Midterm 1 Material Flashcards
1
Q
Give three reasons why we study viruses and sub-viral agents and their respective importance
A
- medical, veterinary and agricultural importance
- some are very common (e.g. cold)
- some are highly lethal (e.g. HIV/AIDS)
- some cause dread, major disruption, economic loss (e.g. Sars-CoV2)
- some infect animals and plants leading to econmic loss (e.g. foot mouth disease) - source of fundamental info about biology and medicine
- flow of genetic information
- mechanisms of DNA rep., transcription, translation, RNA splicing, and protein localization
- cell cycle and cancer
- immunology - industrial and therapeutic applications
- recombination DNA tech (e.g. using reverse transcription to make RNA into DNA)
- gene therapy
- modulation of the immune system
- treatment of cancer
2
Q
Give three reasons why viruses are distinct from other biological entities & name which one is the most important factor
A
- obligate intracellular parasites
- replicate by assembly (most important factor)
- depend of HC for energy, metabolic precursors, translation machinery
3
Q
What is the viral genome comprised of?
A
DNA or RNA, but never both
4
Q
What is the capsid?
A
protective protein shell that consists of smaller subunits, capsomeres that are arranged in a symmetrical pattern
5
Q
What is the viral envelope?
A
- lipid bilayer which contains viral proteins (some viruses, not all) where the membrane belongs to the host membrane
- without the envelope, the virus is no longer infectious (given that it is an enveloped virus)
6
Q
What are the eight steps in the viral life cycle? Name and describe each step
A
- attatchment - viral attatchment proteins bind to a specific cellular receptor
- penetration - getting across the cell membrane
- uncoating - release of the viral genome from the capsid and delivery to the correct cellular location
- transcription - production of viral mRNA
- translation - production of structural and non-structural proteins
- genome replication - production of multiple progeny virus genomes
- assembly - assembling genomes and structural proteins into progeny virions
- release - release of progeny virions by cell lysis (non-env) or budding (env.)
7
Q
What three factors classify viruses as biological systems?
A
- contain a nucleic acid which replicates
- encode more than one protein
- undergo mutations and natural selection (e.g. emergence of covid-19 variants)`
8
Q
What do all viruses rely on the HC for?
A
- biosynthetic precursors (a.a and nt)
- energy
- machinery to translate mRNA
9
Q
What do some viruses depend on the HC for?
A
- enzymes that replicate the viral genome
- enzymes that transcribe viral mRNAs
10
Q
Which viruses have been associated with humans since specification, and how do we know that humans have been associated with viruses since speciation?
A
viruses: herpesvirus and endogenous retroviruses
how: 10% of the human genome consists of retrovirus proviral genomes
11
Q
Which viruses have been recognized since antiquity, and what is one piece of evidence?
A
viruses: polio and smallpox
evidence: ancient Egyptian tablets depicting an injury caused by polio
12
Q
Which viruses have been associated with humans more recently?
A
HIV, ebola, sars-cov2
13
Q
which viruses are zoonotic and when did they jump?
A
HIV-1 -> early 20th century
Sars-Cov2 -> 2019
14
Q
How did the move from hunter/gatherer societies to agricultural societies effect viral spread?
A
increased population density -> greater number of hosts for viruses
15
Q
What are the properties of a virus that can maintain stablitity in a small population vs that of a large population? Name an example of each
A
small population: viruses that establish life-long latent infections (e.g. herpesvirus)
large population: acute viruses (e.g smallpox)
16
Q
Why did variolization come about?
A
Life long immunity from smallpox was evident which led to deliberate immunization because the mortality rate of variolization was much smaller than that of getting smallpox without variolization
17
Q
What is variolization?
A
deliberate infection with variola virus to generate mild disease with protective immunity
18
Q
What are Koch’s postulates?
A
- the organism must always be present in diseased people or animals
- it must be isolated as a pure culture in vitro, say by growth on nutrient agar plates
- the isolated organism must cause the same disease when injected into a healthy host
- the organism must be reproducibly isolated from the diseased test hosts
19
Q
How did vaccination arise?
A
Jenner used cowpox scabs instead of smallpox scabs to deliberately immunize people, which was just as effective as variolization but was much safer
20
Q
What are the three revolutionary changes in our understanding of infectious diseases?
A
- Pasteur discovered that cellular life forms do no arise by spontaneous generation
- Koch discovered that infectious diseases are caused by bacteria
- Beijernik established the existence of acellular infectious agents (viruses)
21
Q
What were Beijernik’s major observations of the agent that disfigured tobacco leaves?
A
- agent passed through filters that retained the smallest known bacteria
- the filtrate could be diluted, but with loss of infective strength
- strength was increased by passage through intact leaves, but not by incubation in extracts of uninfected leaves
- agent is capable of replication, but only within living host cells
22
Q
Name and describe two other early examples of “filterable” agents besides Beijernik
A
Loeffler and Froth: foot and mouth disease of cattle
Reed: yellow fever in humans
23
Q
What is the differences between segmented and continuous viral genomes? Name an example for each.
A
Segmented: multiple nucleic acid segments that make up the entire genome (e.g. influenza A)
Continuous: one nucleic acid sequence (e.g. Sars-Cov2)
24
Q
What is the purpose of the capsid?
A
- Encases and protects the genome
- mediated binding to the HC and entry of naked viruses
25
What are the symmetrical properties of viral capsids?
- capsid is symmetric
- comprised of multiple copies of more than one proteins
- the subunits themselves are asymmetric
26
How is helical symmetry defined? Name a virus that has helical symmetry.
- Length is defined by the length of the genome (not fixed by capsid)
- can be open ended , but some viruses have capping proteins on the end
- Virus: TMV, bacteriophade M13, rabiesvirus
27
What are the properties of icosahedral symmetry?
- 20 trianglular faces
- 12 vertices that have 5-fold symmetry
28
What are the properties of the simplest icosahedral capsids? Name an example of a virus.
- 60 subunits, 3/face
- e.g. canine parovirus
- each subunit contributes to both a face and vertex
29
What is the purpose of having more complex icosahedral capsid?
It uses more subunits which encloses a larger volume
30
How are bigger faces made in complex icosahedral capsid shapes?
Out of groups of smaller traingels, or other shapes that have a 3-fold axis symmetry (e.g hexagons)
31
What is the equation to determine the number of subunits/capsid (N), and what do the terms represent?
N = 60T, where T = triangulation number
32
What values can the triangulation number (T) be?
T = 1, 3, 4, 7, 9, 12
33
Name an example of a virus with a T= 3 capsid, how many subunits make up the capsid, and how are these subunits arranged?
Virus: nodavirus
Total subunits: 180 (60 x T -> 60 x 3 = 180)
Arrangements: 20 groups of 6 (hexons) and 12 groups of 5 (pentons)
34
How does a virus build a capsid if all the capsid proteins are the same?
the virus uses quasi-equivalent interactions to form pentons and hexons
35
How are capsids organized when T>1?
- 12 vertexes made of pentons (60 subunits)
- the remaining subunits (60T-60) are organized into hexons -> faces
- note for that T =4, some of the hexons straddle the boundaries between faces
36
Name a virus that contains combined symmetry
bacteriophage T4
37
Name a virus that contains complex capsid symmetry
Poxviruses -> small pox
38
What is the Baltimore classification system?
An unoffical classification system based of gene expression and replication strategy of viruses
39
Name a ssRNA (+) retrovirus, and does it pack any enzymes in viral particles and why?
HIV, yes it has to pre-package reverse transcriptase because the RNA is reversely transcribed to DNA and the DNA is transcribed to make mRNA, the RNA genome is not used for translation.
40
Name a ssDNA virus
canine parovirus
41
Name a dsDNA virus and does it pre-package it's own ezymes and why or why not?
Adenoviruses, no it uses cellular DNA-dep RNAp
42
Name a ssRNA (+) viruses and does it pre-package its own enzymes, why or why not?
Sars-Cov2, no it only needs cellular machinery to translate it's genome
43
Name a ssRNA (-) virus and does it pre-package its own enzymes, why or why not?
Rabies, yes it pre-packages RNA-dep RNAp because the genome cannot be translated without the positive sense RNA and thus cannot make it
44
Name a dsRNA virusus and does it pre-package any enzymes, why or why not?
Rotavirus, yes it pre-packages RNAdep RNAp because ribosomes cannot denature dsRNA, and thus no protein can be made until the dsRNA is denatured
45
What is the International Committee on Taxonomy of Viruses (ICTV)?
Offical classification system of viruses primarily based on genetic relationships
46
Name four methods that detect virus particles
1. electron microscopy
2. nucleic acid hybridization or PCR
3. immunodetection of viral structural proteins
4. hemagglutination
47
Name a viruses that leads to hemagglutination of eythrocytes?
influenza
48
How do you perform a plaque assay?
1. prepare serial dilutions of virus stock
2. inoculate aliquots of each dilution into susceptible cells
3. overlay with agar to restrict virus diffusion (prevents viral progeny from spreading)
4. count plaques
49
How are plaque assays quanitified?
plaque forming untis (PFU) per mL
50
Why is a viral titre usually less than the number of viral particles?
1. viral particles are defective
2. cells fight back
51
What are a few limitations of plaque assays?
1. not every cell infected with a virus produced progency virions
2. does not work with non adherent cells
3. not all viruses kill cells
52
What are seven characteristics of viral receptors?
1. protein or carbohydrate linked to a protein or lipid
2. can be cell-type specific (HIV -> CD4 and chemokine R) or broadly distributed (e.g. EGFR)
3. influence host-range of the viruse -> tropism
4. serve normal cellular functions
5. some serve as the receptor for more than 1 type of virus (e.g. SA: influenza, reovirus, rotavirus)
6. some related viruses use different receptors (e.g major group rhinovirus -> ICAM1, minor group rhinovirus -> LDL)
7. can play a major role in dictating cell-cell specificity of virus (e.g. CD4 and HIV -> macrophages express more CCR5 co-R)
53
Which co-receptors are used for HIV binding?
CCR5 or CXCR4 are used, with CCR5 required for efficient initial infection of a person
54
Where are G- viral receptors located?
on the outer membrane only
55
Is human CD4 necessary for infection by HIV?
Yes - CD4+ human fibroblasts were able to be infected by HIV
56
How do we know that human chemokine receptors are necessary for entry of HIV?
When human CD4+ mice were exposed to HIV, they were not infected
When human CD4+ and human chemokine receptors+ mice were exposed to HIV, they were infected
Note: most human cells inherently contain chemokine receptors
57
What mutation allows for resistance to HIV infection? Is this mutation recessive or dominant?
32bp del in the CCR5 gene, recessive gene
58
If someone is homozygous for D32 what is their phenotypic response to HIV
" heterozygous for D32 "
homozygous: highly resistant to infection
heterzygous: progress more slowly to AIDS
59
What caused selection for the D32 mutation in humans and why? Who contributed to the spread of the D32 mutation?
selection: Black Death (Y pestis uses CCR5) or smallpox (severity of disease is increased by CCR5 signalling)
spread: vikings?
60
What are the three steps to viral entry?
1. getting the viral genome across the p.m OR get the phage genome across a membrane and a cell wall of bacteria
2. uncoating
- capsids are stable
- must disassemble rapidly in a cell
3. delivery of the genome to the appropriate sub-cellular location
- e.g. nucleus or cytoplasm -> tend to be the most metabolically active
61
What is a common phage receptor located on G- bacteria?
LPS
62
How does bacteriophage T4 inject its DNA?
1. legs bind to the recepor
2. injects in viral genome (DNA)
63
What did the Hershey-Chase experiment demonstrate?
That the protein coat of phages remain at the cell surface while the nucleic acid is internalized; this allowed them to separate cells and the viral capsids to determine that DNA was the genetic material
64
What happens to viral proteins when enveloped viruses enter the HC?
viral proteins become integrated into the HC membrane
65
Describe HIV-1 entry
1. the viral proteins that are involved are gp41 and gp120
2. the viral receptors are CD4 and CCR5 or CXCR4
3. gp120 binds to CD4 -> gp120 undergoes a conformational change that allows it to interact with CCR5 or CXCR4
4. after binding to CCR5 or CXCR4, gp41 is exposed which can trigger fusion
66
How does respiratory syncytial virus enter HCs?
1. viral F fusion protein binds insulin-like GF-1 R
2. triggers a cellular signalling pathway that calls up an internal cellular protein (nucleolin) to the surface
3. F then binds nucleolin
4. fusion and cell entry
67
Which viruses undergo pore-mediated penetration?
picornaviruses - polio and major group rhinoviruses
68
Why do some viruses evolve to do endocytosis as a mode of entry?
To get their genome closer to its target site
69
Why do viruses use clathrin-mediated endocytosis?
it is fast
70
How do enveloped and naked viruses escape the early endosome?
enveloped: fusion
naked: lysis or permeabilization
71
What triggers enveloped viruses to escape the endosome
pH decreasing to 6
72
What are the steps to enveloped viruses escaping the endosome?
1. viral fusion protein is bonded to the host receptor
2. low pH causes unmasking of fusion-peptide
3. host membrane penetration
4. host membrane scission
5. apposition
6. hemifusion
7. pore
73
How does influenza employ low pH of the endosome to uncoat its genome?
1. protons enter virion by M2 ion channels
2. low internal pH displaces M1 matrix protein from NP-RNA complexes
3. nuclear localization signal on NP proteins targets RNP complexes to nucleus
74
How does adenovirus escape the lysosome?
lysis
75
How does reovirus escape the endosome?
local permeabilization of the endosomal membrane
76
How does the genome get delivered to its target?
on microtubules or actin filaments
77
How does herpesvirus deliver its genome?
capsid binds to the nuclear pore complex embedded in the nuclear envelope and the genome is passed through the pore
78
What are the receptors for bacteriophage T4 and what is there normal functions?
receptors: OmpC (protein) and LPS
function: OmpC = porin; LPS = OM structure
79
What are the receptors for bacteriophage lamda, and what are their normal functions?
Receptor = IamB (protein)
Function = maltose transport protein
80
What are the receptors for bacteriophage MS2 and what is their normal function?
Receptor = F pilus (protein)
Function = conjugation
81
What are the receptors for influenza and what is their normal function?
Receptor = sialic acid (SA)
Function = ubiquitous components of extracellular glycosylated proteins
82
What are the receptors for HIV and what is their normal function?
Receptor = CD4 (protein)
Function = helper T cell marker
83
What are the receptors for rhinoviruses and what is their normal function?
Receptor = Icam-1
Function = intercellular adhesion
84
What are the receptors for murine leukemia virus and what is their normal function?
Receptor = cationic amino acid transporter
Function = amino acid transport
85
What are the receptors for SARS-COV-2 and what is their normal function?
Receptor = angiotensin converting enzyme 2 (ACE2)
Function = regulates heart function and blood pressure
86
What are three ways DNA viruses differ from RNA viruses? Describe each way.
1. use DNA-dependent nucleic acid polymerases
- ddDNAP = replicate genome
- ddRNAP = transcription
- can be host or viral origin
- small viruses have greater dependency on HC functions than large viruses
2. many DNA viruses modulate the cell cycle
-resting animal cells do not contain dNTPs or DNA replication proteins (only present in S phase)
- some large DNA viruses make these themselves
- small DNA viruses push the cell into S phase
3. DNA viruses have a lower mutation rate
- ddDNAP has proofreading function, rdRNAP or RT do not
- the biggest viruses all have DNA genomes because RNA synthesis is not accurate enough to copy large RNA genomes with high fidelity
- exception: coronaviruses
87
For the following DNA viruses, list what they require from the host and what the virus supplies in terms of DNA replication machinery: parvo, polyoma, adeno, herpes and pox (describe herpes and pox together)
Parvo
- host supplies = dNTPs, DNAP
- virus supplies = nothing (only replicated in cycling cells)
Polyoma
- host supplies = dNTPs, DNAP
- virus supplies = proteins that drive resting cell into S phase
Adeno
- host supplies = dNTPs
- virus supplies = proteins that drive resting cell into S phase, DNAP
Herpes, pox
- host supplies = ?
- virus supplies = DNAP, enzymes to make dNTPs
88
How are DNA viruses genes expressed?
- not all viral genes are expressed at the same time
- viruses that replicate in the nucleus depend on HC ddRNAP
- E genes have to appear like HC genes
- needs to appear that the genes need to be ts
ASAP
- L genes are only expressed after the genome is replicated
89
What are some characteristics of E genes in DNA viruses?
- expressed before viral replication
- functions: gene regulation, host alterations, DNA replication
- sometimes referred to as intermediate-early or delayed-early (still expressed before replication)
90
What are some differences between how eukaryotic cells utilize mRNA vs prokaryotes?
EUK
- found in nucleus and cytoplasm
- one mRNA: one protein (only one ORF; monocistronic mRNA
- genes contain introns that need to be spliced in the nucleus
- mRNA is capped at 5' end and polyA at 3' end (posttranslational modification, not encoded)
PROK
- found in cytoplasm (no nucleues...remember)
- one mRNA: multiple proteins (polycistronic mRNA; each ORF preceeded by ribosome binding site)
- genes lack introns (no splicing)
91
Why is it necessary to get rid of introns?
1. makes readable transcripts
2. splicing makes the cell know that the transcript is ready to leave the nucleus
92
What is the purpose of a 5' cap and a 3' polyA tail?
5' cap = ribosome magnet
3' tail = prevents degradation and required for a ribosome to translate
93
Describe how the 5' cap on mRNA is recruits ribosomes.
1. ElF4F
- recognizes 5' cap
- recruits 40S subunit
2. when 40S finds the start codon, it recruits the 60S subunit
3. stop codon causes the ribosome to disassemble
94
If a euk mRNA has multiple ORFs on a monocistronic mRNA, which ORF is translated?
only the 5'-most ORF
95
Describe the order of postranscriptional modifications of eukaryotic mRNA
1. capping
- addition of 5' cap (7meG)
2. 3' polyadenylation
- polyadenylation signal on mRNA causes the RNA to be cut downstream of the signal
- NTs downstream of the tail are destroyed
3. splicing
- remove introns
- join exons
96
Describe the properties of SV40
- small circular genome of dsDNA
- replicates in the nucleus
- ts by RNAP II
- rep by host DNAP
- encode genes that cause tumours (T Ag.s)
- discovered contaminating early polio vaccines
- doesnt cause cancer in humans
97
Briefly describe the genomes of SV40
- E genes (small and large T-Ag) contain a powerful enhancer
- each gene contains a polyA signal
- L genes (VP1 -> 3) have repressor proteins on them
98
In SV40 there is only one promoter for two E genes, how are the separate genes expressed?
Alternative splicing
-There are 2 splice donors and 1 splice acceptor
- only one splice at one donor site and one acceptor site
- yields two different possible mRNAs
- splicing is regulated (which mRNA combination is
produced)
The small T mRNA is made when the 3' most splice donor is used (early stop codon is not spliced out)
The large T mRNA is made when the most 5' splice donor is used (stop codon is spliced out)
99
What are the functions of p53 and Rb?
p53:
- activation promotes cell cycle arrest
- blocks replication
- can trigger apoptosis
Rb:
- binds to and inhibits TFs that promote cell cycling
100
How does SV40 push a cell into S phase?
by binding and inactivating host p53 and Rb
101
What does the large T Ag of SV40 do?
- down-regulates the early promoter
- activated the late promoter
- recruits DNAP alpha
- initiates viral DNA replication (helicase activity that melts the DNA at ORI)
102
How are the L genes of SV40 expressed?
1. need to bypass Ibp (repressor protein that sits on the promoter)
- replicate so much that there is no more Ibp to go around, which allows some replicated genomes to express their L genes
2. need T-Ag to bind (TF)
103
Describe the characteristics of adenovirus
- causes the common cold
- linear dsDNA genome
- ts in nucleus by RNAP II
- encodes it's own DNAP
- 7 RNAP promoters (6 early)
- early genes are subdivided into IE (E1a) and DE (E1b, E2 -> 4)
- one major late promoter (MLP)
104
What is the function of adenovirus E1a protein?
- binds Rb -> driving the cell into S phase
- activates the DE genes promoter
-inhibits the E1a enhancer (binds to proteins that bind to the enhancer)
105
Describe the functions of adenovirus DE proteins
1. inactivate host antiviral defense mechanisms
- E1b proteins bind p53 -> blocks apoptosis
- E3 proteins prevent immune recognition
2. viral DNA replication (E2)
3. late gene expression (E1b and E4)
106
How does adenovirus express multiple L mRNAs if there is only one late promoter?
alternative splicing and alternative polyadenylation by host machinery
107
Describe some characteristics of HSV-1
- cause cold sores
- latent in sensory neurons
- linear ds genome (really big)
- IE -> E -> L regulatory cascade
- replicate in nucleus (viral DNAP)
- transcribe in nucleus (host RNAP II)
- most of the 80 viral genes lack introns; does not heavily rely on alternative splicing
108
What is the function of Vhs (virion host shutoff protein) in HSV-1?
degrades cellular mRNAs
109
What is the function of VP16 in HSV-1, and how does it do this?
transactivates IE genes
- this requires the help of host cellular factor (HCF) and Oct-1 (DNA binding protein)
- VP16 has seperate promoter targeting and activation domains
- the activation domian is full of acidic residues and is highly charged
1. HCF can get into the nucleus, VP16 cannot
- VP16 binds to HCF
2. HCF-VP16 binds to Oct-1 using its promoter targeting domain
3. VP16 recruits RNAP II initiation complex using its activation domain
110
Why does VP16 piggy-back on cellular proteins (like HCF) to access IE promoters?
- HIV uses lytic and latent schemes
- lytic = productive virus infection (cold sores)
- latency = quiescent circular virus episome that is maintained in sensory ganglia
- VP16 is required for lytic replication and reactivation from latency
- in neuronal cells, HCF is retained in the cytoplasm (favors latency)
- HCF migrates to the nucleus when neurons are stressed; conditions that favour reactivation
111
How do HSV-1 and poxviruses transcribe all their genes despite not using alternative splicing?
must have one promoter per ORF
112
What are three principles of DNA replication that leads to an end-replication problem?
1. DNAP can only extend
2. can only build 5'-3'
3. strands are antiparallel
113
How does SV40 solve the end-replication problem? Briefly describe it's DNA replication
How: Uses a circular genome that completely avoids the end replication problem
DNA rep:
1. large T binds to ori
2. helicase activity unwinds the ori
3. large T loads the host DNA replication machinery at ori
4. replication proceeds bi-directionally
5. theta-mode replication
114
How did we find that replication in SV40 always started at the same place?
Take replicating genomes and cut each one in the same place. Measure the distance from the cut site to the centre of the replication bubble. Found that the distance was the same for every replicating genome of SV40.
115
What is an important cellular factor needed for SV40 replication and what is its function?
cellular factor: PCNA (proliferating cell nuclear Ag)
function: increases to processivity of DNA rep by clamping onto the DNA to prevent DNAP from falling off
116
How can we find what cellular factors are need for SV40 replication?
SV40 replication can be recapitulated in a test tube using mammalian cell extracts. You only need T Ag from the virus and you can determine what HC machinery SV40 needs.
117
How does phage lamda solve the end replication problem?
1. theta mode upon entry and circularization
- contains cos sites and the end of its genome that are cohesive ends to circularize its genome
2. rolling circle replication
3. terminase recognizes cos sequnce and cleaves it making staggered cuts to regenerate unit length genomes
4. cos cleavage is coupled with DNA packaging
118
Describe rolling circle replication
1. nick one strands
2. displace the 5' end of the nicked strand
3. new synthesis starting for the 3' end of the nicked strand around the circle many times
4. fill in the other strand of the tail
119
Why doesn't it matter in phage lamba and HSV if one newly synthesized genome doesn't have one end fully replicated?
During rolling circle replication concatemers are synthesized, that having only one out of hundreds incompletely synthesized DNA sequence won't be of significance (it wont be packaged)
120
How does bacteriophage T7 solve the end replication problem?
Uses terminal redundancy to regenerate the ends
121
Describe how terminal redundancy solves the end problem
1. the ends of each strand in the dsDNA are the same in the 5'->3' direction, and the two strands are complementary to one another at their terminals, this is called a terminal redundancy
2. each strand is replicated, where the 5' terminal redundancy of the complimentary daughter strand is missing
3. the two daughter genomes aneal together to create a concatemer
4. viral nuclease makes staggered breaks on either end of the terminal redundancy (either at the beginning or end of each complementary redundancy)
5. the 3' ends of each genome are repaired
122
How does adenovirus solve the end replication problem?
Uses a protein primer and inverted terminal repeats (the terminal on one end is complementary to the other terminal on the same strand!)
123
Describe how a protein primer and inverted terminal repeats solve the end replication problem
1. a terminal protein (TP) is covalently bonded by a phosphodiester bond (one on each strand) at the 5' end
2. during infection new TPs are synthesized and they covalently link to a free dC residue. TP-dC serves as a primer.
3. TP-dC will base-pairs with the 3' most G residue of one of the strands
4. the DNAP uses the OH of the TP-dC as the primer
5. elongation of the new strand occurs while displacing the parental strand as a ss molecule
6. the result is a ds duplex and a ss dispalced parental strand
7. the ends of the ss parental strand will anneal (as they are complementary to each other) creating a duplex
8. the duplex looks like the terminal of a complete adenovirus genome and thus another TP-dC will bind and act as a primer and replication begins
124
What are three ways in which adenovirus DNA replication is considered remarkable?
1. one of the few known examples of protein priming
2. replication is completely continous (no lagging strand)
3. two strands of the parental duplex are replicated in different ways
125
What are some major differences of RNA viruses from DNA viruses?
- replicate in the cytosplasm (expection = flu)
- no splicing (expection = flu)
- capping and polyA done by virus (expection = flu)
- need do not need dNTPs or host DNAP
- do not need to push the cell into S phase
- genome and mRNA are made of the same stuff
- must encode its own polymerase RNAP
-rdRNAPs are more error prone that ddDNAP (exception = coronaviruses)
- RNA viruses have a high mutation rate
126
What are some consequences of RNA viruses having a very might mutation rate?
- drug resistance
- immune evasion
- exist close to the error threshold
- population comprises of a quasispecies
127
What are some properties of RNA genomes?
- mostly linear
- some rdRNAP can initiate new polynucleotide chain
- end-problem does not arise
- the sequences at the ends are often very special and are not easily modified
- most are ss genomes
128
Describe phase 1 of RNA genome replication
parental genome to RF (replicative form)
- promoter on genome allows rdRNAP to replicate the genome creating a complement of the virion RNA (the RF)
- the RF will then make the progeny genome
129
Describe phase 2 of RNA genome replication
RF -> RI (replicative intermediate)
- RF contains a promoter
- RF uses a cluster of polymerases to create lots of genome from one strand
130
Why do positive strand RNA viruses build replication compartments?
- make invaginations and replicate in there
- builds a fort to hide from cell and makes it easier to replicate with everything close together
131
What are some properties of picornaviruses?
- naked. icosahedral
- ss (+) genome
- viruses: polio, rhino, hep A
- use their genome as mRNA
132
How does polio cause pathology?
kills motor neurons which leads to paralytic poliomyelitis (paralysis your diaphragm)
133
What vaccines are there against polio?
- live attenuated (oral, Sabin)
- inactivated (injected, Salk)
134
What are some characterisitcs of polio's genome?
- encodes at least 11 proteins but only contains one ORF
- genome itself already encodes a polyA tail
- contains an IRES upstream of the ORF
- contain VPg at the 5' end
135
How is VPg linked to polio's genome?
covalently linked to genome via phosphodiester bond using Tyr
136
How does polio create many proteins if there is only one ORF?
uses proteolysis: protease domains act upon itself while still imbedded in the polyprotein
- used by all picornaviruses and flaviruses (hep C)
- many viruses use a less extensive version of the strategy
137
What are two properties of viral proteases and what are the consequences of these properties?
1. essential
- good target for antiviral drugs
2. highly specific
- most cellular proteins are not cut, but the ones that are cut are for the virus' benefit
138
Give three examples of how viral proteases cut specific cellular proteins that are to the virus's benefit
1. polio 3C cuts and inactivate a host TF (TF 11D) and cellular transcription is shit off
2. polio 2A cuts the translation factor that recognizes the 5' cap on host mRNAs and cellular translation is shit off (polio uses IRES to avoid killing itself)
3. hep C NS3/4a protease cleaves a protein (TRIF) involved in signaling from a TLR which shuts off induction of an anitviral response
139
What are properties of viral IRES elements?
- binds translation factors that attact ribo
- allow cap-independent translation
- most are naturally located at the 5' end but work anywhere if moved using biological methods
140
What is a limitation of the picrornavirus gene expression strategy? And how do other (+) RNA viruses avoid this
Limitation = not easy to produce different amounts of each viral proteins (for most viruses capsid proteins are required in larger amounts that non-structural proteins
Avoid = make more >1 mRNA (e.g togavirus)
141
How does poliovirus generate the RF?
- VPg is a protein primer that is bonded to two UMPs
- the two UMPs on VPg base pair with the last two AMPs in the 3' polyA tail of the postive sense strand
- the negative strand is synthesized
142
How does VPg get uridylated?
Uses the cis-replication element (cre) in the positive strand
- VPg interacts with it and uses the last two AMP's to uridenylate it
143
How does poliovirus generate the RI?
VPg base pairs with the last two AMPs in the negative sense strand (this is not the polyA tail) and clustes of rdRNAP replicate the negative sense strand to yield positive sense genomes
144
Describe some characterisitics of togaviruses and an example
- icosahedral, enveloped
- ss (+) RNA genome
- e.g. sindbis virus
145
What are some characteristics of sindbis virus' genome?
- 5' cap and polyA tail are made by viral rdRNAP
- only the 1st ORF is translated from genomic RNA
- the 1st ORF contains a supressible stop codon
- rdRNAP coding sequences are located downstream of this codon
- the 2nd ORF (structural proteins) is translated from subgenomoc mRNAs
146
Describe the steps of the early phase of Sindbis virus
1. suppressible stop codon regulates the stoichometery of proteins made (less rdRNAP)
2. if the suppressible stop codon is used P123 is generated
- which is a protease and regulates rdRNAP (has imbedded proteolase activity at 3 sites)
3. if the suppressible stop codon isn't used P1234 is generated
- where there is in-cis proteolysis at the 3-4 site to generate P123 + P4 = early form of rdRNAP
4. P123 is cleaved in-trans at late times (after the levels have increased) creating P1 + P2 + P3 + P4
- higher [P123] -> greater frequency of intermolecular collisions
- trans-cleavage
- P1 + P2 + P3 + P4 = late rdRNAP
147
Describe the late phase of Sindbis virus: genome replication
the genome is translated to yield replication proteins which are needed to make negative strands (P123 + P4 = early rdRNAP) and positive sense strands are then synthesized by P1 + P2 + P3 + P4 = late rdRNAP
148
Describe the late phase of Sindbis virus: production of subgenomic mRNAs
subgenomic mRNAs are transcripted from the internal promoter on the (-) strand and then translated to yield a structural polyprotein
149
Describe some characteristics of coronaviruses
- helical capsid, enveloped
- ss (+) RNA genome
- 10-20% of common colds are caused by endemic coronaviruses
- pathogenic: SARS CoV1, MERS, SARS CoV 2
- structural proteins = S, N, M, E
- non structural proteins = Nsp's
150
What was the believed intermediate host of SARS CoV 2?
Pangolin
151
Where did SARS CoV 2 originate and spread?
- Wuhan China 2019
- initial cases non-randomly clustered around the Huannan Seafood Maket
- current best guess = transmission from animal -> humans happened in the market
152
What is the natural host of SARS CoV 1 and 2 and what are the closest genetic relatives of SARS Cov 2 and which one is the closest?
natural host = bats
relatives:
- RaTG13 isolated from a horseshoe bat
- BANAL-20-52 from related bats (closest to SARS CoV2 in RNA sequence and biological properties)
153
What is SARS CoV 2 binding protein and what is the host receptor and which genetic relative of SARS CoV 2 matches this the best?
Receptor binding protein = spike (S)
Host receptor = ACE2
Relative: BANAL-20-52 RBD matches closley (RatG13 is divergent here)
154
How is the S protein of SARS CoV 2 activated for fusion?
Cleavage at the S1-S2 junction by host proteosomes
- in endosome by cathepsin L
- at the cell surface by TMPRSS2
155
What does SARS CoV 2 contain that makes it divergent from bat relatives
12nt (polybasic) insert that creates a novel furin cleavage site (FCS) at the S1-S2 junction, this provides an alternative pathway for S1-S2 cleavage
156
Describe SARS CoV 2 entry through endosomes, and the consequences with this route
1. S protein binds to ACE2 R
2. virus is endocytosed into a vesicle
3. the vesicle fuses with an endosome
4. capthesin L cleaves the S1-S2 junction
5. fusion
consequence: this process can be blocked with drugs that increase pH of endosomes and lysosomes which will inhibit cathepsin L activity
157
Describe SARS Cov 2 entry through direct fusion at the cell surface and the consequences with this route
1. S protein binds to ACE2 R
2. host cells that express TMPRSS2 will cleave the S1-S2 junction
3. fusion
consequence:
- much more efficient for CoV 2 than CoV 1 due to the polybasic insert (pre-cleavage of some S molecules by furin within the virus-producing cells decreases the number of TMPRSS2 hits required)
- drugs will not block CoV 2 replication in cells that express TMPRSS2 (e.g. respiratory epithelial cells)
158
Why do variants of SARS CoV 2 bear mutations that enhance infectivity in humans?
- virus did not arrive on the scene fully adapted to humans
- emerging variant continue to improve spread, replication, and immune evasion
- e.g the furin cleavage site is sub-obtimal and is enhanced by mutations present in some variants (delta)
159
Why are most of the vaccines for SARS CoV 2 target the S protein?
Abs that bind to S interfere with ACE2 binding, fusion or both
160
Where does SARS CoV 2 replication and transcription occur and which proteins are responsible?
DMV that are derived from the ER, induced by nsp4 and nsp6
161
In SARS Cov2 how is ORF1a and 1b translated?
via a -1 ribsomal frameshift, so more ORF 1a proteins are translated than ORF 1b
162
What protein is transcribed in SARS CoV 2's ORF1b?
rdRNAP
163
Describe the -1 ribsomal frameshift and what this does for SARS CoV 2?
-1 ribosomal frameshift:
- mRNA contain a pseudoknot that can provide a barrier to ribosomes; most of the time, the ribosome will translate as usual, but sometimes it will backtrack one nt and then resume translating
- not the same as a suppressible stop codon
SARS CoV2:
- allows the translation of ORF1b from gneomic RNA
- ensures that ORF1a products are more abundant that ORF1b proteins
164
How does SARS CoV 2 translate its genes that are downstream of ORF1?
Makes subgenomic RNAs
165
Name and describe the function of some of the Nsp's in SARS CoV 2
Nsp1: shuts off host protein synthesis
- binds 40S subunit and blocks the mRNA entry channel (5' leader of viral mRNA can unblock this)
- causes destruction of all cellular mRNAs (5' leader protects viral mRNAs)
Nsp12: rdRNAP
Nsp13: RNA helicase and 5'triphosphatase
Nsp14: 3'-5' exonuclease
Nsp15: endoribsonuclease
Nsp16: 2'-o-ribose methyltransferase
166
Describe how the subgenomic mRNAs of SARS CoV 2 are made
1. there are TRS present at the 3' end of the leader and just upstream of each mRNA body
- TRS = 6-7nt consensus sequence
2. when the rdRNAP hits the TRS, it has the option to translocate to the leader TRS, carrying the nascent strand with it and continue
3. this makes multiple different (-) strands that will make different subgenomic (+) strands
167
Describe acute respiratory disease
- fluid-filled alveoir cellular infiltrate - macrophages and neutrophils that destroy epithelial tissues in an attempt to kill pathogens
- high level of inflammatory cytokines and chemokines, including TNF and IL-6 (cytokine storm)
- weak or delayed IFN response
- predisposing conditions: hypertension, obesity, diabetes, coronary artery disease (predispose to inflammation)
168
What are the two distinct innate antiviral pathways that viruses induce and how do these differentiate pathogenic from non-pathogenic CoV?
1. IFN response (type I and III)
- block virus repolication within infected cells
2. production of inflammatory cytokines and chemokines (IL-6 and TNF)
- recruit and activate immune effector cells
- pathogenic CoV does not induce much IFN but do activate pro-inflammatory chemokine and cytokine production
- non pathogenic CoV induces high IFN activity
169
Describe how a cytokine storm is created
strong viral suppression of host IFN response -> failure to suppress viral replication -> high viral load -> induction of cytokine storm
170
Describe host innate virus detectors and how SARS CoV 2 suppress it and what drug can help treat Covid 19
TLRS and RIG-1 activate NF-kB which produces and inflammatory response and IRF3 which promoted IFN synthesis. SARS CoV 2 proteins (e,g, Nsp15) suppress the activation of IRF3. Dexamethasone inhibits an inflammatory response mediated by Nf-kB
171
Describe some conditions that both (-) ssRNA and dsRNA viruses most adhere to
1. must transcribe their genome before any viral proteins can be made in the infected cell
2. must package their rdRNAP into the virion
- mRNA synthesis is often accomplished within the virion core and (+) strand mRNAs extrude into the cytoplasm
172
What are some characteristics of (-) strand RNA viruses?
1. includes:
- some of the most feared pathogens (ebola, pandemic influenza, measles, and rabies)
- some of the most common pathogens (endemic influenza)
2. divided into viruses that exhibit:
- monopartite/continuous genomes
- polypartite/segmented genomes
173
Describe the properties of VSV
- member of Rhabdoviridae
- helical capsid, enveloped
- ss (-) RNA genome
- pathogen of cattle, pigs, horses
174
Describe some characteristics of the VSV genome
- 5 genes, seperated by intergenic regions
- one promoter at the 3' end
- 5' cap added by rdRNAP
- polyA tail added by RdRNAP
- (-) strand genes: 3' N ... L 5'
175
How does VSV yield 5 mRNAs from one promoter?
1. rdRNAP starts transcription at the 3' end of the (-) strand
2. after rdRNAP is done transcribing the N gene, it will let the (+) strand go, but rdRNAP doesn't have to, it has a choice:
- fall off too
- continue on
3. if rdRNAP continues on it will transcribe the next gene and when it is done it will let the (+) strand go and the rdRNAP again has a choice to fall off or continue going
4. this process repeats itself, therefore N will be the most abundant and L will be the least abundant
176
In VSV how are the mRNAs polyadenylated?
polyadenylation:
- a stuttering reaction where the rdRNAP slips back and goes forward at a run of 7 UMPs (intergenic region) in the template
- used by most, if not all, (-) RNA viruses
termination:
- occurs after about 200 AMP residues have been added
177
What is one problem with VSV genome replication and how does it overcome this?
Problem: Transcribed (+) sense mRNAs cannot be used as a template because polyA and termination reactions at the intergenic regions prevents the production of full-length (+) strands
Solution: at low concentration of N protein, mRNA production occurs. At a high [N] this covers the (-) strand genome which allows for transcription/replication of full length transcripts, by blocking polyA and termination
178
What are some characteristics of paramyxoviruses?
- includes measles and mumps
- genome organization and overall expression strategy are similar to VSV
- most of the mRNAs encode just one protein
- in some paramyxovirus one mRNA is edited to produce two proteins
179
Describe editing of P mRNA in Mumps virus
- +2 frameshift
- unedited mRNA codes for V protein
- edited mRNA with 2 extra GMPs codes for P protein
- the extra G residues in the edited mRNA are added via a stuttering reaction at a run of C residues in the template
180
Describe some characteristics of influenza A
- genome goes to the nucleus
- orthomyxoviridae
- genome consists of 8 separate RNA segments (chromosomes)
- 3 of the segments generate 1 mRNA encoding 1 protein
- 3 of the segments generate at least 2 mRNAs by alternate splicing
- 2 segments use other means of increasing coding capacity
181
How do viral transcripts of influenza gain their 5' cap?
Cap snatching:
1. a 5' cap is added by a host enzyme to a nascent host pre-mRNA
2. flu RdRNAP has endonuclease activity and it snatches a host 5'cap + 25-30nts which shuts of cellular translation
3. RdRNAP uses the 5'capped olgionucleotides a as a primer for (+) strand synthesis
182
What are some problems that influenza faces when replicating its genome?
flu mRNAs cannot be used at templates for the genome because:
- contain a 5' non viral extension (the capped oligio from cellular mRNA)
- contain a 3' polyA tail that is not part of the genome
- lack sequences downstream of the polyA/termination site
- some are spliced and thus lack introns
183
How does influenza virus (+) mRNAs become polyadenylated?
stuttering reaction by rdRNAP
184
How does influenza replicate its genome?
1. replication involves a switch from transcription to synthesis of full length direct copies of each genome segment:
- "primer-independent" transcription
- read-through the polyA/termination sites
- mediated by an NP protein
2. the resulting template strands can be used to make progeny genomes
185
What is the importance of HA and NA in influenza virus?
- major targets of the immune response to influenza
- each are encoded by a genome segment
- if the NA and HA segments of pigs or birds are put into the background of a virus capable of replicating in humans (re-assortment) the antigenic structure is greatly altered an existing immunity becomes irrelevant (antigenic shift)
186
How does re-assortment of influenza occur?
1. old viurs: most human populations are immune (seasonal flu)
2. human virus and bird virus both co-infect cells
3. the segments get shuffled between the two viruses because they can't tell the difference between the same segments -> re-assortment
4. new virus (antigenic shift): no pre-existing immunity in human population (pandemic)
187
What is the difference between antigenic drift and antigenic shift?
1. Ag drift = accumulation of a series of minor genetic mutations in HA and/or NA that are selected for
2. Ag shift = "mixing" of genes from influenza viruses from different species
188
How did 1918 antigenic shift of influenza A occur? How was this deduced?
likely direct bird-human transmission (deduced from re-constructed sequences "molecular archaeology")
189
What are the most predominant cases of influenza A in the world
H3N2, except H1N1 in south asia
190
Why don't most bird influenza viruses efficiently infect humans or spread from human to human?
Avian sialic acid has an alpha2,3 linkage to galactose; whereas most human sialic acid has an alpha2,6 linkage (exception = ciliated epithelial cells in the respiratory tract). Therefore human-human spread requires the avian virus to recognize alpha2,6 linkages.
