16-17 Virus: Genes and Genomes

Question 1

What did Hershey and Chase experiment revealed?

Answer 1

2 bacteriophages
Protein labeled with radioactive sulfur
DNA labeled with radioactive phosphor

only for DNA labeled bacteriophages the radioactivity was predominantly in cell pellett and radioactivity detected in next generation

so DNA is the carrier of genetic information

Question 2

4 types of polymerases

Answer 2

DdDp
DNA synthesis
3’-5’ exonuclease

DdRp
transcription RNA Polymerase II
multisubunit enzymes 8-14 subunits

RdDp
RT for Retroviruses, Retroposons
RNaseH to digest away RNA from DNA template

RdRp
RNA synthesis
Viruses only, every RNA Virus

Question 3

Difference between DNA and RNA Polymerase

Answer 3

DNA Polymerases with proofreading so one mutation/10^9

RNA Polymerase no proofreading so one mutation/10^4

Question 4

How do RNA polymerases are specific for RNA or DNA ?

Answer 4

e.g. RdRp of Poliovirus has GDD catalytic triade with D238 that binds OH of ribose and not H of deoxyribose, is involved in Mg2+ and RNA synthesis

one aa for specificity

Question 5

What information is encoded in viral genome?

Whats not encoded?

Answer 5

Gene products

Regulatory signals
Replication of viral genome
Assembly/Packaging
Replication cycle timing
Modulation of host defense
Spreading to ther cells and host

Secondary structure important

What’s not encoded?
centromer/telomer/histones

no genes for proteins involved in energy prodction and membrae synthesis

No genes for complete protein synthesis machinery, but some RNAs and proteins involved in translation may be present

Question 6

Genome size bp
Human
Plants
Fungi
Bacteria
dsDNA
RNA Virus
ssDNA
Viroids
largest virus for humans

Answer 6

Human 10^9
Plants 10^10
Fungi 10^7
Bacteria 10^6
dsDNA Viruses 10^5
RNA Viruaes 10^4
ssDNA smaller than RNA Viruses
Viroids 10^3

largest for humans: Poxvirus 5x10^5
largest in general: Pandoravirus 2x10^6

Question 7

Genome structure of Viruses
DNA
RNA
Other characteristics
Biggest difference between DNA virus and eukaryotic genome?

Answer 7

DNA
linear, circular, ss, ds

RNA
same but no ds circular RNA

other characteristics
segmented
gapped
+/- strand only important for RNA Viruses
ambisense strand (genes on + and - strand)
attached proteins
cross linked ends of dsDNA
DNA with covalently attached RNA

Biggest difference between DNA virus and eukaryotic genome?
Eukaryotic: Chromosomes with Nucleosomes/Histones, highly packed

Question 8

DNA Synthesis in general
Which direction?
Bond formation?
Starting sequence?
Mechanism
Ori in different species
All enzymes

Answer 8

Which direction?
always 5’->3’ direction for each strand
leading strand continusly by DpDp III
lagging strand in small sequences by DdDp I

Bond formation?
Formation of phosphodiester bond from 3’OH of nucleotide with 5’OH from phosphor

Starting sequence?
Starts at ori sequence in genome building replication fork

Mechanism
Helicases unwind DNA complex
ssDNA binding proteins stabilize the replication fork

leading strand continously by DdDp III
lagging strand uses random 120 nucleotide RNA primers, produces okazaki fragments and these fragments + primers being removed and okazaki fragment ligation

Ori
multiple in eukaryotes, single in prokaryotes, also in DNA Viruses and for Plasmids

All enzymes
Topoisomerase
Helicase
DNA Primase
DNA Polymerase 1 und 3
Ss DNA binding proteins
Okazaki fragments, RNA primer and DNA ligase

Question 9

DNA Viruses
Where does replication occur?

Answer 9

Where does replication occur?
mostly nucleus of host cell except for Poxviruses

Question 10

DNA Viruses
Symmetry of capsid
enveloped or naked?
Genome architecture
Balctimore class

Answer 10

DNA Viruses: symmetry, envelope, genome, Baltimore class and family name

Question 11

dsDNA genomes
Family
mRNA production
replication dependent on….

Answer 11

Familiy
Polioma, Papiloma; Adeno, Hepndna, Herpes, Irido and Pox

mRNA production
only possible with dsDNA, uses -strand DNA copied by DdRp to form +strand mRNA (coding strand)

replication dependent on….
-mostly enters host nucleus (except Polio)
-cell cycle dependent
-some require host cell polymerases, some have their own like Adeno, Herpesvirus, HepDNA and Poxvirus

Question 12

Circular dsDNA Viruses
example

How does replication inititation work, mechanism based on one example, viral protein characteristics/domains

other examples of viral proteins doing the same?

Answer 12

example
Polyoma (SV40)
Papilloma
Hepadna (gapped)

How does replication happen, mechanism based on one example

SV40 has a protein called large T antigen that can recognize the oriR:

LT antigen: hexamer that binds DNA with A/T rich region follwed by specific sequence to initiate DNA synthesis

also inactivates tummor supression proteins like p53 to enter S phase promoting DNA synthesisx

Helicase: unwind DNA
central: patch, that binds refulatory region
3rd domain: interaction with host cell proteins

twelve copies (2x6) assemble to form tube, hole is just big enough to encircle DNA double helix

other examples of viral proteins doing the same?
Papillomavirus E1
Adenovirus Rep68 (linear)
Herpesvirus UL9 (linear)

Further mechanism
Rolling circle:
Elongation with bidirectional replication, then 2 full dsDNAs present, but catenated, after synthesis topoisomerase allows seperation

Question 13

Linear dsDNA Viruses
Family
What’s a problem regarding replication and how is it solved ? Give examples

Answer 13

Family
Adeno, Herpes, Irido, Pox
What’s a problem regarding replication and how is it solved ?
5end problem, lagging strand how to connect okazaki fragments (last RNA primer)

Solutions
Self Priming Circulazation: Host DNA Ligase 4 leads to circulazation, then like dsDNA, after finishing
e.g. Herpes

Self priming partial circulazation (terminal loops) form covalently joint ends; oriR near end to synthesize new 5’-3’ strand, DNA is complementary to itself xo forms hairpin structure, full replication, then again closed genome
e.g. Poxvirus

Protein Priming:
own Polymerase where protein serves as 3’ end for synthesis, pTP (terminal protein) binds to each end to displace the non synthesized strand
e.g. Adenovirus

Question 14

Gapped double stranded DNA Viruses
Family and example
Baltimore class
General mechanism
What serves as a template for DNA genome production?

Answer 14

Family
Hepadna, HBV
-DNA with complete genome and +DNA incomplete, RNA primer von +DNA strand
Baltimore class
7

Mechanism
gapped dsDNA subsequently filled to form covalently closed circle DNA called cccDNA that serves as template for mRNA and subgenomic RNA production

gapped dsDNA has protein on one 5’end and RNA primer on other end, only -DNA is fully complete

Infection and DNA repair to form cccDNA
Pregenomic RNA synthesis
Assembly of core and first primer shift to form replication complex, own polymerase
Reverse Transcriptase forms -DNA
RNAse H digests pg RNA
DNA synthesis from -DNA via RNA primer

What serves as a template for DNA genome production?
subgenomic +RNA (produced out od cccDNA) serves as template for Reverse Transcriptase to form DNA genome using -DNA to form gapped dsDNA

Question 15

Single stranded DNA Viruses
Baltimore class
circular or linear?
basic problem

Answer 15

Baltimore class
2

circular or linear?
linear: Parvo B19 parvovirus (5th disease)

Filling the gap, Dispalcement synthesis, rearrangement, displacement synthesis, enlargement of concatemer, then site specific cleavage

circular: Circo: TT virus (ubiquitous guman virus)
dsDNA synthesis by host DdDp
Viral replication protein creates Nick
rolling circle to produce ssDNA that can
capsulated
or used to produce another dsDNA

Basic problem
RNA only be made from dsDNA template, so first you need DNA synthesis, mostly DdDp from host needed

replicate in nucleus

Question 16

RNA Polymerases
3 types and characteristics

Answer 16

RNA Polymerase 1
rRNA (60% of cellular RNA)
nucleus

RNA Polymerase 2
mRNA (processing, CAP, polyA, splicing)
neucleoplasma

RNA Polymerase 3
tRNA, 5S rRNA

Question 17

RNA Synthesis
Mechanism

Answer 17

Initiation
Nucleus: RNA polymerase recognizes the recognition site to bind to promoter, seperation of DNA into single strand for start of template strand (non coding) in 3’ to 5’ direction (at 3’end), so mRNA is +strand

Elongation’
pre-mRNA nucleotides paired wirth complementary based of template strand using uracil instead of thymine (5’->3’ direction for mRNA, 3’->5’ for template strand)

Termination
terminator signals RNA Polymerase to stop and release from DNA, after seperation DNA comes back together, pre-mRNA released

Question 18

RNA Synthesis
Initiation more in detail

Answer 18

Tata Box bound by TFIID/Tata binding protein
RNA Polymerase 2 complex binds to Initiator sequence

Part of the complex:
Transcription factors
Tata box binding protein
enhencer binding proteins (region far away)
silencer binding proteins
also DNA bending proteins that can bend DNA to make distal region able to be bound by enhencer proteins + complex

RNA Polymerase binds
DNA opens up
Initiation complex at 3’end of non coding strand

Question 19

RNA Processing
3 steps and some characteristics

Answer 19

pre-mRNA
exons/introns and nothing at 3’ and 5’ end

1. 5’ capping
2. 3’ polyA
3. Splicing

also 2 untranslated regions

cap: metylated guanine added to 5’ end by 5’5’ phosphodiester linkage, m7G by transferase, addional bases can be methylated

polyA: Enzyme complex that put A at 3’ end
Virus encode polyA in genome

Splicing: Introns cut out and Exons ligated together; Introns form laraiat complex through snRNPs

Question 20

mRNA rules

Answer 20

requires dsDNA template
+sense
ribosome ready
synthesis in 5’->3’
pol 2 binds 3’ of non coding end
there is no -strand RNA in eukaryotes, only in viruses
there is no large dsRNA (except secondary structures)

Question 21

Untranslated regions and their characteristics

Answer 21

5’
50-70 nt, often secondary structures that must be unwound to allow passage of ribosome and influences translation efficiency

3’
can regulate translation initiation, translation efficiency, mRNA stability

polyA tail which is necessary for efficient translation and stability (half life longer if long polyA)

Question 22

Translation
Machinery
Viral Initiation and examples
Difference between eukaryotic and bacterial/virus mRNA and advantage for virus

Answer 22

Machinery
ribosome with initiation , elongation and termination factors

PolyA binding protein
5’ cap binding elongation factors
other factors

before AUG start, 48S initiation complex primed via cap/protein or IRES, then elongation by 60S rRNA, termination at stop codon

Viral Initiation and examples
Cap dependent: HIV, Influenza
Protein dependent (VPg): Norwalk Virus
Internal ribosome entry site dependent: Poliovirus
secondary structure that can start internal translation, often multiple IRES to make multiple genes

Difference between eukaryotic and bacterial/virus mRNA
bacteria: multiple ORF to produce more proteins on one mRNA (polycistronic)

there are proteins that can bind to stop codon to inhibit fall off or IRES possible

advantage: similar number of proteins which is good for virus assembly

Question 23

RNA genomes
4 types and examples
rules for RNA Viruses
form
what about ambisense?

Answer 23

ssRNA (+)
Picorna, Calici, Flavi, Toga, Corona
genome that is translation ready but needs -RNA as a copy template to produce more +RNA for genome synthesis

ssRNA (+) DNA intermediate
Retrovirus
ssRNA is template for -DNA intermediate by RT, then dsDNA in host genome (Provirus) that will be used as a template to get more +RNA for tranlation and genome

ssRNA (-)
Orthomyxo, Filo and Arenaviruses, Bunya, Paramyxo, Rhabdo

not translation ready, so RdRp from virus particle needed to produce +ssRNA that can be used for translation or -ssRNA for genome

dsRNA
Reo- and Birnaviruses
make +ssRNA for translation and genome

Rules
always RdRp, -ssRNA even needs to transport it within particle to produce +ssRNA

genome must be synthesized end to end with no loss of nucleotide sequence

Always transcription into +ssRNA necessary

Form
dsRNA linear
ssRNA linear or circular

what about ambisense?
does exist: Coding genes on both strands

Question 24

dsRNA genomes
facts and examples

Answer 24

facts and examples
often segmented

Reovirus like Rotavirus have 1-12 segments, large (often polymerase, medium and small sequences)
Birnavirus have 2 segments

not translatable by ribosomes

Question 25

Difference between +RNA and -RNA regarding virus genome structure

Answer 25

+RNA
is naked with secondary structure formation
No RdRp in capsid
not that stable
infectious
Helicase: unwinding

-RNA
is ribonuceloprotein associated because it needs to protect from host, these complex prevent basepairing
RdRp in capsid
stable against RNases
not infectious

Question 26

RNA Viruses characteristics (picture)
Symmetry
Enveloped
Genomic architechture
Baltimore
Example

Answer 26

Question 27

+ssRNA genome
problem and how to solve with (example)
subgenomes?
advantages

Answer 27

Problem
RNA synthesis and protein synthesis at same template, so regulation of transciption and translation is necessary

Poliovirus protein synthesis at ribosomes, RNA synthesis at membrane vesicles, so space seperation, membrane vesicles also to protect from host

Subgenome
Togavirus can also produce RNA subgenomes for mRNA synthesis, especially for proteins that virus needs a lot

advantage
also advantage to regulate protein synthesis, more subgenomic sequences = more protein

Question 28

-ssRNA
2 types
What’s the rule of 6?
What’s special about Influenza virus and -ssRNA viruses regarding mRNAs?

Answer 28

unimolecular
polycistronic ssRNA with certain sequences in gene that are termination sequence, also nucleoproteins can inhibit some of these sequences tp form full length strand
e.g. VSV

seperation depending on how much nucleoprotein bound to -ssRNA, if a lot template used for genome replication

Rule of 6
e.g Paramyxovirus

number of nucleotides always multiple of 6 because during replication, these viruses are dependent on nucleoprotein molecules that each bind to 6 nucleotides

segmented
e.g Influenza replicates in nucleus (splicing of some RNA synthesis products)

each segment has own polymerase bound to it
**
What’s special about Influenza virus and -ssRNA viruses regarding mRNAs?**
Cap snatching, steal cap from host mRNA and out it on their own mRNA for efficient translation

Question 29

Ambisense RNA Viruses
example

Answer 29

example
e.g. Bunya and Arenaviruses

use both strands for translation

Question 30

What’s special about Retroviruses?
Mechanism

Answer 30

ssRNA (+) DNA intermediate
Retrovirus
ssRNA is template for -DNA intermediate by RT, then dsDNA in host genome (Provirus) that will be used as a template to get more +RNA for tranlation and genome

also they contain 2 copies of RNA genome with polyA and cap but it will never be translated

Mechanism
ssRNA bound to nucleocapsid proteins, late assembly protein and enzymes like Integrase and RT in particle

Question 31

Reverse Transcriptase
domains

Answer 31

Polymerase domain that produces cDNA from RNA

Nuclease (RNaseH) domain that cleaves RNA in hybrid

Polymerase Domain that produces dsDNA from cDNA

ssRNA into ds cDNA

Question 32

David Baltimore
What did he discover?

Answer 32

RdRp in VSV
Reverse Transcriptase of Retrovirus
Baltimore system of virus classification
Development of infectious recombinant clonme of RNA virus

Question 33

DNA Family Virus and an example

Answer 33

Parvovirus: B19 causing 5th disease

Circovirus: TTV and TTV like mini virus, chronic but no disease

Polyomavirus: Merkel Cell PyV: cancer, BK virus

Papillomavirus: can cause cancer

Adenovirus: flu like symptoms

Hepadnavirus: HBV

Herpesvirus: HSV1,2 and Herpes zoster

Irido: Fish natural host, Mimivirus biggest virus

Poxvirus: Smallpox by Variola major/minor, Vaccina Virus

Question 34

Polyadenylation of RNA Viruses

Answer 34

can be encoded on viral genome and not non-template dependent like for eukrayotes, only polyA singnals encoded

+RNA: encoded by viral genome as polyA tract
-RNA: poly-U tract

sometimes: reiterative copying of short U sequence in template strand e.g. VSV