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What is the mechanism of Viral entry for NON ENVELOPED Viruses?
Receptor mediated endocytosis in a clatherin coated vesicle. Acidifcation from the viral proteins will triggers virus genome release.
What is the only difference between nonenveloped and enveloped viral entry for receptor mediated endocytosis?
In a nonenveloped virus, acidification causes the clatherin coat to degrade and the capsid to degrade to release genome. In an enveloped virus, acidification causes the FUSION of two membranes which releases the genome.
What is the mechanism of viral entry of ENVELOPED viruses?
There is Receptor mediated fusion with the plasma membrane that has no Acidification and that leaves the viral envelope as a patch on the plasma membrane once fused and receptor mediated endocytosis that has acidification to cause fusion of the two membranes.
What is acidification?
Change in pH.
What is a coreceptor?
a Receptor required ALONG with the main receptor for the next steps of viral infection. Usually additional cell surface proteins (HIV has CD4 and CCR5 depending on what its infecting)
What is an alternative receptor?
A receptor that gets used if the main receptor is not present. It has a lower effeciency (Measles vaccine uses a different receptor than its wildtype version).
What is syncytia formation?
Cell to Cell Fusion (of adjacent cells). Its the only way to avoid having to use a receptor for entry.

For this to work the cells must be in close contact and the virus has to be able to cause membrane fusion.
What is a virus that enters by plasma membrane fusion?
Pseudorabies virus (part of the herpesvirus family, not rabies virus)
If a virus does not require acidification how will it enter the cells?
receptor mediated fusion with the plasma membrane.
What is the first step of bacteriophage entry?
A WEAK INTERACTION between tail fiber protein and receptor.
What is the second step of bacteriophage entry
A STRONG interaction between tail pins and outter membrane.

This causes compression of the sheath and the tail tube can penetrate through the cell wall.
What is the third step of bacteriophage entry?
A VIRAL PILOT PROTEIN will enable the DNA translocation through the inner membrane.
What is a way to experimentally bypass using a receptor?
TRANSFECTION. For example, the VZV (chicken pox) can be given to suseptible cells that can take up the infectious DNA by non-specific cellular internalizaiton. You can then visualize this happening at a time progression using flourescent stain for glycoprotein genes.
WHich part of the viral cycles are generalized? Which are specific?
You can generalize early and late stages. BUt the middle stages are specific for each.
What are the late stages of viral replication?
Helical nucleocapsid assembly or icosehedral nucleocapsid assembly, formation of viral envelope and the budding of enveloped viruses.
What are the steps of Helical Capsid Assembly?
1. A two layer disc forms from capsomers.

2. The two layer disc attracts the viral RNA and it binds to it.

3. VIral RNA loops through the central hole.

4. Disc rearranges to form lockwasher shape and the RNA is threaded through as the helix forms.
What are the proteins that make up the Procapsid (for icosehedral viruses)?
coat proteins: surround capsid

pilot proteins: needed to infect other cells

scaffolding proteins: fill the inside of the capsid to make the shape of it before DNA is put in.

portal protein: where the genome will come out in the mature virion infection.
What happens when Immature procapsid is ready to assemble?
MATURATIONAL PROTEASES trigger conformational changes, scaffolding proteins come out, genome is either inserted into the empty capsid or the capsid assembles around genome (like polio virus).
How is a viral membrane formed and released?
The viral glycoproteins are made just like regular glycoproteins (through the GOlgi) and transported to the cell membrane where they are inserted.

The viral capsid will migrate when ready to the virus modified membrane and then bud into a free infectious virus.
Where does a virus get its lipids for the membrane?
All Lipids are derived entirely from cellular membranes, not from the new viral membrane.
Budding out occurs from which membranes?
can occur from either the nuclear or plasma membranes (herpes). Most RNA viruses ONLY bud from the plasma membrane.
What acts as the 'glue' to bring viral capsids to membrane areas?
The matrix, or tegument, proteins (the ones on the outside of the capsid)
What can be formed on accident during the budding process?
Empty capsids can bud and also dense bodies of aggregated matrix proteins can bud.
What are the nature of viral receptors usually used?
They are usually cellular transmembrane glycoproteins.
How specific is the interaction of the virus and the receptor?
It is HIGHLY Specific.
How do you grow virus in Vitro in primary cells (from living tissue)?
1. Mince tissue from mouse
2. Add collegenase, filter and pellet.
3. wash pellet (cells) and differentially centrifuge
4. Put cells into the culture dish with medium for dense cell clumps
5. Treat with trypsin and discard any loose cells so you only have the dense clumps.
How do you progress from a primary cells to a cell line?
To make a cell line, you want immortalized cells so you would to get cells from a tumor.

Therefore you put the primary cells back into an animal and then you would isolate and culture cells from tumor or mass. Furthur changes in cellular genes will lead to a continuos cell line that cannot be put back into an animal.
What are the two main outcomes possible when a virus gains entry into a cell culture (or cells)?
1. Abortive or Nonproductive infection: the cell lacks some element that is important for viral replication or virus has defective gene product. MAY LEAD TO LATENCY (depending on the virus)

2. Productive Infection: new infectious virus is produced.
If a virus cannot find a receptor in a cell culture, what possible outcome will come it?
It will not have any outcome because no entry will occur.
What are the three fates of the cell following virus infection?
1. Cell survives (persistent infection).

2. Cell dies by necrosis (inflammation response)

3. Cell dies by apoptosis (programmed, organized shutdown).
What is the process of necrosis?
Chromatin clumping, swollen organelles lead to disintegration which releases intracellular contents that illicit a inflammation response.
What is the process of apoptosis?
Cytoplasm condenses, organelles become compartmentalized which leads to apoptotic bodies that are eaten by the phagocytic cell.
What is the cytopathic effect of a virus?
Its the idea that a cell does not look the same after it has been infected with a virus.

Ex: change in properties of actin microfilaments in HSV-infected cells.
What is the most common way to titer a virus (determine # of infectious units)? How do you perform this?
Virus plaque assay.

Ex: you can get a 6 well plate grown with a monolayer of cells days before you infect those cells with your virus. You remove the growth media and You plate dilutions of your virus onto the plate (you do this in duplicate). After about an hour they absorb in and you can add media that has a solidfying agent in it so cells can only infect neighboring cells. A while later (24h to a week depending on virus) and then you remove the agar and add a stain that stains cells uninfected bright and leaves holes where the virus infected.
If you had 12 pfu on a 10^-7 plate what was your original titer if you plated 200ul?
convert pfu/well to pfu/ml:

12 x 5 (200 ul goes into 1000 ul 5 times) = 60 pfu/ml

Then times that by your dilution:

60pfu/ml x 10^7 = 6 x 10^8 pfu/ml
How can you measure the tranforming activity of a virus?
Tranforming activity can be measured as 'foci' of cells that have lost contact inhibition. Foci are piled up cells, rounded up on top of other cells.
How oculd you determine the titration of a virus that does not form plaques well or transform well?
A endpoint Dilution assay (quantal assay)
How would you perform a quantal assay?
You would get a 24well plates and make up viral dilutions (which have an endpoint: no infectious virus). You plate the virus into each well of cells and you see using antibody what viral infections were made.

You get the percent infected wells and you log those on the Y axis against the log dilution and from this you can extrapolate the dilution for 50% infected wells.
WHat are the two ways to purifying viruses?

Which one is better for studying membrane bound viruses?
Equilibrium density centrifugation and differential centrifugation

Equilibrium is better for membrane bound because the lipid membrane will band at a certain density.
HOw does equilibrium centrifugation work?
You have a tube with a sucrose gradient that you have made. You add your sample onto the top and the virus will sink down until it hits its own density in the gradient. You then can punch a hole in the bottom of the tube and take fractions unti you get to your virus.
When does the virus come off in differential centrifugation?
The virus will pellet at about 100,000 times the force of gravity with the small ribosomes and organelles.
How can you tell if viral proteins are in equimolar proportions?
By an SDS gel. You run it out and then have band intensity compared to migration distance (MW) and if you see that the LOWEST one is 1/4 (if there were 4) of the BIGGER protein, then they are equimolar. If they have no proportions then they are not equimolar.
If you had a ratio of 1 to 5 what would it mean (in a sds gel)?

What about 20 to 3?
The proteins are associated with the vertices.

The proteins are associated with the edges.
What protein composition will you see in a complex virus like Adenovirus or HSV capsids?
You will see no clear pattern. You may see a subset of proteins in a complex that are equimolar but usually not.
What can you find out from Northern Blotting? How would you do this?
You can characterize the viral mRNA expressed during infection by probing the blot with sections of RNA/DNA probes from part of the whole genome.

EXPRESSION PATTERNS CHANGE WITH TIME (like 3 hrs. gives one pattern and at another 5 hours you will get a different patterns of the SAME PART OF THE GENE).
What 3 things should a cloning vector have?
origin of replication, selection gene, cloning site.
Explain how complementation may function with viruses?
If you had two mutant viruses, and the defect is in different genes then grown in a cell they could complement each other and end up with the same genotype as the parents. If the defect are on the same gene, no virus will grow at all.
What is the frequency of recombination with there is a double infection of cells?
There is a LOW Frequency of recombination between the two genomes under permissive conditions.
How do you measure complementation?
You could find the complementation index:

yield of infectious virus from double infections/yield of infectious virus from single infections at the RESTRICTIVE conditions (which is usually no virus at all or very low yield.
How do you measure recombination?
Recover the virus from a double infection under the PERMISSIVE conditions and look for the PROPORTION of viruses showing the WILDTYPE Genotype (or the double mutant phenotype. )
How does Marker Rescue methodology to engineer specific virus recombinants work? What viruses does it work best for?
Marker rescue works for large DNA viruses. It works by recombination to 'map' where a mutation lies.

Once you have a virus you can double transfect that virus and viral fragments and see if recombination occurs between the two. IF recombination occurs between the two to produce some Wildtype and some new recombinants then you know that that gene fragment you put in must have contained a mutation.
What are the differences between +, -, and dsRNA viruses?

Whats the Baltimore scheme of classification?
A +sense RNA does not need to encode its own transcriptase to make mRNA while the -sense RNA and the dsRNA have to have the viral transcriptase to make mRNA before translation can occur.

Baltimore scheme of classification is how the RNA are separated into +,-, and ds.
What are the two stages of Replication in +RNA viruses?

What is the template for the RI-1?

What is the template for the RI-2?
The two stages are RI-1 and RI-2. They use Replicases to do this.

The template in the RI-1 is the same sense virion RNA. (to make the opposite sense RNA)

The template in the RI-2 is the opposite sense RNA genome. (to produce a same sense intermediate that will be used for expression of RNA the same sense as the virion.
What is common to all RNA viruses?
They all must encode their own polymerase because the cell they infect will not have RNA dependent RNA polymerase activity.
What is the prototype for picornaviridae?

How many types are there?

How is it spread?

What other viruses are picornaviridae?
THe poliovirus is the prototype. There are three serotypes (Type 1,2,3) which differ in antigenic properties.

It is spread by fecal contamination. It is only able to infect primates!

Hepatitis A, coxsackie A and B, echoviruses, rhinoviruses.
How many ORF does Polio have? How long is it and where does it start?
It has one long ORF that is about 7700nt long. It starts at about 743nt.
What is Vpg?
Important protein that links the the viral genome by a Tyr residue at the 5' end of the RNA. It also acts as the initiator of RNA replication
What is the function of the untranslated region of the ORF?
It contains an IRES (Internal ribosome entry site). Its function, since the 5' end does not have a cap, is to act as a place for the small ribosome to land.
What proteases are important in polio replication? Where do they act?
2A (that comes from P2) and 3C (that comes from P3). 2A acts to autocleave P2 and P1 and then also works to cleave the P3 intermediates into 3C and 3D.

3C works to autocleave P3 and P2 and then also to cleave the P1 intermediate into the precapsid parts.
What does P1 translate to by the end of cleavage?

What does P2 and P3 translate to by the end of cleavage?
P1 ends up being the capsid proteins (VP1-4).

P2 code for replicases, P3 codes for Polymerases and Vpg.
Where does the polio replication cycle occur in the cell?
In the cytoplasm.
How does a virus make sure the cell only makes its proteins in the polio replication cycle?
It shuts off host cell protein synthesis by cleaving the host translation factor eIF4.
Whats the receptor for polio?

What kind of entry does it use?
ICAM, receptor mediated endocytosis (non membrane)
What are the steps in the assembly of the poliovirus virion?
1. P1 is cleaved from the whole protein through the autocleavage of 2A. P1 is cleaved again by 3C into VP0, VP1, and VP3 which then come together to form a PROTOMER.

5 PROTOMERS go to PENTAMER. 12 PENTAMERS become the PROCAPSID. The RNA is inserted and a final cleavage of VP0 into VP2 and VP4 locks the capsid and genome tightly into the mature VIRION.
What type of capsid does the flaviviridae have?
Enveloped, Icosehedral
What virus is the flavi related to?
How is flavi considered seasonal?
It has a lot of mosquito-borne agents (west nile, encephalitis, etc) that would be in a seasonal climate while there are also arboviruses that cause disease all year long in the nonseasonal areas.
What is the prototype for the flavi virus? How big is it?
yellow fever virus, 10,000nt.
Does the yellow fever virus have a 5'cap and 3'tail?
It has a 5'cap but no polyA tail.
How is yellow fever like polio? How is it different?
Both have a single ORF that is processed by viral-encoded proteases. Yellow fever has an E (envelope glycoprotein) and an M (single integral membrane protein) that it will translate to.
What is NS5 in Yellow fever? What is the NS4B?
NS5 is what fights back against immuneresponse. NS4B is a polymerase.
What other hepatitis virus is a flavivirus?
Hepatitis C.
What type of virus is togaviridae? What does it encode?
It is an enveloped, icosehedral virus that encodes subgenomic mRNA.
What is the prototype for togavirus? What is another type of togavirus?
The prototype is Sindbis which isnt very deadly, but RUbella is also a togavirus that is associated with arthritis and neurological complications.
How big is Sindbis virus?
It is 11,000nt.
How many ORF does sindbis have? What mRNA modifications does it have?
It has 2 ORF (the first is longer than the second) and it has both 5'cap and 3'tail.
What does ORF1 code for? What does oRF2 code for? (for sindbis)
ORF1= nonstructural (replicase genes)
ORF2=structural genes
How is the ribosome able to make its proteins from two ORF's in sindbis in proportions that it needs?
It has a suppressible stop codon after ORF1 that can be stopped or not stopped depending on what the virus needs.
What is the subgenomic mRNA in sindbis virus?
It is th genes that occur at the towards the 3'end that make up the ORF2.
What are the early stages of Sindbis infection?
It goes in by receptor mediated endocytosis, the fuses with the membrane of the vesicle and releases genomica RNA. Then the 49s genome RNA serves as mRNA and the first ORF gives rise to NONSTRUCTURAL precursors proteins (that become helicase, capping enzymes).

It will either translate the whole two ORF's or it will only do the first one. The shorter one is more abundant.
Why does sindbis virus encode its own capping proteins?
Because the cells capping proteins are in the nucleaus and all this is happening in the cytoplasm.
How is the RNA replicated in Sindbis virus?

How is the subgenomic RNA made?
It is made by the RI-1 and RI2 intermediates.

With the RI-2, there are two modes to make the RNA: you can have full size genomes (49s) or you can have internal replicase starts which will make the subgenomic RNA that is 26S.

Both 49 and 26S are capped and adenlyated.
WHich portion of the genome in sindbis makes the structural proteins?
The subgenomic RNA (26s)
In Sindbis, how does the cleaved protein get into the ER?
The protein is cleaved at the N terminus which creates a new N terminus that contains a signal sequence for the inserting of itself into the ER like any other cellular protein.
What happens to the Sindbis proteins inside the Golgi?
There are further cleavages that produce the E and M proteins in the end.
Study what each of the viruses (+sense) affects...
Study :)
What is the only group of +sense viruses that have a helical nucleocapsid?
What +RNA virus has the largest RNA genome? How big is it
Coronaviruses. >30kb.
What types of viruses are Coronaviridae?
Mild respitory infections, (large number of serotypes in common colds)--SARS is an example.

Coronaviruses have a different effect depending on the animal its in.
what kind of virus is the coronavirus?
Its an enveloped, helical nucleocapsid.
WHat RNA modification does the Corona virus have?

How many ORF?
It has both 5'cap and 3'tail.

It has 5 ORF.
In corona virus what are the five major ORF's translated from?
They are translated from the seven 3' COTERMINAL SUBGENOMIC RNAS, each with have a common 5' leader sequence (this is done by skipping certain portions of the orginal mRNA)

This gives all the subgenomic RNA's a look like they have been spliced, except this is all happening during transcription.
What is the mechanism of producing subgenomic RNA in the coronavirus?
It is not that known. It involves Polymerase jumping (skipping) over certain areas. It always keeps the common leader (5'end) and the common 3'end.
In Coronavirus replication what is the first ORF translated into?
Into the polymerase and capping/methylation proteins which help to make RI-2.
What entry does Coronavirus use?
Receptor mediated fusion with the membrane.
How are nested set of subgenomic RNA's made in the coronavirus replication cycle?
Using several INTERNAL sites that it can jump to using the mius template of RI-1.
What are the envelope proteins in coronaviruses?
E1, E2, and E3.
What is unique to +sense plant RNA viruses?
They have multiple genome segments that are SEPARATELY ENCAPSIDATED (instead of the entire genome being in one particle, it will be separate so that all particles must infect to get the whole virus).

Also, some plants encode CELL-TO-CELL Movement PRoteins.
What are three examples of plant +sense RNA viruses?
TMV (helical, one segment)
CMV (icoseh., two segment)
BMV (icoseh., three segment)
For plants, what RNA modifications do they have?
at the 5' end, some have the Vpg or cap structure, and some have a polyA tail but some have a tRNA like sequence at the 3' end that may protect from degradation.
What type of virus is a leviviridae?
Its a +sense bacteriophage that are male specific (QB, MS2, R17)---They target the pillus of male bacteria.
What RNA modifications does Levivirus have?
NONE!! (the fundemental differences between eukaryotes and prokar.--they have no machinery to recognize the cap so no cap)
How big is lentivirus QB genome?
very small <2kb...It encodes only three proteins.
What are the three proteins that the mRNA of the levivirus code for?
A Protein, coat (with a suppressible stop codon), and a replicase.
What is required for replication in a levivirus?
Suppression of termination at the stop codon.
What is the difference in translation between prokaryotic virus with ORF and a eukaryotic one with ORF?
A bacteriophage's whole genomic RNA is translated without stop because bacteria have internal start signals to interact with the ribosome. A eukaryotic virus cannot have more than one ORF read.
What is the first thing you replicate as a levivirus?
The replicase. This is acccumulated in early phase (from the -sense RI-2).
What is the temporal regulation of QB translation?
The secondary structures of infecting genome have ribosome blocks to the start sites of protein A and coat proteins.

Once a nascent strand is made later, these blocks are absent.
What does coat protein accumulation in QB cause?
inhibition of replicase translation. (part of the temporal regulation of these proteins)
What is the start site for the maturation protein A in QB virus?
It is GUG instead of AUG.
What stage of virus infection is the Incubation Period?
The early stages.
What is viremia?
A name for when the virus is spread in the circulatory system. This is common. The virus can be a free virus or absorbed into the surface of cells.
How is tissue tropism determined?
By both viral and host factors (receptor dist., intracellular interactions, inflammation..)
What is the innate immunity response?
Its the early acting immunity. It includes tissue inflammation, marcrophage destruction, fever, interferon production.
What is the adaptive immunity response?
The long lived response that required the maturation of both B and T lymphocytes (effector T cells to recognize antigens, Helper T cells to drive B cell maturation).
What are the two later stages of viral infection?
spread to other individuals and the fate of the host
What often plays a role in the spread of a virus?
Where the virus accumulates.
What do many acute infections often involve (2 things)? What sometimes will happen?.
RECOVERY of the host and CLEARANCE of the virus.

Sometimes, the virus will become a latent infection.
What are chronic diseases suspected to involve?
They are suspected to involve PERSISTENT virus infections (diabetes, rhemeatoid arthritis)
What are the 3 things that classify viruses?
1. Virus genome (dna/rna, ss or db)

2. Virion structure (icosahedral/helical and envelope/naked)

3. Baltimore scheme of classification (how a virus produces RNA--DOES NOT APPLY TO DNA VIRUSES).
What is the icosahedral stucture?
20 faces. 12 vertices. 30 edges.

There is 2fold, 3fold, and 5 fold symetry when you rotate it.
What are the individual protein units of a capsid called?
What are the roles of Pol 1, Pol 2 and Pol 3?
Pol 1 does rRNA, Pol II does mRNA, Pol III does Small RNA's.
What is the difference between a general and specific transcription factors?
general transcription factor is TFIID which binds to any promotor site to for the preinitition complex.

Specific transcription factors bind to enhancers, upstream sequences from TATA box for example.
What do small nuclear RNAs (snRNA's) do?
They form SNRP's that can recognize 5' splice site and 3' splice site and form splicosome complex which can remove the introns and bind the exons.
What is an R-loop?
Its a technique that was used when they discovered the introns in Adenovirus. Its the looping out of ssDNA that showed the introns were removed.
How does a prokaryote terminate transcription?
It can do it by Rho dependent (where a rho factor protein recognizes a stop sequence). or it can be Rho-independent where the RNA will loop at a certain point which will dissassociate the RNA.
How many ORF's are POSSIBLE in mRNA?

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