membranes. transport of genome to appropriate size)

what biophysical property was used to isolate viruses in the past

replicates in hosts, not in broth

what is a conformation of RdRp

resemble a right hand consisting of palm, finger, and thumb domains with the active site located in the palm

what is a subgenomic RNA

shorter than genome RNA templates

what does it mean that virus is asymptomatic

showing no symptoms -- not causing harm

what was the first known viral receptor

silica acid -- for influenza virus

What type of genome does HPV have?

small non enveloped dsDNA

what viral properties do we use to classify them

specific nature and sequence of nucleic acid in virion, symmetry of protein shell (capsid), presence

What is the function of the late genes?

structural function

what is a subunit and how does it differ from a structural unit

subunit is a single polyprotein chain and a structural unit is a unit from which capsids or nucleocapsids are built (included one or more subunits)

what are syncytia? inclusion bodies?

syncytia is rounding of the infected cell, fusion with adjacent cells. Viral glycoprotein mediates fusion of infected cell with neighboring cells leading to formation of multi-nucleate enlarged cells (usually a result of expression of viral fusion protein at host cell membrane during viral replication. Inclusion bodies are elementary bodies, nuclear cytoplasmic aggregates of stable substances, usually proteins (typically represent sites of viral multiplication in cell and usually consist of viral capsid proteins) **not all viruses cause CPE**

what is the name for the characteristic blisters that form from having a varicella infection

teardrop vesicles arise from the starting maculopapular rash

What type of viral particle does VZV have? What is the name of the structure it forms?

the VZV has a single copy of linear dsDNA and forms a icosahedral structure with a nucleocapsid

how is the capsid different from nucleocapsid

the capsid is the protein shell surrounding the genome and the nucleocapsid is the nucleic acid AND the capsid together

What triggers the production of the late genes for HPV?

the differentiation to the granular epithelial layer

what is a viron

the early stage of a virus (inanimate phase, not replicating)

What are the differences between the vaccines available for HPV?

the effectiveness -- one is 2 valent vaccine, one is 4 valent vaccine, and one is 9 valent vaccine (most effective)

what is a viral envelope? where does the viral envelope come from

the envelope is the lipid bilayer and is virus specific**always derived from host membrane**

what experiment lead to the discovery of RdRp origin

the experiment led by Baltimore -- monitoring the synthesis of viral RNA using radioactive nucleotides with polio virus (conclusion: there must be an enzyme responsible for viral RNA synthesis)

which of the following methods for studying virion structure provides the best resolution

x-ray crystallography

are these endogenous retroviral sequences of any benefit to humans

yes they have been repurposed to fight modern viruses 9produce proteins that block the receptors through which new viruses invade)