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243 Cards in this Set

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  • Back
What are the four classifications of viruses that require RNA templated mRNA synthesis as a first step?
1. Monopartite genome
2. Multipartite genome
3. dsRNA genome
4. Circular ssRNA genome
What is the distribution of Rhabdovirus?
Very broadly distributed in nature (animals, plants, insects)
What is the prototype for Rhabdoviruses and what does it cause?

What is another rhabdovirus?
Vesicular Stomatitis Virion (VSV)

It causes severe, self-limiting disease in cattle horses and pigs (economic importance)

Rabies (most important rhabdovirus human pathogen)is also one of these.
What do each of the 5 genes in the Rhabdovirus code for?
N = nuclear proteins
P = polymerase complex protein
M = membrane protein that acts as a glue to hold the virus core and lipid envelope together
G = glycoprotein spikes that are needs to bind virus to receptor and help in membrane fusion
L = catalytic subunit of the polymerase complex. Also has a coding for methylating/capping enzymes
What are the functions of the P/L complex in Rhabdoviruses?
1. Associate with the helical nucleocapsid in the virion RNP core
2. Produce 5 capped and polyadenylated mRNAs (it does all the capping,etc)
3. Replication of the RNA
What is meant by transcription competent virus cores?
That the virus has everything ready for transcription which is carried in with it (i.e. P/L complex is taken in with it)
What is meant by the transcription process is seqeuntial?
That each transcript is made in order (N and then NS,etc) and its initiated at the 3' end.
WHich two mechanisms in rhabdovirus are slightly different than the hosts two mechanisms?
Capping and Polyadenylation (stuttering occurs)
What is replication coupled to in the rhabdovirus?
Its coupled to assembly of nucleocapsids
What kind of template is required for all synthesis carried out by the P/L complex?
Requires encapsidated templates (N-RNA complex)--the N protein never becomes dissassociated from the RNA!!
What is the gradient transcription of the rhabdoviruses?
It is the gradient transcription of each gene. There is a probability that at each gene junction that the polymerase will fall off and this makes it so that by the time it can make it to the L transcript, it hasnt really made L all that much. N is made much more effeciently and more often than the L (last lipid protein).

Process of REINITIATION at each gene junction is only 60% effecient.
Where is the G protein made in VSV? How does VSV shut off transcription and translastion of the host?
G protein is translated by membrane-bound ribosomes and transported through the GOlgi.

VSV shuts off host machinery by having the M protein go into the nucleus of the cell and block cell nuclear-cytoplasmic transport functions.
What are the steps to the replication and release of VSV from the host cell?
Once there is enough N protein accumulated, replication of the (-) sense strand will occur, coupled with the N protein assembly.

As the polymerase proteins are tranlated, they assemble with the progeny nucleocapsid (N-RNA).

G protein comes from the golgi to sit on the cell membrane and the M protein mediates the association of the progeny nucleocapsid to cell membrane with modifications by the insertion of the G spikes.

Budding then happens from the cell membrane to release the mature virus.
What is the polymerase called during transcription? What is it called during synthesis of mRNAs?
Transcriptase during transcription. Replicase during replication.
What are the methods of capping and polyAing in VSV?
mRNAs are capped (CIS ACTING FUNCTION) as the the RNA chain is being made by the P/L complex (it cannot cap it if its all complete, must be capped as its being made).

At the end of each gene the p/L complex will stutter on a stretch of about 7 U residues to add polyA tail.

Once PolyA'ed the transcript is released and the polymerase will reinitiate downstream or go back to the N gene.
What are the 4 subgroups of Paramyxoviridae?
Paramyxoviruses (Sendai, influenza 1,2)

Rubulaviruses (mumps)

Morbilliviruses (measles, canine distemper, rinderpest)

Pneumoviruses (respiratory syncytial virus RSV)
What type of virus are Paramyxoviridae family?
They are monopartite family of RNA viruses needing RNA templated mRNA synthesis as their first step.
WHat virus, which has a vaccine, still is a major cause of childrens death in the world?
Measles (morbillivirus)--paramyxoviridae family
What does mumps cause in children and post pubescent children?
in children relatively benign, but in postpubescent children it can cause severe reproductive damage
What does RSV cause?
mild cold symptoms in adults, but can lead to fatal epidemics in newborns.
How are the G protein of the VSV and the F and HN proteins of the paramxyo different?
The G protein has fusion and receptor activity but no neuraminidase, while the paramyxo has to have F for the Fusion and then HN for the binding to receptors.
What are the two transmembrane glycoproteins of the Paramyxo?

What does neuraminidase do?
F is the fusion protein and HN is the hemagglutinin which has neuraminidase. Neuramindase cleaves the syalic acid off of the host cell in late stages of budding so that when the virion buds it does not get "stuck" back onto the host cell receptors.
What is the shape of the paramyxo virus? How big is the genome?
Its roughly spherical, rather pliable and soft. It has a RNP core.

It is about 15,000 nt.
What does the P/L complex do in paramyxo that it does not do in rhabdo?
In the P gene, it will stutter somewhere in transcription and will add extra noncoding G residues which will allow for extra proteins to be made out of the same gene. (These proteins are Y, W, Z).

It also makes different proteins by using different internal start sites in the genes.
What are the four virus families that are neg. sense and have a monopartite genome?
Rhabdovirdae, paramyxoviridae, filovridae, bornaviridae
What are the filoviridae viruses and what do they cause?
Marburg (transmitted from Green Monkeys) and Ebola (unknown natural resevoir) both cause very sever hemorrhagic fevers with high mortality rate.
What are the bornaviridae virus and what does it cause?
bornavirus, causes lots of neurological symptoms in many vertebrate animals. Also an association with mental illness in humans.
How is bornaviruses different from other mononegaviruses?
It replicates in the nucleus and some mRNA's are spliced.
How do gene organization and replication compare in Filo/Borna and Rhabdo/Para viruses?
gene organization is similar in all four groups. In filo/borna replication and splicing occurs in the nucleus (both have nuclear involvement) and rhabdo/para doesnt.
What are the three families of neg sense multipartite genome?
Orthomyxoviridae, bunyaviridae, arenaviridae
What type of viruses does the family orthomyxoviridae have?
Influenza (8 segment genome) A,B, C and thogoto-like (6 or 7 segments)
What are the serotypes of influena and which is most dangerous?
There are three: A, B, C and usually A is most infectious, sometimes B and C has never really been a problem.
What causes a seasonal epidemic with influenza?
New flu variants that are due to changes in virus surface protiens (HA and NA)
What is antigenic DRIFT?
Its the gradual accumulation of mutations in HA and NA which allow for the virus to overcome immunity from a previous strain.
What is antigenic SHIFT?
Its the reassortment of genome segments in cells infected with two different viruses that have mutations in HA and NA that are different. When the two viruses exchange segments you get a SUDDEN (not gradual as in drift) change that can cause a PANdemic (worldwide epidemics)
How can antigenic shift cause the creation of a new hybrid virus?
It can result from the infection of pigs which have receptors for avian and human viruses to make a virus that is a hybrid between the two (bird flu)
What is the shape of influenza?
Roughly spherical, smaller than paramxyo
What is the prototype for orthomyxoviridae?
What is the history of influenza antigenic changes? How is it measured?
Fig. 16.7. History of influenza is shown by measuring the degree of antigenic changes. The major drops indicate major shifts. There have been three major shifts leading to three major pandemics in 1946, 1957, 1968....
What is the genome size of influenza?
There are 8 segments packaged as helical RNPs with a total genome of 14 kb.
What are the two distict surface proteins of influenza? How does this compare to rhabdo and para?
Neuraminidase and hemagglutinin. The hemagglutinin is like the G protein of rhabdo in that it has its fusion and receptor activity. This is unlike para viruses that have a fusion protein and hema and neuro together as another protein.
What do the eight segments code for in influenza?
Six of the segments code for one protein. Two of the segments code for 2 proteins each (thru splicing in the nucleas).
Where does transcription and replication take place in influenza?
IN the nucleas. Once all 8 segments are released in cytoplasm they immediately go to the nucleas for trans/repli.
In influenza, how do the RNA segments get transported across nuclear membrane?
They have SIGNALS for transport which are very similar to signals used by host.
What is replication in influenza coupled to? What is the primer used?
Replication is coupled to encapsidation and requires no primer.
HOw many subunits does the influenza polymerase have? What do the subunits do?
3 units which lack Capping activity but one unit has an endonuclease activity that is required for cap stealing.
What is cap stealing?
The influenza viruses do not encode their own capping/methylating enzymes and so it has a unit of the polymerase which has an endonuclease activity that will go in and cleave 10-15nts from the 5' end of nuclear mRNA's and steal the cap to put it on the viral mRNA.

All 5' ends are heterogenous since they are not their own cap.
What viruses are under Bunyaviridae? How many segments?
LaCrosse, Hantaan. 3 segments.
What is one of the largest known virus families? How many subgroups and serotypes does it have?
Bunyavridae, it has 5 distinct genus subgroups and greater than 300 serotypes.
What is the vector for most of the bunyaviridae? What do these viruses cause?
Most are arboviruses (insect vector). Hantavirus (hantaan) use rodents

They mostly cause encephalitis or hemorrhagic fever.
How many genome segments does bunyaviridae have? What are they?
3 segments: L, M, and S (there is no accessory P protein in bunyaviridae).
In LaCrosse where does cap stealing take place? What about polyAdenylation?
Cap Stealing takes place in the cytoplasm. No polyadenylation occurs in bunyaviridae virus.
What is replication coupled to in the lacrosse virus?
encapsidation of RNA.
What unique processes are commonly observed in translation of Bunyavirus?
The M segment will get proteolytically processed into three proteins (G1, G2, NS) and also there will be an alternate use of the reading frames of the S segment to make two proteins (NS and N)
What are the two surface glycoproteins encoded for in bunyaviridae?
G1 and G2.
What is the ambisense coding stratedgy? What viruses use this?
It is usually in the S segment of bunya and arena viruses. It is the encoding of proteins from both minus and plus sense strands.

The 5' end (left side) will be (+) sense and have the GP protein code and will not be able to be made until the whole virion genome is replicated. Once replication occurs then the GP proteins can be made after transcription back to (+) sense.

On the right side is the (-) sense that codes for the NP proteins can be made as soon as it goes into the cell and the mRNA from it gets made. It does not have to wait for replication to occur.
How come the GP proteins have to wait for replication to occur to be made in ambisense coding?
Because the GP proteins are carried in on (+) sense RNA and it is encapsidated, there is nothing to translate it immediately. It must be replicated to (-) sense and then transcribed to (+) sense without encapsidation to be able to be translated.
What is the hairpin present for in the ambisense coding?
It serves as a termination site in translation of GP proteins.
Which specific viruses and which of their segments use ambisense coding stratedgy?
Phleboviruses (s segment) and tospoviruses (M an S).

Also some arenaviruses.
How many segments does arena Viridae have? What is the nature of the virus?
2 seg., enveloped.
How large is the arenaviridae? What do these viruses cause
Very large group and they cause severe hemorrhagic fevers.
What are the most common carriers of arenavirus?
Rodents, who often shed large amounts of virus
What is the prototype of arenaviridae? What does it cause?

What are the other two arenaviridae?
Lymphochoriomeningitis virus (LMCV). It causes mild flu like symptoms in mice and humans and causes a persistent infection in infant animals but is usually fatal in adults(the immune response kills the animal).

Other two are junin and lassa fever.
Does Arenaviridae have P/L complex?
It has only an L polymerase without an associated P protein.
In LMCV, do the viral mRNAs have caps and PolyA tails?
They have caps (from host mRNA) and NO polyA tail.
In LMCV, what is the products of primary transcription of L and S segment?
From the L segment you get mRNA for the L polymerase, from the S segment yu get the mRNA for the NP protein.
In LMCV what is the products of the transcription of the antigenome segment?
Fromthe L segment you get mRNA for the Z regulatory protein. From the S segment you get the mRNA for precursor glycoproteins which are cleaved into GP1 and GP2.
What virus family has dsRNA genome? What kind of virus is it?

Whats the prototype and what does it cause?
Its Reoviridae. its part of the (-) sense viruses that need RNA templated mRNA synthesis as a first step.

THe prototype is reoviruses(respiratory orphan virus) and causes no disease in humans.
What is the distribution of Reovirus?
There is large distribution:

vertebrates: reovirus, rotavirus, colorado tick fever

invertebrates: cytoplasmic polyhedrosis virus

Plants: wound tumor virus

Bacteria: phage6
What do rotaviruses cause?
They are the leading cause of gastroenteritis and diarrhea associated with early childhood death.
What is the morphology of reovirus?
Its a nonenveloped naked virus that has 2 very tough icosehedral shells that are wrapped around each other. Inside the core their are 10-12 segments of dsRNA.
What RNA modifications does the reoviral RNA have?
The pos. sense has a cap and NO tail and the neg. sense has neither
What is the inner core of the reovirus? and what proteins does it contain?
Its lamda1, lamda2, and sigma2 and they contain the enzymes required for mRNA synthesis (transciptase, helicase, capping activities).
What is special about the S segment in reovirus?
It has two overlapping reading frames that are expressed through alternate initiation sites.
What are the gene segments named in reovirus?
L segments (3)which make polymerase, M (3)and S(4)which code for structural proteins.
How does the reovirus avoid exposure of its dsRNA to the cell?
It has transcription take place in the core and mRNAs are made into dsRNA's once they are in the capsid. Therefore NO dsRNA is ever exposed to the cell :)
What is the replication cycle of Reovirus?
The sigma1 on the outershell binds to a receptor for receptor mediated endocytosis.

VIrus core is relased thru partial uncoating and within the core you make the mRNA transcripts. Once capped, these mRNA transcripts are released into the cytoplasm for translation or encapsidation.

Once TEN distinct mRNAs are encapsidated together, there is the synthesis of the (-) strand to make it dsRNA.

The virion then goes thru morphogenesis and gets all the proteins that were translated in the cell to become a mature virion.
What virus neg. sense viruses has circular ssRNA?
Hepatitis delta virus (HDV)
What is HDV? How is it spread and what does it cause?
Its a subviral pathogen that cannot infect on its own. It has to have coinfection with hepatitis B virus.

It is spread through blood contamination and causes liver damage.
What unique genome does HDV have?
small covalently-closed circular ssRNA of negative sense. It is 1.7kb and has high base-pairing content.
What does the HDV genome encode?
It encodes a SINGLE nucleocapsid protein (the delta antigen) from the (+) sense antigenome.
HOw many glycoproteins envelope HDV? Where do they come from?
Three that are derived from HBV.
WHat is the shape of HDV? What is this similar to?
Its rodlike shaped. Its similar to plant viriods.
How is viral RNA synthesis of HDV carried out? WHy is this unusual?
Its carried out by host cell RNA pol II. This is unusual because pol II usually only transcribed DNA only.
Are HDV viral mRNAs capped and polyA'd?
Yes to both.
Once the nucleocapsid enters the cell where is it transported to in HDV?
To the nucleas.
What are the 2 sites that are on the HDV genome? What are they there for?
Self cleavage site and RNA editing site.

Self cleavage site (catalyzed by the ribozyme activity) is there to cleave the (+) antigenome circle so that the mRNA for the small delta antigen can be translated.

The RNA editing site is used by a cellular enzyme to change a UAG stop codon to a UGG tryp codon which will give the LARGER delta antigen.
What are the short and long delta antigen for in HDV?
THe short delta antigen is required for replication. The long Delta is for suppressing replciation and promoting assembly.
What are viriods?
Infectious agents associated with disease that are found ONLY in plants.
Why are viriods similar to viruses but NOT viruses?
They have small covalently closed circular ssRNA that is highly base paired BUT they do not encode any protein and therefore are not considered viruses.
How is RNA replicated in viriods?
It is replicated by cellular RNA polymerases. And the genome has ribozyme activity....
How many groups are there of retroviruses? What are the groups based on?
7 groups based on genetic relatedness

3 groups based on pathogenesis

2 groups based on gene numbers (gene arrangement)
What are the three groups of retro viruses that are based on pathogenesis?
Oncornavirus-usually benign
Lentivirus-long incubation followed by severe disease
Spumavirus-foamy appearance of infected cells, benign
What are the two groups of retroviruses based on gene numbers?
simple (encodes genes required for replication only)

Complex (encod additional genes regulating interaction with host)
Do retroviruses kill the cell?
most do not kill the cell. infected cells usually shed viruses for weeks, months, years.

HIV is an exception that kills cells.
What kind of virus is retrovirus classified as?
An RNA virus (since it packages RNA)
How does the retrovirus differ from other RNA viruses?
It does not copy anything from RNA. It goes into the cell and immediately wants to be DNA. It inserts itself (INTEGRATES) its DNA at certain points in the genome and gets copied by the HOST cell machinery for DNA.
What do oncornaviruses do in the cell?
They will stimulate the infected cell replication (causing cancer). THis is usually slow and inapparent though.
WHat is a gag protein? What does it stand for?

How does it vary in retroviruses?
Its the capsid structure of the retrovirus. It stands for 'group antigen'.

It varies in different groups of viruses. Sometimes its icosehedral and sometimes more complex shapes
Which type of viruses are diploid? Whats that mean?
The retroviruses are diploid meaning that their are two copies of the entire genome.
What three proteins make up the Pol gene in retrovirus? What are they associated with?
Reverse transcriptase, protease, and integrase are all associated with the capsid core.
How are core enzymes derived in the oncornavirus?
They are derived by cleavage from the Gag-Pol precursor before the release of the mature particles (this is because they are required for activity)
HOw is the gag-pol precursor generated in the retrovirus?
Through two different mechanisms:
1. Supression of the stop codon (like sindbis)
2. Ribosomal framshifting (slipping)
Are retroviruses capped and polyA'ed?
Yes, by cellular enzymes
What three essential genes are always included in the genome of retroviruses? What else is included and where?
Gag, Pol, Env. and also untranslated sequences at the 5' and 3' ends.
What are the untranslated terminal sequences called in retroviruses? What do they contain? Why are they necessary?
They are called Long Terminal Repeats (LTR) and they contain CONTROL ELEMENTS that are required for generating PROVIRUS and synthesis of viral genomes and mRNAs (Pol II recognizes these control elements)
What does the PROVIRUS contain?
Provirus copies have the LTRs that were derived from the reverse transciption process in retroviruses?
What is the Genome of a Retrovirus? What does each gene letters stand for?

R= seen on both the 5' and the 3', its the terminal repeat sequence (LTR) that is 20-250 bases). combined with U3 and U5 they contain the Transcriptional Control signals for provirus.

U3, U5= LTRs 75-200bases.Contain Control Signals.

Leader = 50-400bases, contains the genome packaging and mRNA splicing signals.

Gag:prot:pol:int = make up the Gag-Pol precursor

prot= protease that generates the pol and int proteins from the gag-pol precursor

env= envelope proteins that are always tranlated from spliced mRNA

PP= short polypurine tract also used for reverse transcriptase.
How does the PB work in the retrovirus?
PB stands for the primer binding site. The retrovirus will take a cellular tRNA (AA on it doesnt matter) and it will bind its 3' end to the PB site and therefore serve as a primer for the DNA polymerase.
What are the two oncornaviruses that have a simple genome and can cause tumors, but do it slowly?
Avian and Murine Leukosis
What separates Rous sarcoma and Mouse Mammary tumor from the avian and murine leukosis? What kind of virus are they?
The RSV and MMTV cause tumors MUCH more rapidly and they have additional v-onc genes (v-src in RS and v-sag in MMTV)

They are oncornaviruses (Retrovirus)
What does Human T-Cell Leukemia have in addition to the simple retroviruses stuff? What does this do?
It has addiitonal activator proteins called Tax and Rex that stimulate cell division and metabolic activity to make it a SLOW transforming virus.
How does the Lentivirus genome differ from regular retrovirus genome? Whats an example of LEntivirus?
Encodes SEVERAl other genes through a complex splicing pattern which are important in regulating HIV replication during the LATENT phase.
What are the steps of retrovirus replication?
1. Receptor mediated endocytosis.

2. Parital uncoating, initiation of reverse transciption to produce dsDNA.

3. dsDNA is transported to nucleas, along with integrase.

4. dsDNA is integrated into genome and then transcribed as either full length transcripts or spliced versions that can be transfered to the cytoplasm

5. once in the cytoplasm translation occurs or the progeny rna are taken for the capsid assembly and then released by budding.

How many "jumps" are involved in cDNA synthesis?
2 jumps :)
Which parts of the genome are necessary for cDNA synthesis?
U3, U5, R, pb, pp
What are the important things to know about cDNA synthesis in a retrovirus?
1. A cellular tRNA binds to the PB site near the 5' end to serve as a primer.

2. Since synthesis can only occur 3 to 5', you only get to begin with a short strand called MINUS SENSE STRONG STOP.

3. THe RNAH will degrate RNA strand that does not have any compliment to it.

4. There is a FIRST reverse transcriptase JUMP where the small MINUS strand will jump to another peice and the R's will anneal together.

4. RNAH comes again to remove any extra RNA genome, except for the PP site that will displace the tRNA and begin to prime the PLUS strand called PLUS STRAND STRONG STOP.

5. SECOND jump occurs so that the PB ends of paritally ds cDNA anneal and the cynthesis will continue in both directions to complete the duplex.
What does the PP in retroviruses do?
It is the primer for the plus sense DNA synthesis (it displaces the tRNA)
What do the spliced and unspliced transcripts get translated to in the retrovirus?
unspliced = translated into GAG and GAG-POL proteins OR assembled with nucleocapsid proteins.

spliced = env mRNA and other viral genes specific to different retroviruses.
When does the FINAL maturation of capsids occur in retrovirus? What does this final maturation consist of?
Final maturation it occurs only FOLLOWING budding and release from infected cells.

Maturation is complete when there is CLEAVAGE to generate reverse transcriptass.
What are the three basic mechanisms that oncornaviruses use to "tranform" the cell?
1. Action of a v-onc gene to rapidly tranform viruses.

2. LTR activation of normal c-onc genes that will alter the normal cellular control.

3. Increased relase of growth-promoting cytokines from infected cell which will alter the nomral growth control on non-infected cells.
How do c-onc genes regulat cell growth?
Directly and indirectly
What are some examples of c-onc genes?

What does p53 have to do with viruses?
1. Growth hormones and their receptors

2. G protein

3. Protein kinases

4. Specific transcription factors (e.g. myc, p53)

**p53 is a major negative growth regulator often targeted by DNA tumor viruses.**
How does HIV enter the body?
Through genital or rectal mucosa or direct injection into bloodstream.
Where is the CD4 receptor present?
On lymphoid cells (macrophages, helper t cells, and a few other types of cells)
Does HIV Require a coreceptor?
Yes, For macrophages it requires different co-receptors (CCR5)and for Tcells it requires CXCX4)
What does HIV usually target?
Usually targets macrophages (M TROPIC virus) and those replicating variatns give rigse to T TROPIC virus.
What cells can carry a latent proviral forms of HIV to lymphatic tissues where a virus can continually replicate?
How do most eukaryotic transposable elements move? W
MOve via RNA intermediates:
Retroelements or retroids.
What are retroelements or retroids?
They are sequences that use reverse transcrptase to propagate. They are evolutionarily related to retroviruses?
Retroelements played a major role in what?
In the shaping of evolution of eukaryotic genomes (>20% of higher eukaryotic genomes is related).
What is the yeast Ty1 retrotransposon related to? How is it related? What elements in Drosophila follow this patterN?
Retrovirus. They both have ribosomal frame shifting to make gag-pol fusion protein. The Ty1 element codes for RTase and RNase H.

Copia and Gypsy in drosophila also have this activity.
What are the different types of hepadnavirus? What are hepadnaviruses?
THey are DNA viruses. Two are hepatitis B and cauliflower mosaic virus in plants.
What are hepadnavirus related to? How are they similiar? How are they different?
They are related to retroviruses because they encode and incorporate a reverse transcriptase.

They are different in that the mature capsids contain a DNA genome and they do not integrate. It is maintained as an episome: an extra genomic small molecule.
Are Hepadnaviruses hard to study?
Yes they do not replicate well in tissue culture. Also, they establish often lifelong persistant infections in their hosts.
What is Hepatitis B correlated with?
Hep B infection is correlated with incidence of hepatocellular carcinomas. The mechanism of carcinogenesis is not clear.
What is the core called for Hep B? What does it contain? What is the enveloped virion called?
Core is called HBc capsid protein and contains DNA, RTase, and 3 membrane associated proteins. The enveloped virion is called Dane particles after who discovered it.
What causes the DNA to be circular in a hep B virus?
The overlap between the 5' end of plus and 3' end of minus strands maintain the genome as a circle.
What type of genome does hep B have?
3.2 kb that is partially double stranded.
Where is the RTase located on the hep B virus?
Its covalently bound to the 5' end of positive sense strand. (the polio virus has a protein bound to it like this)
What do the four ORFs code for?
Core, polymerase, S (envelope proteins) overlap different ORF's and using different start sites the mRNA is made into the fourth protein.
Is their splicing in Hep B?
No! They have a common polyA tail but there are different start sites.
What are the steps to replication of HepB virus?
1. Entry by fusion
2. Partial uncoating
3. RTase completes genome synthesis
4. Genome is tranported to the nucleus and the host enzymes ligate gaps to make the genome circular (an episome) that has associated host histones. NO INTEGRATION!!
4. Episome is transcribed by host enzymes.
What does the large mRNA called in hep B? What does it code for?
Its called C-mRNA and it encodes core protein and pol by riosomal frameshifting.

It has repeated sequences at both ends that are required for assembly with core protein and RTase.
What about cDNA synthesis is the hep B virus?
It is incomplete for + strand and takes place in immature cores. It requires RNase H as for retroviruses.
What viruses use reverse transcriptase?
Badnaviruses (plant DNA virus)
Cauliflower MOsaic virus
Hepatitis B virus
All Hepadnaviruses
All Retroviruses (onca, spuma, lenti)
What kind of virus is cauliflower mosaic virus? What size is its genome
Its a hepadnavirus and it is 8 kb with 3 ss breaks.
How many proteins are encoded in the CMV?
Five proteins from the large 35S RNA and 1 from the small 19S RNA
What is another class of virus that uses RTase that is not mentioned in detail in the slides?
What are the three catagories of genomes with DNA? What viruses are in each group
1. small genome: papova and parvo and geminivirus.

2. medium genomes: adenovirus, t-odd bacteriophage (T3, T5, T7 etc)

3. Large genomes: herpesvirus, baculovirus, poxvirus.
What is the man difference of RNA and DNA viruses?
DNA viruses usually (except Pox) do everything in the nucleas and not the cytoplasm.
What are the two families of papovavirus?
polyomavirus: SV40, polyoma

papillomaviruses: warts, some human cancers.
What is the morphology of the papovavirus?
They are naked icosehedral capsids with covalently closed circular genomes.
What are the three specific viruses under the papovavirus?
SV40 (no human pathology)
BK and JC which are polyomaviruses, they also infect humans for life with no pathology unless the person is immunocompromised...
How many capsid proteins are in SV40? What are they called?
VP1, VP2, VP3
In SV40, what does the control region include?
Ori, early promoter/enhancer, and late promoter (goes in opposite direction)
In SV40, the genome is packaged with WHAT once made in the cell?
It is packaged with host histones so that the replication stuff can recognize it when it goes into a cell (looks like chromatin)
HOw are early and late transcripts made in SV40?
Early transcripts are began with the early promoter (with a TATA box and single start site).

Late transcripts begin about 60-80 bp in with no TATA box.
What protein in SV40 is made and required for DNA replication. What does it bind to?
T antigen is made which binds to the ori reigion to begin replication.
What kind of symmetry in SV40 is required for DNA replication?
Dyad (it recognizes binding sites that are the same sequence whether read in one direction or the other)
IN SV40 where does the early and late transcript terminate?
At the polyadenylation signal for both strands (they terminate in the same region)
What should you know about the SV40?
It is a circular closed genome that has only two transcripts read in reverse direcitons. One direction makes the T antigen (big) and small (when spliced). One direction makes the capsid proteins.
How do you get the small and big TAntigen in SV40?
Through alternative splicing.
What are the function of 80 kDa TAg in SV40?
It is multifunctional:
1. Activates cell division by binding to P53 and Rb.
2. Binds to ori
3. shuts off early transcription
4. Activates late transcription.
What does the small (20kDa) T Ag do?
It serves a host range function meaning it allows the virus to replicate in certain cell types.
What does the early and late units encode in SV40?
Early is the T-antigens.
Late are the capsid proteins.
How does SV40 encode the capsid proteins?
Through alternative splicing (thru two spliced mRNAs)
What is agnoprotein? When is it made in SV40?
It is implicated in host range functions and is translated from very late mRNA.
What are the four steps of SV40 DNA replication?
1. The large T Ag will bind to the ori and initiate synthesis by melting and separating genome.

2. Host DNA polymerase copmlex synthesizes viral DNA with leading and lagging strands.

3. Host topoisomerase and resolvases separate interlocked circles.

4. The replicated DNA associates with HOST histones before encapsidation.
What is the difference between productive and abortive SV40 infectioN?
If SV40 is put into a non-natural cell type it will be non-permissiive (abortive) in that DNA replciation will be blocked in some way.
In SV40, what is the difference between stable transformation and transitory tranformation?
Stable tranformation is when by a rare recombination event, the genome is integrated into the host so that it will always make the T ag so that cell division is always 'on'.

In a transitory transformation, no integration takes place but the temporary concentration of T Ag causes stimulation of cell division.
What is the difference in tumor causing properties of polyomavirus and papillomaviruses?
polyomaviruses cause tumor through unnatural settings. Papillomaviruses cause benign tumors as a consequence of NAtural replciation.
Are the tumors caused by papillomavirus benign or malignent?
The serotypes are usually benign but some will cause persistent genital infections in women.
What are HPV16 and 18 correlated with?
They are correlated with cervical carcinoma.
Why are papillomaviruses difficult to study?
They replicate poorly in cultures cells.
What does the control region of HPV have in it? Whats the name of this control region?
Its called LCR and it contains the promoter/enhancer/ori.
HOw is termination of late and early transcripts different in SV40 and HPV?
In SV40, they end on the same polyadenlyation site. In HPV, the early and late mRNAs terminate at DISTINCT cleavage/polyadenylation sites.
Which two viruses use extensive alternative splicing to make new transcripts?
HPV and SV40.
in HPV are the ealry and late transcripts use and early and late promoter?
No they use the same promoter (same direction!)
What is the morphology of the HPV virus?
7.2 kb, circular.
In wart formation, where does HPV induce the DNA replication?
At the basement epithelial cells.
How is a wart formed by HPV?
THe HPV will induce replication in the basement epithilial cells (which are nonpermissive cells).

Then the enhanced DNA replication will lead to local cell differentiation with the production of keratin and these new differentiated cells are fully permssive. These cells begin to SHED the virus which causes the benign tumor assocaited with a wart.
How is growth stimulation done in wart formation?
Through E5, E6, and E7 which interact with host tumor suppressor proteins Rb and p53.
Does the HPV virus integrate?
No it remains as an episome?
What is oncogenicity?
Its when further mutation and integration of a viral genome (like HPV into the cervical epithelium) can lead to FULL CELL TRANFORMATION.
What is the morphology of parvoviridae?
Its a naked icosehedral virus of 5 kb.
Does parvo have ssDNA or dsDNA
What do paroviridae infect?
They infect warmblooded animals like cats and dogs where it destroys the cells dividing in the immune system.

It also infect INSECTS which is unlike many of the viruses we have studied.
What does parvoviruses require to divide?
It requires Actively dividing cells for replication. (they cant cause the cells to replicate like SV40 and HPV due through their antigens)
What is the prototype of parvovirus and what does it do?
Adeno-associated virus (AAV). it is considered defective because it relies on co-infection with adenovirus or herpes virus to help stimulate the host cell division.
Why does AAV have potential as a human therapeutic vector?
Because its genome only integrates at ONE SPECIFIC site in the human genome (chromosome 19)
Does AAV integrate its DNA or stay as an Episome?
It integrates at one specific site!
What two proteins are made in AAV? How are they made and what do they do?
Capsid (codes for capsid proteins) and REP (codes for protein necessary to make hairpin loops in dna synthesis and required for integration.

These are made by 3 families of 3' co-terminal spliced transcipts.
How is replication unique in AAV?
Replication uses host cell DNA polymerase (not unique)
BUT priming relies on HAIRPIN LOOPS formed by the terminal inverted repeat sequences on the ends of the linear genome.
What is the morphology of adenovirus?
Its a medium genome dsDNA virus that is naked, icosehedral
What does adenovirus infect? What does it cause?
It infects humans and other mammals and usually causes mild flu-like respiratory disease in humans and in some cases causes gastrointestinal symptoms.
What does adenovirus have in common with papovaviruses?
They both have long replication cycles and their abortive infection can lead to cell tranformation.
Why is adenovirus now being studied as a therapeutic agent?
Because if its ease of manipulation by genetic engineering (large enough genome, very easy to grow, robust growth).
Why is adenovirus considered complex?
It has a lot of proteins in non-equimolar proportions
Does adenovirus have histones on it? Why not?
No it doesnt. It has a core protein that acts like histones to condense the genome into a chromatin-like structure.
What kind of genome does Adenovirus have?
Linear, ds genome of 30 kb.
What is bound to the 5' end of adenovirus?
A viral terminal protein is covalently bound to the 5' end.
In adenovirus, what do the inverted terminal repeat sequences serve as?
They serve as the orgins of replication.
What is the genetic map for adenovirus?
It is a map that divides the genome into a 100 map unit. It shows the complex splicing pattern made by using host pol II.

**Look on chart for some of the proteins that are made from this**
What polymerase transcribes VA RNA? What does VA RNA do?
Host pol III (not pol II!). THis stops the activiation of host cell defenses.
What serves as a primer for adenoviral DNA replication?
Its a terminal protein precursor that has a cytosine covalently bound to protein.
Does adenovirus use host or viral DNA polymerase? How does this affect the DNA synthesis?
It uses its own viral DNA polymerase.

This makes it so that viral dna synthesis is continuous. There are no Okazaki framents as you would see for host polymerase.
What happens to the terminal precursor protein at the end of adenovirus DNA replication?
It is cleaved during packaging
What are the two mechanisms that are used for adenovirus DNA replication?
Type I mechanism: when the viral DNA polymerase copies genome from BOTH ends (starts replication on ssDNA that was displaced and the strand circularizes).

Type II: viral DNA polymerase copies genome from ONE END (ds DNA becomes displaced as a single strand)
How is adenovirus unique in priming?
There is NO RNA priming!! It just uses its protein to prime DNA synthesis!
What does the TYpe II mechanism rely on?
Terminal inverted repeat dequences that allow displaced ssDNA to circularize and provid a ds end for second strand copying.
WHat does herpesviridae infect? What do they cause?
All vertebrates and some invertebrates. They cause primary infections generally with mild symptoms (unless immunocompromised). They can establish a latent infection
What are teh three subgroups of herpesviridae? What are examples of each?
1. alpha: neurotropic (HSV1, VSV)

2. Beta: lymphotropic (CMS, HHV6, HHV7

3. Gamma: lymphotropic (Epstein-Barr, HHV8)
What does EBV do? What kind of virus is it?
Its a large dna virus under herpeviridae.

It is a co-carcinogen for Burkitt's lymphoma and for nasopharyngeal carnicoma.
What is HHV-8 the contributing factor for?
For development of Kaposi's sarcoma, which is seen in AIDS patients.
What is the morphology of herpesviridae?
Its a enveloped, complex, icosehedral virus with a tegument layer.

It has linear dsDNA that becomes circular when in infected cells.
How many trancripts and ORFs does herpesviridae encode? Is there splicing of mRNAs?
It encodes about 100 transcripts and >70 ORFs. Few of the mRNAs are spliced but most encode a single ORF.
How is the transcription map of HSV-1 set up?
It has two regions: small (s) and Large (L) and two repeats: Rs Rl, Us Ul
What is meant by the cascade of gene expression in HSV-1?
In HSV it is not as simple as early and late genes, but there are very early genes (immediate early) that are required for the expression of the next set of early genes, and those are required for the middle genes, etc. to the very late genes.
alpha then beta then gamma genes
In HSV what does the immediate early gene expression require?
It requires a specific transcription enhancer protein that is carried in the virion: TIF (VP16).
What does VP16 (TIF) associate with?
It associates with the host oct1 which is a transcription factor.
What kind of transcriptional proteins does HSV-1 use?
At the early stages it uses its host machinery a lot but as the cascade goes on, there are some immediate-early proteins that are transcriptional activators that are required throughout the productive cycle.
How many ori sites are their in HSV Replication?
Three (two in Rs and one in Ul)
What are the steps to HSV Dna replication and assembly?
First, early proteins must be made. THese are ori-binding protein, helicase-primase, ssDNA binding protein, DNA polymerase.

Then the ori-binding protein binds at two spots on one of the ori sites.

Through bidirectional replication (like normal cellular replication), it uses the helicase and primase activities.

The circularized DNA becomes nicked at a replication fork to produce a "rolling circle intermediate" which generates many concatemers.

The maturation/encapsidation proteins then bind to and cleave packaging signals on the dna at "a" sites.

When the virus buds through the virus modified nuclear membrane, the tegument proteins are added in nucleus.
What cells do the HSV infect? What does latent infection mean for the early gene expression?
HSV infects neurons. The latent infection means that the early gene expression is SUPPRESSED.
In HSV latent infection, what does the viral genome associate with? What does it form?
It associates with histones to form a stable episome.
What is the only thing expressed by HSV in a latent infection?
Only the LAT family of transcripts (a single promoter in Rl).
What causes the HSV to go from latent to productive infectioN?
Reactiviation by stress or some other stimuli induces the limited expression of early genes and production of infectious particles.
What is different about EBV latency?
In B lymphocytes, it requires more viral expression (11 genes). Latency genes activate host DNA replication.
What does HSV-1 Favor? Where does it establish the latent infection?
lip and facial area. Establishes latent infection in trigeminal ganglion.
What does HSV-2 Favor? Where does it become latent?
Genital mucosa. It becomes latent in sciatic nerve ganglia.
What does HSV do to counter host immune response?
It encodes several proteins:

1. 47 inhibits MHC class 1mediated antigen presentaiton

2. ICP34.5 blocks interferon action

3. viral proteins also block apoptosis.
What is virus with the largest known genome?
Where does the poxviruses replicate at? What does this mean for what proteins it encodes?
In the cytoplasm of infected cells. (RARE for a DNA virus!!)

This means it encodes its own replication machinery AND its own transcription machinery.
What diseases are the small pox virus responsible for?
Variola major, variola minor and several animal poxviruses.
When was the last case of small pox? Why is it such a bad virus?
It was in 1977 until there was a vaccine. Its bad because its easily spread and highly resistent to desication. It does not cause a persistent infections and is cleared unless organism dies.
Since people cannot study small pox, what can they use to study it?
Vaccinia virus. ITs a popular expression vector for vaccination studies.
What does the poxvirus encode that causes rapid proliferation of neighboring cells?
It encodes a protein related to epidermal growth factor.
What is unique about the genome of poxvirus?
Its a dsDNA genome that has a unique closed ends (hairpin loops) and long inverted terminal repeats.
What does pox viral replication require? Are their ori sites?
Requires nicking of DNA, but mechanism of priming is still unclear because there are no ori sites.
How long does the cycle of infection of pox virus take in cultured cells?
Only 14-16 hours.
What are the steps to vaccinia virus replication?
1. The virus enters through receptor mediated endocytosis.

2. The virus is partially uncoated and this partially uncoated virion catalyzes early transcription using virus core-associated enzymes.

3. Early enzymes cause full uncoating and initiate intermediate gene transcription that encodes DNA replication enzymes.

4. The intermediate gene products trigger transcription of late mRNAs that encode virion proteins and assembly factors.
What do Large bacteriophages (T2, T4, Todd) replication do they follow?
THey are like the pox virus LARGE genome...