Midterm 2 Flashcards
1
Q
Genus of influenza?
A
Genus: Alphainfluenzavirus - Species: Influenza A virus (IAV) Genus: Betainfluenzavirus - Species: Influenza B virus (IBV) Genus: Gammainfluenzavirus - Species: Influenza C virus (ICV) Genus: Detlainfluenzavirus - Species Influenza D virus (IDV)
2
Q
What are the general characteristics of influenza virus?
A
Size of particle = 80-120nm
Size of core = 9nm
Replication location = nuclear
Genome type = - sense RNA
3
Q
What is unique about IAV?
A
Infects many mammals & birds
- Main human species for epidemics/pandemics
- Pigs & birds are important reservoirs (virus changes in reservoir)
18 haemaglutinin (H) & 11 neruaminidae (N) serotypes
Subtypes:
- A (H1N1)
- A (H3N2)
4
Q
What is unique about IBV?
A
Infects humans only - Mainly children Not as sever as IAV Serotypes aren't distinguishable Two lineages: - B/Yamagata - B/Victoria
5
Q
What is unique about ICV?
A
Infects humans only
- Doesn’t cause disease (only one)
Genetically & morphologically distinct from IAV & IBV
6
Q
What is unique about IDV?
A
Infects cattle mostly
- No infection in humans
7
Q
What are clades & subclades?
A
Groups of viruses with similar genetic changes (nucleotide/amino acid changes) w/ a single common ancestor
8
Q
Is influenza virus pleimorphic or not?
A
Highly pleiomorphic
Mostly spherical/ovoid
- Many other forms, including filamentous
9
Q
What is the structure of influenza virus?
A
Outer surface = lipid envelope w/ glycoprotein spikes
- Spikes = 80% trimer (haemagglutinin (HA)) & 20% tetramer (neuraminidase (NA))
Inner surface of envelope = matrix protein lining
Revised architecture: has several host exosomes
10
Q
What glycoprotein is the trimer in influenza viruses?
A
Haemagglutinin (HA)
11
Q
Which glycoprotein is the tetramer in influenza viruses?
A
Neuraminidase (NA)
12
Q
Which virus is highly labile?
A
Influenza:
- Half life = hrs
- Not resistant to soaps, drying…
13
Q
What is the nomenclature of influenza virus?
A
Group/species/geographic origin/isolate #/isolate #/year of isolation/virus subtype
Ex. A/human/Prague/1/56(H2N1)
14
Q
What species is every subtype of influenza virus found in?
A
Birds
15
Q
How many segments are there of the influenza virus genome?
A
8 viral segments - Each has 3 polymerase polypeptides - Since has 8, potential for re-assortment Packaged into the core Called the RNP (RNA + nucleoprotein) - Each segment (RNP) in helical form
16
Q
What is the function of the first 3 segments of influenza virus?
A
Transcriptases
- 3 polymerases
17
Q
What is the function of segment 4 in influenza viruses?
A
Haemagglutinin
18
Q
What is the function of segment 5 of influenza virus?
A
Nucleoprotein (binding, transcriptase, transport)
19
Q
What is the function of segment 6 of the influenza virus?
A
Neuraminidase (releases virus)
20
Q
What does the HA bind to?
A
Sialic acids
21
Q
What happens when the HA binds to the sialic acids (SA)?
A
Virus internalized by receptor-mediated endocytosis into the endosomes
22
Q
How does membrane fusion & release of RNPs of influenza virus occur?
A
Cleaved HA to allow fusion protein binding
- Cleaved from HA0 by cellular protease to HA1 & HA2
- Cleaved HAs form a disulfide bond-linked complex
Low pH
- HA protein undergoes conformational changes leading to exposure of HA-2 fusion peptide
- Facilitates low-pH-induced fusion of viral envelope w/ endosomal membrane
- Causes influx of H+ ions via M2 ion channel, leading to release of viral RNPs into cytoplasm
23
Q
Why is NA required?
A
To remove SA from cell membrane to allow virus to leave cell surface
Otherwise stuck to cell
24
Q
How is the +RNA synthesized for influenza replication?
A
-RNA moved to nucleus
- Allows +RNA to be synthesized
Requires cap for translation
- Influenza polymerase does “cap-snatching transcription mechanisms”
+ sense capped RNA moved to cytoplasm
25
What is the PA-PB1-PB2 complex?
Localized in nucleus
PB2 binds 5' methylguanosine cap for RNA
PA endonuclease cleaves 10-15nt downstream of host pre-mRNA
- Initiates polymerization by RdRp of PB1 using vRNA as the template
26
How is influenza virus replicated?
HA binds to SA on cell membrane
Endocytosis & acidification (pH5 or below) activates membrane fusion & release of genome
3 polymerases from cause "cap-snatching" to produce +mRNA occurs in nucleus
Protein is synthesized w/ PA-PB1-PB2 in cytoplasm
-RNA is replicated in nucleus
Assembly occurs in cytoplasm
NA cuts SA from cell membrane to prevent virus from "sticking"
27
What does NP concentration determine?
If mRNA or viral genome RNA is made
28
What is the interferon synthesis of influenza replication?
virus inhibits innate immune responses so less resistant to infection
NS1 protein inhibits NFKB transcription pathway
29
What are the different ways of influenza prevention during replication?
Amantadine: blocks uncoating
Relenza or Tamiflu: blocks budding
Leptomycin B: inhibits polymerase
30
What animal did IAV derive from?
Wild birds
HA recognizes different forms of sialic acid depending on species
- Difficult to overcome barrier between species transmission
Needs additional adaptations for human-human transmission
31
What is the glycoprotein of birds vs humans?
Birds = a2,3 linkage
Humans = a2,6 linkage
Pigs = a2,3 or a2,6
- Mixing pot: allows passage of reassorted avian viruses to humans
32
What is the peak month of influenza?
January, February, March
33
What are the normal ages of influenza mortatlities?
```
Young peaks (below 10)
Old peaks (exponentially increases after 20-30)
```
34
What influenza pandemic had a strange age peak?
1918
| Age 15-55
35
What is an example of an eidemic?
Ebola virus spread between 3 African countries between 2014-2016
36
What is an example of a pandemic?
IAV H1N1 (Spanish flu) in 1918
37
How is influenza spread?
Airborne transmission/aerosols (less than 10um in size) (sneeze, cough, laugh, exhale)
- Sneezing generates 40,000 particles between 10-100um (droplet or aerosol
- Most common
Droplets (coughing or sneezing)
- Propelled short distance
- Comes in contact w/ others conjunctiva, mouth, or nasal mucosa
Contact transmission
- Direct (body-body)
- Indirect (contact w/ intermediate objects)
38
1deg vs 2deg infection:
1deg = necrosis of ciliated cells (regenerate after 7 days)
- Fever, chills, muscle ache, headache, prostration, anorexia
- Lasts 3-7 days
- Nasal congestion & malaise last whole time mostly
- 50% are asymptomatic but contagious
2deg = bacterial pneumonia
- Death
- Very rare
39
How does the cell-based vaccine against influenza work?
Gives 50-80% protection against HA & NA
Decides which HA serotype to use for the next year
- Produced in embryonic eggs
- Usually right 1/2 times
- Efficacy = 30%
Creates attenuated vaccine strain from attenuated strain & new virulent strain
40
What is the difference between antigenic drift & antigenic shift?
Antigenic drift: changes in proteins by genetic point mutation & selection
- Ongoing & basis for vaccine change each year
- Error rate = 1/2000-10000 not
- Will allow binding when antibodies were previously blocking
Antigenic shift: changes in proteins through genetic reassortment
- Produces different viruses not covered by "normal" vaccine
- Usual cause of pandemics
- Usually caused by intermediate host (pig) w/ both human & avian
41
How to prevent/treat influenza?
```
Act to block matrix protein (M2)
- Prevents pH decrease
- Results in inhibition of RNP release
- Ex. Amantadine
Neuraminidase inhibitors
- reduce spread of virus between cells by stopping SA from allowing virus to leave membrane
- Ex. Tamiflu, Zanamivir
- Resistance emerging
Vaccines
- Trivalent = best guesses
- Quadrivalent = new (contains 1 A virus & both B virus lineages
- recombinant HA (rHA): shortens production time
```
42
How does rHA vaccines work?
HA sequence is cloned into baculovirus
- Expressed by insect cells
- Shortens production time from 6-8 months to 4-5 months
43
What Influenza strain seems to have disappeared since covid began?
```
B/Yagamata
Due to public health & lab responses:
- Travel restrictions
- PPE
- Social-distancing
```
44
What could be used to more rapidly construct attenuated viruses to be used as an antigen?
Reverse genetics
| Predicts antigen-encoding sequences via genomic information
45
What are the conventional vaccines of influenza?
Live-attenuated vaccines
Inactivated vaccines
Split-virion influenza vaccine
Subunit vaccine
Virosome influenza vaccine
* Differ in activity, antigen component, & structural organization
- Impact immunogenicity & protective efficacy
46
What re the nucleic acid vaccines (nonconventional)?
Viral vector vaccines
DNA vaccines
mRNA vaccines
47
What are the advantages/disadvantages of live-attenuated vaccines?
Use weakened (attenuated) form of the virus
Approved for use in multiple countires
Advantages
- Capable of replicating in host cells
- Creates stong & long-lasting immune response
- Immune response involves CTLs, B cells & Th cells
Disadvantages
- Temperature sensitive
- May trigger disease in immunocompromised individuals
48
What are the advantages/disadvantages of inactivated vaccines?
The dead version of the virus
Approved for use in multiple countries
Advantages:
- Well-established technology & simple to manufacture
- Stable
- No live components, so no risk of disease
Disadvantages:
- Not as strong as live-attenuated vaccines
- Booster shots required
- Induce B cell-induced humoral immunity only
49
What are the advantages/disadvantages of split-virion influenza vaccine?
Splitting agents/detergents & conditions are selected to disrupt viral envelope & virion particles
- Preserve HA & NA, M & NP proteins
Approved for china trivalent & quadrivalent inactivated influenza vaccine
Advantages:
- Concentrate & increase levels of active antigenic proteins in each volume of the vaccine (maximum antibody production)
- Safer than inactivated b/c no components of virion particle preserved that can cause side effects
Disadvantages:
- Mainly induce B cell-induced humoral immunity
50
What are the advantages/disadvantages of subunit influenza vaccines?
Prepared by extraction, purification, & concentration of HA & NA antigenic proteins from the split-virion vaccines
- Cloned into protein expression plasmids, then transferred into cells to produce antigen
Common trivalent vaccines
- Influvac, Agrippal
Advantages:
- Good immunogenicity
- Safe & tolerable in children, adults, pregnant women, & infants
Disadvantages:
- Low immunogenicity (need high dose)
- Expensive
- Mainly induce B cell-induced humoral immunity
51
What are the advantages/disadvantages of virosomes influenza vaccine?
Consists of HA, NA, & viral phospholipids
- Maintains membrane fusion & cell-binding capabilities
Epaxal & Inflexal
Advantages:
- Increased immunogenicity compared to subunit/split-virion vaccines
- For all age groups & immunocompromised individuals
Disadvantages:
- Mainly induce B cell-induced humoral immunity
52
How can whole influenza vaccines be inactivated?
Chemical inactivation:
- Formaldehyde (protein cross-linking or RNA cross-linking)
- Beta-propiolactone (BPL) (irreversible alkylation of nucleic acid bases)
Radiation inactivation:
- Gamma irradiation (genome destruction; no protein effects)
53
How do viral vectored influenza vaccines work?
HA gene RT & insertion into Adv shuttle plasmid
Co-transfected to cell lines along w/ Adv backbone plasmid
Purification of replication-defective Adv containing HA
Vaccinate
Influenza HA protein production & recognition by APCs in body
54
What viruses use viral vectored vaccines?
```
Influenza
Arenavirus
Baculovirus
NDV
Herpresvirus
```
55
How do DNA vaccines work?
Plasmid injected into muscle cell
Plasmid moves to nucleus
Transient expression in host cell occurs
Antigen produced
- Stimulates B & T cell responses
- T cell response by: plasmid added to APC or apoptosis of somatic cells
- T cell response by: somatic cells secreting antigens or T helper cells
56
What are the advantages of DNA vaccines?
Stimulate both B & T cell response
Improves vaccine stability (DNA is stable)
DNA resists temperature extremes
- Storage & transportation are easy
No infectious agent
Plasmids easily manufactured
Sequence can be changes easily in lab
- Easy to respond to changes in infectious agent
* could've worked against Spanish flu in 1918
57
How can DNA vaccine be delivered?
```
Gene gun
Epidermal powder immunization
Jet injecter
Tattoo device
Microneedles
```
58
What are the two categories of mRNA vaccines?
Non-replicating mRNA (NRM)constructs
| Self-amplifying mRNA (SAM) constructs
59
What is the non-replicating mRNA constructs structure?
Cap structure: linked via a triphosphate bridge to first transcribed nucleotide
- Essential for efficient translation
- Blocks 5'-3' exonuclease mediated degradation
UTRs: important for maximizing gene expression/efficiency
ORFs
3' poly-A-tail: crucial for translation & protection
* No sequence for self-induced replication
60
What is the self-amplifying mRNA constructs structure?
```
Cap structure
UTRs
ORF
3' poly-A-tail
Replicase: RdRp for self-replicating
```
61
How do mRNA vaccines work?
1. Formulated in lipid nanoparticles for protection & cellular uptake
- Uptake via membrane-derived endocytic pathways
2. mRNA released into cytosol
3. Translated:
- NRM translated by ribosomes to produce protein of interest that undergoes subsequent post-translational modification
- SAM translated by ribosomes to produce replicate machinery for self-amplification of mRNA; then produce protein of interest that undergoes post-translational modification
4. Expressed proteins are:
- Secreted
- Trans-membrane
- Intracellular
5. Innate/adaptive immune response detects proteins via MHCI
62
What are the advantages of mRNA vaccines?
Can be picked closer to actual flu season
- Removes guess work & potential mutations in eggs
Flexibily
- Same production process no matter change of flu strain
- Only need to replace ORF
Rapid response
Induce T-cell mediated immune response better
63
How many influenza pandemics have there been?
10 in 300 years
Major ones:
- 1918-1920 = 50-100 million deaths
- 1830-1832 = smaller population, but as severe
64
What features are needed for a new influenza pandemic to occur?
Novel strain that is:
- Readily transmitted
- Genetically unique (no preexisting immunity)
- Increased virulence
65
What was different about the 1918 pandemic influenza?
H1N1 strain:
50-100 million out of 1 billion died
Killed mostly healthy young adults (strange)
- Most flu's kill below 3years or above 65 years
- Life expectancy dropped to 38/39
- Pregnant women deaths ranged from 23-71%
More deaths in US than any war
Multiplied faster & created more lesions than a more common virus
- Caused cytokine storms
66
What are cytokine storms?
1. Viruses infect lung epithelium to produce viruses & release cytokines/chemokines
2. Cytokine/chemokine activated macrophages & infected dendritic cells lead to increased immune response & beginning of cytokine storm
3. Released chemokines attract more inflammatory cells to migrate from blood vessels, amplifying the cytokine storm
Result: lung & other organ damage from inflammation
67
What happened during the avian influenza?
Worldwide in poultry in 1983-84
High pathogenicity
- 90% mortality
Transmission: farm to farm via clothing (fecal route)
- Survived long periods at low temps
Migratory waterfowl = natural reservoirs
* most avian influenza stains are not highly pathogenic & don't impact wild birds
- Chicken don't have RIG-I that stops infection
68
What is RIG-1?
Detects viral RNA & viral transcriptional intermediates
69
What was the 1997 avian to human transmission?
H5N1
Fecal oral route from waterfowl to chickens
Fecal oral route from chickens to humans (live poultry markets)
- 18 cases, 6 deaths
Believed to have been changed via antigenic shift of:
- HA from goose
- NA from teal
- Internal genes from quail
*No human-human transmission
70
What are the symptoms of avian influenza?
Poultry:
- Diarrhea, incoordination, lack of energy, sudden death
- 70-100% mortality
Human:
- Fever, malaise, muscle aches, sore throat, cough
- 50% mortality
71
What are the reasons for transmission of the avian flu from bird to man?
Poor sanitation of wholesale markets
Chicken markets close to residential areas
No central slaughtering facility for chickens
Practice of slaughtering chickens at retail outles
NO system to monitor importation of chickens into Hong Kong from mainland
Poor hygiene at local chicken farms
72
How to spread avian flu?
Banned all chicken imports
Slaughter all Hong Kong chickens
Clean-up markets
73
What was the impact of the avian influenza?
```
100 million birds died/killed
Inadequate compensation to farmers
- Discouraged reporting
Poultry & poultry products are food staple
- Provide 30% of protein intake
H5N1 elimination is unlikely
- Large spread (control varies/country)
- Backyard flocks
- Wild birds infected
```
74
What is a neoplasm?
Also called tumor
The uncontrolled, abnormal growth of cells or tissues in the body
Can be benign or malignant
- Only malignant neoplasms are cancers
75
What are the main differences between benign & malignant neoplasms?
```
Metastasis (spread)
- Benign = no; malignant = yes
Encapsulation:
- Benign = common (fibrous capsule); malignant = none
Growth rate:
- Benign = slow; malignant = rapid
Vascular growth:
- Benign = no; malignant = yes
Functional?:
- Benign = retain functions; malignant = dysfunctional
```
76
What is a polyp?
A soft, fleshy mass growing out of the skin or a mucosa
- Risky (can be cancerous)
Three types:
- Sessile: if polyp lacks a stalk
- Adenomatous: if polyp contains some glandular growth (66% of colon polyps)
- Pedunculated: polyp has a stalk
77
Tumor terminology:
```
Benign: -oma (originates in epithelium)
Malignant: -carcinoma (originates in epithelium) or -sarcoma (originates in nerve, bone, muscle)
- 90% of cancers are carcinomas
Exceptions (all malignant):
- Lymphoma (lymphoid tissue tumor)
- Myeloma (bone marrow tumor)
- Hepatoma (liver tumor)
- Melanoma (tumor of melanocytes)
- Leukemia (white blood cells)
```
78
What are the components of a tumor mass?
1. A population of neoplastic cells
2. Stroma (connective tissue & vessels supplied by the host) for support & nourishment
* microenvironment contains tumor-associated cells (fibroblasts, immune cells, macrophages)
79
How many people die from cancer per year?
10 million
2/5 Canadians will develop cancer
- 1/4 will die
80
What is PYLL?
Potential years of life lost
How many more years a person would live if they didn't die of cancer (death is 75 years)
- Cancer rises exponentially with age (avg = 65-69)
81
What is the main cancer diagnosis in males & females?
Males: prostate cancer
Females: breast cancer
82
What are the factors of cancer survival?
Age
Females have higher chance (66% vs 62%)
Lung & pancreatic: steep decline in first 3 years
83
What factors influence cancer incidence?
Socio-environmental influences (location)
Genetic influences
If women has child, decreases risk of breast cancer
Social factors:
- Diet
- Health care availability
- Sunbathing
84
What is a countries HDI?
Average achievment in 3 basic dimensions of human development:
- Long & healthy life (H)
- Decent standard of living (D)
- Knowledge (I)
* high HDI countries expected to have highest increase in cancer incidence
- DUe to growth & aging of population + risk factors
85
What viruses are known to be carcinogens?
```
Epstein-Barr virus
HBV
HCV
HIV type 1
Human papillomaviruses
Human T-cell leymphotrophic virus type 1
Kaposi sacroma-associated herpesvirus
Merkel cell polyomavirus
```
86
How does radiation affect cancer?
UVB ray: damage DNA
- Associated w/ 90% of skin cancers (including melanomas)
UVA ray: Penetrate more deeply into skin
- Play role in skin cancer formation (especially premature skin aging)
UVC: completely absorbed by ozone layer
Radon: lung cancer in those who work in mines
Electric & magnetic fields (from appliances): no contribution
Radio waves: no link
Nuclear radiation: ionized molecules
- Carcinogenic
87
What chemicals cause cancer?
Benzene: leukemia
Arsenic containing pesticides: lung cancer
Polychlorinated biphenyls: liver & skin cancers
Asbestos fibers: lung cancer & mesothelioma
88
How does pollution effect cancer?
Long-term exposure to high levels increases lung cancer risk by 25%
89
Infectious agents cause how much of human cancers?
30%
| Include viruses, helicobacter, & helminths
90
What types of viruses are linked to cancer?
```
Mostly DNA viruses
HPV (sexually transmitted types): linked to cervical cancer
- Plus 25 other types
HBV & HCV: liver cancer
HIV: Kaposi's sarcoma & lymphoma
Retroviruses: cancers in animals
```
91
What are the the general properties of cancer?
Disregard external & internal signals that regulate cell proliferation
Avoid apoptosis
Circumvent programmed limitations to proliferation
Genetically unstable (amplification, deletions, rearrange)
- Karotypes will show triploid or quadraploid #s of chromosomes
Invasive: can escape from normal host tissue
Can survive & proliferate in foreign sites
Immune evasion
Growth-factor independent
92
What is invasion of cancer?
direct extension & penetration by cancer cells into neighboring tissues
93
What is oncogenesis?
The process of healthy cells becoming cancer cells
94
What is senescence?
The process where cells stop dividing & enter permanent growth arrest w/out cell death
95
What is an oncovirus (or oncogenic virus)?
A virus that can cause cancer
| * oncogene: altered gene that can help make a cell cancerous (mutated gene involved in cell growth or division)
96
What is a tumor suppressor gene?
Gene that normally prevents formation of a cancer
Losing this function increases likelihood that cells become cancerous
* opposite of oncogene (which gains function for cancer)
97
What is an oncolytic virus?
A form of immunotherapy that uses viruses to infect & destroy cancer cells
98
What causes cancer?
Acquired mutations (most common)
- Occur from damage to genes
- Not found in every cell
- Caused by carcinogens
Germline mutations (less common) (called inherited cancer)
- Account for 5-20% of cancers
- Occurs in sperm/egg cell
- Passes from parent to child (can pass between generations
- During growth, mutation is copied into every cell
Permissive tissue
- Cells that allow viruses to replicate
99
What types of genes can mutate to cause cancer?
Oncogenes/proto-oncogenes:
- normal function is cell growth & division
Tumor suppressor genes:
- p53 mutation stops its function
- Common cancer is retinoblastoma (Rb)
- Breast cancer gene (BRCA1/2) mutate
DNA repair genes:
- Fix mistakes in other genes that can happen when DNA is copied (can fix oncogenes & tumor suppressor genes)
- MSH6/2 recognize mismatched nucleotides
- MLH1 is a tumor suppressor gene that repairs mismatched DNA; heterodimerize w/ PMS2 to form MutL alpha to repair
100
What is the major cause of death of patients with cancer?
Metastasis
| Causes 90% of deaths
101
What is metastasis?
Physical changes help cells assume a motile phenotype
- Causes the downregulation of epithelial marketers & upregulation of mesenchymal markers
EMT: epithelial-mesenchymal transition allows tumor cells w/ ability to leave primary tumor to metastasize to distant organs
- Use TGF-beta & Wnt/beta-catenin signaling pathways for transcriptional reprogramming
Once at distant organ, undergoes mesenchymal-epithelial transition (MET)
- Stromal cells in tumor microenvironment (TME) help create the metastatic niche
- Stromal cells are TAMs (tumor-associated macrophages) or CAFs (cancer-associated fibroblasts)
102
How do tumor cells use the tumor microenvironment?
TME is the normal environment that surround/feed a tumor cell
Normally the TME does immunosurveillance
- Failure of this causes neoplasia to become cancer
- Evade & eliminate immune system by regulating own antigen machinery
- Continually mutates to escape immune system
- Regulates surrounding cells to facilitate tumor growth & escape immune system
103
Macrophage M1 vs M2 in cancers?
M1 is more in early stages of cancers
- Tumor suppressor that act in TME to stop cell growth
- Recruit cytotoxic T cells & NK cells
As cancer cells hijack immune system, M2-like population increases
- Malignant cells secrete M2-like cytokines to recruit more monocytes & M0 macrophages to differentiate into M2
104
What are the characteristics of the TME?
Poor nutrient
- Cancer cells acquire nutrients from TME (high demand)
- Take in lipids, amino acids, glucose (energy), glutamine (biosynthesis of nucleotides & amino acids)
- Poor nutrients = more suppression of T/NK cells
High acidity
- Aerobic glycolysis generates energy
- Secretes lactic acid
Hypoxia (no oxygen)
Immunosuppressive microenvironment
105
What is an epigenetic landscape?
Heritable phenotype changes that do not involve alterations in the DNA sequence (changes in DNA methylation, histone modification, chromatin remodeling...)
Cancer genomes cause dysregulation of them
- Altered landscapes contribute to initiation & progression of cancer
106
What can alter ncRNA expression?
Amplification, deletions & mutations
- Change function
ncRNAs deregulation is increasing cancers
107
What does autosomal dominant mean and how does it relate to cancer?
Only one mutated allele is necessary for mutation
| Oncogene is autosomal dominant
108
What are the different types of oncogene?
Cellular oncogene = c-onc
Viral oncogene = v-onc
- Feature of tumor viruses that induces proliferation
Proto-oncogene = p-onc
- Activated by mutation to c-onc
- Encode proteins involved in normal cell function
109
What can cause a proto-oncogene to convert into an oncogene?
Deletion or point mutation
Regulatory mutation
Gene amplification
Chromosomal rearrangement
110
What are the four classes of oncogenes?
Class 1: oncogenes that mimic growth factors to induce cell proliferation (rare)
- Ex. Sis: secreted protein that mimics platelet-derived growth factor (PDGF)
Class 2: oncogenes that come from mutations of cell-surface receptors, resulting in an overactive or constitutive protein-tyrosine kinase (PTK)
- Ex. erbB: an epidermal growth factor (EGF) receptor
Class 3: intracellular transducers (four types)
Class 4: transcription factor oncogenes (activate pathways)
- Ex. colorectal cancer
111
What is autocrine growth stimulation?
Ability of cancer cells to produce & respond to their own growth factors
Has become central concept linking oncogene & growth factor
112
What is the EGFR mutation?
Class II oncogene mutation
Ranges from extracellular domain point mutations & deletions to deletions in cytoplasmic tail of receptor
Prevent teathering of domain II & IV creating an continuously active EGFR conformation
113
What are the four types of oncogene transducers?
Class III oncogene:
1. Protein-tyrosine kinases: add phosphate to specific tyrosine amino acids
2. Protein-serine/theronine kinases: add phosphate to specific serine or threonine amino acids
3. G-proteins: trimeric GTPases that bind GTP to become active as signal transducers
4. Phospholipase C (PLC): activated by certain G-proteins to trigger inositol phospholipid signaling pathway
114
What is the process of class IV of oncogenes?
Activate transcription facotrs
Accumulate transcription factors in the nucleus
Drive tumor-promoting target genes' transcription
Inflammation, proliferation, metastasis, EMT...
115
How many oncogenes are required to transform a primary cell?
at least 2
First causes immortalized cell (non-tumor)
Second causes transformed cell (tumor)
* normally, Hayflick limit stops multiplication of cells (after 40-60 divisions)
116
What is cellular senescence?
An anti-cancer mechanisms that prevents proliferation of damaged or dysfunctional cells
Cells will multiply 40-60x before entering senescence phase
Exceptions:
- Germ line
- Stem cells
- Tumor cells
117
What happens during the senescence phase (replicative senescence)?
```
Irreversible arrest of cell proliferation (all)
Resistance to apoptosis (some cell types)
Altered function (all but function differs by cell type)
```
118
What causes cellular senescence?
```
Telomere shortening (cell proliferation)
DNA damage
Oncogene expression
Supermitogenic/stress signals
*DREAM complex triggers senescence
*All are potential cancer-causing events
- Normal cells w/ tumor suppressor mechanisms go through cell senescence & apoptosis (called acute senescence)
- Immortal cells (precancerous) go through transformations
```
119
What are p53 & RB?
At the heart of the two main tumor-suppressor pathways to control cellular responses to oncogenic stimuli
120
What is SASP?
Senescence-associated secretory phenotype
In embryo & placenta, cells secrete signaling molecules
Promote morphogenesis
121
What happens with acute senescent cells during immune dysfunction?
Are chronically present
| Have decreased ability to stabilize p53 for apoptosis
122
What are the potential outcomes for damaged cells?
1. Completely repaired
2. Excessive damage causes apoptosis, senescence, or oncogenic mutation
3. Cell division increases risk of fixing DNA damage w/ oncogenic mutation
4. Senescent cells can't be readily replaced (numbers increase w/ age)
5. Senescent cells secrete factors to alter/inhibit ability of neighboring cells to function, resulting in dysfunctional cells
6. Senescent cells can stimulate proliferation/progression of nearby premalignant cells
123
What phase of the cell cycle do cancer cells not enter?
G0 (nondividing cells)
| Defects in cell cycle regulation = cancer cells
124
What are the regulators of the cell cycle?
Positive regulators: promote progress of the cell to the next phase
Cyclins & cyclin-dependent kianses (CDKs)
- Responsible for progress of the cell through the various checkpoints
p53: a tumor suppressor & principle cellular responder to various stresses (guardian of the genome)
125
What does active p53 do?
DNA repair
Cell cycle arrest (restore genetic integrity)
Apoptosis
Senescence
Ferroptosis (eliminate unrecoverable cells)
* low levels = cell cycle arrest & senescence
* high levels = apoptosis
126
What is pRB?
A tumor suppressor that negatively regulates the G1-S checkpoint
- Restains E2F (transcription factor that activates cell cycle genes)
Phosphorylation of pRB by CDK causes release of E2F, activating transcription of genes needed to proceed through the checkpoint
127
What are the outcomes of DNA damage?
Cancer: from mutations, chromosomal aberrations, or aging
DNA repair systems
Cellular senescence: blocked transcription/replication
128
What is depurination & deamination?
The most frequent spontaneous chemical rxns known to create DNA damage
Depurination: hydrolytic removal of guanine or adenine from Carbon 1 of deoxyribose in DNA strand
Deamination: hydrolytic removal of amino groups from guanine, cytosine, or adenine
- Removal from guanine is most common
- Can create high mutagenic DNA bases
- Stress causes additional deamination products to be fromed
129
What are DNA adducts?
Covalent modifications of the DNA that result from exposure to specific carcinogens
130
What is oxidative DNA damage?
When endogenous ROS (a normal byproduct) attacks DNA leading to damage
Creates:
- 8-oxo Guanine
- Thymine glycol
131
What type of radiation causes DNA damage?
Ionizing radiation
- Induces direct DNA damage & indirect` damage through radiolysis of water
Well known mutagen & carcinogen
Efficient energy source & widespread use
132
How much do DNA shorten with each mitosis?
50-200bp are unreplicated
| - Small stretch can't be copied by Okazaki fragments
133
What is the function of telomeres?
To protect chromosome ends from being recognized as damaged DNA by cellular DNA damage response (DDR) machinery
- Starting length = 10-15kb; after 50-60 replications = 3-5kb
Repetitive DNA sequence: TTAGGG
Shelterin complex is remodeled into T-loop configuration
134
What is the shelterin complex?
protects chromosome ends from DNA damage signaling by ATM, ATR, & PARP1 and from DNA repair signaling by c-NHEJ, alt-EJ, & HR
- Includes telomeric repeat binding factor (TRF1,2), TRF1-interacting nuclear factor 2 (TIN2), repressor activator protein 1 (RAP1), TPP1, & protection of telomere 1 (POT1)
135
What is telomerase?
Enzyme w/ RNA & protein components
Adds telomeric repeat DNA to 3' overhang to stop shortening of telomere in "immortal" cells
Three components:
- Telomerase reverse transcriptase (TERT): catalytic protein w/ transcriptase activity
- Telomeric RNA component (TERC): provides RNA template for telomeric DNA
- Dyskerin (DKC1)
Expressed by:
- Germ cells, early embryonic cells
- Very few stem cells
- 80-90% of cancer cells
136
How is telomerase reactivated?
Activate DDR & gain oncogenic changes (p53/RB loss)
- Bypass senescence
- Continue dividing toward very short telomeres that initiate crisis (M2)
Loss of cGAS-STING/autophagy pathway
- Cell evades death, bypass crisis, & enters third proliferation barrier (M3)
- M3 is the last barrier to cancer development: cells that escape M3 achieve immortality
Enhanced chance of acquiring additional driver mutations & activating TMM (telomere maintenance mechanisms)
137
What are telomerase inhibitors?
Selectively kill cancer cells
Immune response
Direct telomerase inhibitors
138
What are the categories of oncogenic viruses?
Retroviruses
- Human T-lymphotropic virus type 1 (causes T-cell leukemia)
RNA tumor viruses
- HCV (causes hepatocellular carcinoma)
Small DNA tumor viruses
- HBV (causes hepatocellular carcinoma)
- Merkel cell polyomavirus (causes merkel cell carcinoma
- Human papillomavirus (causes multiple reproductive organ cancers)
Large DNA tumor viruses
- Epstein-Barr virus (causes many)
- Kaposi's sarcoma-associated herpesvirus (causes many)
139
What viruses cause only animal cancers?
Marek's disease virus (lymphoma in chickens)
- Caused by herpesviruses that affect young
- Virus concentrates in feather follicles & can survive months as dander on floors
- High mortality
- Females more affected
140
What is the main differences between RNA & DNA tumor viruses?
RNA: exert effects through growth signaling pathways, turning them on in absence of stimuli
- Activates oncogenes
DNA: Sequestering proteins that control cell proliferation (RB, p53) to shift cells into S phase
- Negate tumor suppressors
141
Where do oncogenic viruses target?
Most (even unrelated ones) target pathways involved in cell proliferation or evasion of immune response
Directly or indirectly contribute to development of a cancerous cell
- Byproduct of infection
142
What are the common features of human oncogenic viruses on tumor cells?
Don't initiate productive infection within tumor cells
| Some can't replicate as a result of changes in cancerous cells
143
Retroviruses:
RNA viruses that RT their genome into DNA before integrating it into host cell chromosome
Contain essential genes:
- gag: encodes for core & structural proteins (matrix, capsid, nucleocapsid proteins)
- pol: encodes for RT & integrase enzymes
- env: encodes for envelope glycoproteins used for attachment
Oncogenic retroviruses encode a viral oncogene that become integrated into host chromosome w/ RT DNA
144
Examples of oncogenic retroviruses?
Retroviruses were first oncogenic viruses discovered:
- Avian leukemia viruses
- Rous sarcoma virus (RSV)
145
What human gene becomes an oncogene to retroviruses?
```
src
Promotes cell proliferation, invasion, & survival
About 20% of the genome
Increases the virulence of the virus
- Though not essential for replication
```
146
What are the classes of oncogenic retroviruses?
Acute transforming retroviruses
- Encodes homologs of cellular oncogenes
- Get oncogene via integration from cellular genome w/ proto-oncogene
- Cause tumors within days
- Not in humans, but in birds & mice
Nonacute retroviruses
- Induce tumor formation through insertional mutagenesis
- No viral homologs
- Activate proto-oncogenes or inactivate tumor suppressor genes
- Takes years be/c chances of integration are low & require multiple attempts
147
What are the types of nonacute retrovirus insertion?
1. Insertional activation: promoters in retrovirus LTRs induce transcription of proto-oncogenes via upstream integration
2. Viral enhancer elements: induce transcription of proto-oncogenes from distance
3. Insertional inactivation: retrovirus integrates in sequence of tumor suppressor gene, stopping functional allele from integrating
148
How is homology between protein/DNA sequences defined?
Terms of shared ancestry
| Not of function
149
What is the function of kinase?
Activate proliferation pathways within the cell
150
What is the function of transcription factors?
Turn on genes involved in cell replication
151
How does HIV cause cancer?
A retrovirus
| HIV cause immunosuppression, which allows cancer cells & oncogenic virus to grow
152
How does Human T-lymphotropic virus type I (HTLV-I) cause cancer?
Retrovirus
Causes Adult T-cell leukemia (ATL)
Infects CD4+ T cells
Long clinical latency:
- Suggests T-cell transformation occurs after series of cellular alterations & mutations
Tax (a TF oncogene) causes human T cell transformation
- Effects events that progress the cell through the cell cycle checkpoints (allows the virus through)
- Inactivates p53 (so can't stop cell cycle progression)
- Tax is suppressed by viral accessory gene products (Rex)
Not associated w/ homolog or insertional activation
153
How is HTLV I transmitted?
Through infected bodily fluids
- Risk factors: unprotected sex, injecting drugs, transplant
No vaccine, no treatment
154
How does HCV cause cancer?
RNA tumor virus (enveloped, +RNA)
Infects hepatocytes
Slow development
- Cirrhosis in 20 years (scarring of liver)
Causes 50% of hepatocellular carcinoma (HCC)
Cirrhosis is associated w/ development of cancer (HCV is indirectly involved due to cirrhosis development)
- ROS that damage DNA are generated during cirrhosis, introducing mutations that lead to cancer
HCV effects multiple cellular proteins to induce cellular transformations (directly contributes to oncogenesis)
155
How does HCV replicate?
Attach to hepatocytes
Enters cytoplasm by endocytosis
Genome released from capsid and translated by one large ORF
- Via host ribosomes
Translated into polyprotein of 3k amino acids
Processed by viral & host proteases to create 10 viral proteins
- 4 proteins (Core protein, NS5A/B, NS3) affect pathways involved in oncogenesis
156
How do the four HCV proteins affect oncogenesis?
Core protein:
- Increase expression of telomerase
- Inhibits apoptosis
- Blocks tumor suppressor p53 to induce proliferation
- Interferes w/ tumor suppressor pRB, leading to cellular proliferation
NS5A:
- Inhibits apoptosis by stabilizing proteins
- Block tumor suppressor p53 to induce proliferation
NS5B:
- viral NRA polymerase
- Interferes w/ tumor suppressor pRB, leading to cellular proliferation
NS3:
- Stops innate immune response (inactivates host cell factors that can block viral replication)
157
What are the three small human DNA viruses?
HBV
Merkel cell polyomavirus
Human papilloma virus (HPV)
- Only "high-risk" HPVs
158
What are the general characteristics of HBV?
Small DNA tumor virus
Partially double-stranded
Use RT to get DNA from RNA
Liver is major site of replication
159
The oncogenesis of HBV:
```
Up to 25% of infections become chronic
10-25% of chronic carriers can develop hepatocellular carcinoma (HCC)
- Long latency period (decades)
Chronic infections lead to liver damage
- Increased hepatocyte proliferation
- Increase concentrations of radicals
- Causes mutagenesis
Associated w/ fibrosis & cirrhosis that lead to cancer due to inflammation
Protein X (1 of 7 HBV proteins)
- Implicated in oncogenic process
- Stimulates cell proliferation pathways, interferes w/ DNA repair mechanisms, represses p53 promoter & binding
Vaccine: 95% effective
```
160
How many people die/year from HCC?
500k
161
What are the general characteristics of Merkel cell polyomavirus (MCPyV)?
Small DNA tumor virus
First human polyomavirus associated w/ cancer
Found in 80% of Merkel cell carcinoma (MCC) biopsies
Completely dependent on host cell for replication
162
What are the general characteristics of merkel cell carcinoma (MCC)?
Rare & aggressive skin cancer
Can metastasize to lymph nodes & other organs
Occurs in older & immunocompromised individuals
- Infects 80% of adults by age 50
Infects Merkel cells (basal layer of epidermis) that form synapse-like contacts w/ enlarged nerve terminals
163
How does merkel cell polyomavirus cause cancer?
Causes tumor antigen expression
- Inhibits tumor suppressors pRB & p53, allowing cell progression
Integrated genome contains truncating mutations in LT that disrupt helicase domain
- Results in replication-incompetent mutant form of LT
T antigen induces transcription of E2F
- Necessary for turning on essential S phase genes
164
General characteristics of HPV:
```
Small DNA tumor virus
Double-stranded
Falls into two groups:
- Low risk
- High risk
```
165
What is the difference between low risk & high risk HPVs?
Low-risk: causes no disease
- Can cause warts
High-risk: can cause several types of cancer
- 14 types
- HPV16 & HPV18 are responsible for most HPV-related cancers
- Type 16 causes 50% of cervical cancers
166
What are the genes of HPV16?
Early genes:
- E1: DNA helicase (initiates DNA replciation)
- E2: enhances binding of E1
- E4 & E5: unknown function
- E6: enhances degradation of p53 protein
- E7: binds RB protein to increase cell cycling
Late genes:
- L1: major capsid protein. Create virus-like particles (VLPs) that are highly immunogenic
- L2: minor capsid protein. virus entry, move viral compoenets, DNA binding, capsid formation, stability
167
How does HPV replicate the cell?
Normal: keratinocytes are terminally differentiated & don't carry any significant DNA replication
W/ HPV:
- E7 binds Rb, resulting in release of E2F TF
- E2F participates in expression of a variety of genes in cell cycle
- Keratinocytes are convinced to divide
- Would normally cause p53 to activate, but E6 degrades p53
- E6 & E7 inhibit type 1 IFN signaling
168
How is cervical cancer diagnosed & prevented?
Papanicolaou test (Pap smear)
- Significantly reduces mortality rates of cervical cancer
Cervical warts are pre-malignant & removed (freezing or surgically)
Vaccination
169
What are the characteristics of Epstein-Barr virus (EBV)?
Large DNA virus
| Herpesvirus
170
What cancers does EBV cause?
Burkitt's lymphoma
Hodgkin lymphoma
Post-trnasplant lymphoproliferative disorder (PTLD)
- Behaves like fast-growing non-Hodgkin lymphoma (NHL)
- B-cell lymphomas
- Caused by immunosuppression post organ transplant
Diffuse large B cell lymphoma (DLBCL)
- Most common, fast-growing NHL
- Can arise outside of lymph nodes
171
How does EBV cause cancer?
Infects B cells, turning them malignant
may turn epithelial cells into epithelial malignancies (NPC or GC)
Can infect NKT cells to form NKT cell lymphoma
172
What are the steps for EBV latency
1. Infection leads to latency III growth program (proliferation & expansion of infected B cells)
2. Latency III program results in productive "lytic" infection of B cells
3. Cells enter germinal centers
4. Latency II program: induces differentiation to memory B cells
5. Cells leave & enter memory B cell pool
6. Latency 0 program: memory cells are maintained w/ no productive infection
7. EBV protein expansion is silenced
8. Latency I program: occasional memory proliferation to maintain population (rotates between latency 0 & I)
173
What are the general characteristics of Kaposi's sarcoma-associated herpesvirus (KSHV)?
Large DNA tumor virus
Usually asymptomatic
Causes Kaposi sarcoma
- Cancers develop in immunosuppressed individuals (AIDS, transplant)
174
What proteins are encoded by KSHV?
Chemokines: macrophage inflammatory factors
Signaling molecules: v-interferon regulatory protein
Cell cycle: v-cyclin D
175
How does KSHV survive & proliferate?
G-protein-coupled receptor (GPCR) is located in infected endothelial cells
- Essential for triggering Kaposi' sarcomagenesis
- Release angiogenic growth factors that recruit adjacent endothelial cells via paracrine mechanisms (enhanced by ORF74)
- Those cells are latently infected with KSHV, further promoting cell proliferation & survival
176
What are the pathways for KSHV inhibition of cell checkpoints?
Inhibits Rb
Inhibits p53
Viral interferon regualtory factors prevents interferon from repressing c-myc oncogene
177
What is oncolytic virus therapy?
Called virotherapy
Immunotherapy w/ engineered viruses to fight cancer
Highly-promising
- Ex. against glioblastoma multiforme (GBM) (brain tumor)
Minimal side effects
Can be combined w/ other therapies (ex. check-point blockades or chimeric antigen receptor (CAR))
178
What is needed of the viruses for virotherapy?
1. Preserved potential for an active viral life cycle
2. Tumor tropism
3. Tumor-selective conditional viral replication (causing lysis & release of virions)
179
What are the mechanisms of virotherapy?
Oncolytic viruses replicate in malignant cells only (naturally or after genetic modification)
- Normal cells are unaffected due to viral clearance
Viral replication + induction of cell death pathways leads to lysis (oncolysis)
- Released virions infect new tumor cells
- Induces release of tumor-specific & virus-specific antigens, PAMPs, & DAMPs (leads to T cells)
T cell-attracting chemokines are released via inflammatory response
- T cells migrate toward tumor cells
180
How can oncoviruses in virotherapy be modified?
To encode transgenes (cytokines or antibodies)
| - Ensuring specific delivery to TME & further stimulation of immune response
181
What is RNA interference?
Also called post-transcriptional gene silencing (PTGS)
A cellular mechanisms that selectively negates/knocks out/knocks down/silences the effect of any gene by destroying the mRNA
- Destroying mRNA stops improper protein synthesis (the cause of most diseases) & silences the target gene
Triggered by dsRNA (one strand is identical to target mRNA sequence)
Natural process
182
What does RNAi do in plants?
Forms the basis of virus-induced gene silencing (VIGS)
| - Suggests an important role in pathogen resistance
183
What does RNAi do in mammals?
Long dsRNA cause interferon response
- Not a true component of mammalian antiviral response
Stem cells have retained capacity to elicit an RNAi response
184
What is the difference between RNAi & decay?
The degradation products generated during RNAi have an added role to target the next round of transcript degradation
185
How does mRNA decay occur?
Constant process that removes mRNA that has completed translation
- Recruitment efficiency determines longevity
Begins w/ poly-A-tail using deadenylases
Then removes 5' cap via decapping enzymes
Degradation of mRNA body via exonucleases follows
186
How does RNA interfere w/ mRNA decay?
Instead of exonucleases degrading mRNA, RNA takes on a dsRNA conformation
- The substrate for DICER-family proteins to process dsRNA into small interfering RNAs (siRNAs)
187
What are the steps of RNAi?
1. Processing of long dsRNA by RNase III Dicer into siRNA duplexes
2. Loading of on of the siRNA strands on an argonaute protein (AGO) w/ endonucleolytic activity
3. Target recognition through siRNA basepairing
4. Cleave target via AGOs endonucleolytic activity
188
Why is RNAi important?
Protect ourselves from viral attacks
Guards our genes from interruption by jumping genes
Regulate gene expression
Technology revolution in research, biotechnology, & medicine
189
What do the differences between miRNA & siRNA lead to?
Different therapeutic approaches of:
- Mechanisms of action
- Physicochemical properties
- Delivery
- Clinical applications
190
What are the differences between siRNA & miRNA?
siRNA:
- Target: highly specific w/ one mRNA target
- Purpose: provide viral defense & genome stability
miRNA:
- Target: inhibit translation of multiple mRNA targets
- Purpose: endogenous gene expression regulator
191
How do miRNA & siRNA co-exist?
Both share protein components & are created via similar process
Mammals have four Argonaute proteins (AGO1-4)
- All bind miRNA & siRNA
- AGO2 cause endonucleolytic cleavage of cognate RNAs, big part of RNAi
Mechanism overlap
Functional divergence of pathways
- Arthropods: separate Dicer & AGO pathways
- Nematodes: Same Dicer & AGO pathway but pathways separate due to complex system
- Mammals: Single Dicer for miRNA. Functional RNAi requires high Dicer activity, enough dsRNA substrate & suppression of interferon response (rarely met)
192
What factors are needed for functional RNAi in mammals?
High Dicer activity
Enough dsRNA substrate
Suppression of interferon response
* Need all 3 (rare)
193
What are the phases of RNAi?
Initiation: generation of mature siRNA & miRNA
- miRNA initiation comes from genes in nucleus
- siRNA initiation coes from dsRNA in cytoplasm
Execution: silencing of target gene. Degradation or inhibition of translation
194
How is miRNA created?
Transcribed from endogenous gene as pri-miRNA (using RNA pol II)
- Primary miRNA is long w/ multiple hairpins
- Imperfect internal sequence complementarity
Cleaved by Drosha into pre-miRNA
- Precursor miRNA has 70nt imperfect hairpins
- Exported from nucleus
Cleaved by Dicer into mature miRNA
- 19-25nt
195
How is siRNA created?
```
Dicer cleaves long dsRNA into siRNA
- dsRNA comes from exogenous sources
- 21-23nt
Amplification occurs in worms & plants
- Allows RNAi to persist
```
196
What are the three protein components of RNAi?
Drosha
Dicer
Argonaute
197
What is Drosha?
```
Called the microprocessor
Works w/ cofactor DGCR8
Processes pri-miRNA into pre-miRNA
- Leaves 3' overhang
- Cleaves one helical turn (11bp) away from basal segments & two helical turns (22bp) away from apical loop
A nuclear RNAse III enzyme
Identifies pre-miRNA by:
- Hairpin terminal loop size
- Stem structure
- Hairpin flanking sequences
```
198
What is Dicer?
```
A cytoplasmic RNAse III enzyme
Cleaves dsRNA or pre-miRNA
- Cleaves dsRNA at termini or internally (rare)
Functional domains:
- Putative helicase
- PAZ domain (3' anchor)
- Tandem RNAse III domains
- dsRNA binding domain
Works:
- dsRNA terminus bound by PAZ domain
- Works as a ruler (defines length of small RNA by distance between PAZ domain & RNase III cleavage sites
- RNase III domains (2 of them) form a single processing center to cleave one strand of dsRNA
```
199
What are Argonaute proteins (AGO)?
Forms RNAi effector complex (or RNA-induced silencing complex (RISC))
- Works by loading small NRA onto protein
Binds both ends of siRNA
Domains:
- PIWI domain: has an RNase H-like fold & provides the endonucleolytic "slicer" activity
- MID domain
- PAZ domain
200
What are the steps of AGO interaction with RNAs?
1. AGO exposes nucleotides 2 to 5 for initial target pairing
2. Promotes conformational changes that expose nucleotides 2-8 (seed domain) & 13-16 (supplementary domain) for further target recognition
3. Central & 3' "tail" regions of siRNA provide a helical geometry required for catalysis
* Cleaved by PIWI domain (need full complementarity
201
How is dsRNA introduced in plants & worms for RNAi?
Introduction of dsRNA is sufficient for RNAi to occur
- Methods of introduction: In vitro transcription, chemical synthesis
- Easy for C. elegans (feed E. coli expressing dsRNA, soak in dsRNA
In other organisms:
- Methods for introduction: transfection or microinjection
202
What does RNAi do in plants?
Functions as defense against transposable element & virus activity
Plays role in development by regulating some genes
- Need efficient generation of RNAi to avoid degrading important genic mRNA
203
How can dsRNA be formed in plants other than introduction?
Cleaved mRNA fragment can form template via RdRp protein
- Starts second cycle of RNAi & producing secondary siRNAs
Used to degrade similar mRNAs (post-transcriptional silencing)
204
What do viruses do to RNAi?
Evolved viral silencing suppressors (VSRs)
- Block the RNAi mechanisms by inhibiting functions
- Needed for successful viral infection & most viruses contain at least 1
205
What are the ways for siRNA synthesis?
First choose siRNA target site, then use one of the following methods:
1. Chemical synthesis
2. In vitro transcription
3. Digestionof long dsRNA by RNase III family enzyme
4. Expression in cells from an siRNA expression plasmid or viral vector
5. Expression in cells from a PCR-derived siRNA expression cassette
* 1-3 require in vitro preparation
* 4-5 require introduction of DNA-based vectors & cassettes
206
What is considered for siRNA generation?
1. Target site: find 50-100 nt site from ORF; avoid UTRs & SNPs
2. Length of siRNA: smaller are best for mammalian cells as longer can induce mammalian immune response
3. Specificity checking: to decide method
4. Nucleotide content of siRNA: want 30-50% GC
- Lower = unspecific & weak binding
- Higher = unwinding badly
* Important: Need low internal stability at 5' end of antisense stand to properly unwind
207
What are the ways of siRNA delivery?
1. Nanoparticle encapsulation: encapsulated in synthetic lipid bilayer resembling cell membrane that fuses to actual cell membrane, causing endocytosis
- PEG-lipids: improve nanoparticle properties
- Cationic & Fusogenic lipids: enables cellular uptake
2. Electroporation: cells exposed to short pulses of high voltage, allowing siRNA permeability
- Holes reseal after
3. Calcium phosphate transfection: siRNA mixed w/ phosphate buffer & calcium chloride to form precipitate of calcium phosphate & siRNA that is pipetted into mammalian cells
- Cells take up precipitated siRNA after awhile
4. Hydrodynamic injection: rapid injection of duplex siRNA into mice for in vivo gene silencing
- Needs a large volume (difficult)
5. Viral vector: retrovirus & lentivirus vectored delivery of siRNA
- Use pol III promotor (U6) & miRNA precursor to generate siRNA in cells
208
Advantages of vector-based siRNA?
1. More effective than synthetic siRNA for inhibition of gene expression
2. Very stable & easy to handle
3. Stable cell lines can be established
4. Inducible system can be established
5. A knock-out mouse line can be established using transgenic siRNA method
6. Unlimited supply & cost-effective
Disadvantages: Takes days & can cause inflammatory response
209
What are the functional uses for RNAi?
1. Viral gene therapy
2. Silencing cancer-associated genes
3. Heterochromatic domain formation (used for assembly of heterochromatin @ centromeres)
4. Analysis of gene function (knock-down specific gene expression to analyze loss of function)
- Target delivery of siRNA via chemical modifications or viral vector
210
What are the types of therapeutic siRNAs?
Three delivery systems:
1. SNALP
2. siRNA-GalNAc conjugate
- Binds high avidity to ASGP receptor (in liver)
3. TRIM
211
Why is the GalNAc-siRNA conjugate system considered the best?
Simpler in structure
Cheaper to manufacture
Better tolerated
Can be subcutaneously administered
212
How is siRNA delivered via nanoparticles?
1. Nanoparticle formulation administered by IV
2. NP must circulate, reach tumors, & move through tumor to contact cancer cells
3. Must engage surface of cancer cells & be endocytosed
4. NP releases siRNA into cytoplasm
5. siRNA engages RNAi pathway
6. RISC
7. Some engage DICER
213
Challenges of siRNA application:
1. Long-term and immunogenicity of nanocarreir (anaphylactic response)
2. Non-specific liver uptake in systemic siRNA delivery (can lead to liver toxicity)
3. Heterogeneity of disease (cancer) (one siRNA can't work for multiple mutations)
4. Long process of bench to bedside transition
5. Long-term safety of siRNA (high probability of off-target effects)
6. Off-target effect (can activate innate immune response)
214
What are the general characteristics of microRNAs?
```
Translational silencing
Highly conserved
All from hairpin precursors
Most are intergenic
Some are intronic
Some are clustered
Can regulate up to 20-50% of mRNAs
5 mRNA targets per miRNA (thousands of proteins)
```
215
What does miRNA regulate?
Transcription factors
- Regulate the regulators
Control timing of stem-cell division & differentiation
- lin-4 (higher lin-4 = longer life) & let-7 (first human miRNA)
216
What are viral miRNAs?
```
Most in herpesvirus family
- Viral host interaction
- Viral pathogenesis
- Virus life cycle
Also found in:
- Retroviridae
- Orthomuxovirdae
- Flaviviridae
- Coronaviridae
Groups: dsRNA, +RNA, -RNA
Processed by Drosha
```
217
How are viral miRNAs identified?
Computational approach
- Predict 2deg structure of pre-miRNAs
- Results in false positives
Sequencing of cloned small RNA molecules
218
What is the role of viral miRNA in RNA viruses?
```
Unknown due to:
- Smaller genome than DNA viruses
- Most replicate in cytoplasm
- Processing of miRNAs from viral genome lead to genome instability in virus
General function is viral replication
```
219
What was the first discovered DNA virus miRNA?
```
EBV
- Two clusters
Contribute to EBV-associated malignancies via:
- Accelerating cell proliferation
- Controlling apoptosis
- Silencing tumor suppressors
- Promoting invasions & metastasis
- Inducing MET
- Immune evasion
```
220
What are the applications for viral miRNAs?
1. Biomarkers for disease
- miRNA allows faster detection (BART)
2. Therapeutics in malignancies
- BART miRNAs tightly associated w/ carcinogenesis & progression
2. Plant disease control
- Challenges but successful (costly, governmental approval, public perception)
- Used when there are no other options
221
What are piwi interacting RNAs (piRNA)?
Regulate piwi proteins (needed for spermatogenesis)
PIWI proteins contain three domains:
- PAZ
- MID
- PIWI
Processed from single-stranded RNA precursors (different from siRNAs & miRNAs)
- Processing doesn't require Dicer
222
What are long non-coding RNAs (lnRNA)?
Functions by binding DNA or RNA in sequence specific manner or binding proteins
Four archetypes:
- Signal archetype (molecular signal)
- Decoy archetype (moves away other regulatory RNAs or proteins)
- Guide archetype (directs localization of ribonucleoprotein complexes)
- Scaffold archetype (has structural role as platform for assembly)
