Lecture 11-12 Flashcards
(32 cards)
Socio-historical importance of adenovirus
- 60k original research articles
- Causes colds, acute respiratory disease, not the biggest cause of colds, discovered in the 50s
- Nothing to do with adenoids, just discovered because contaminated adenoids and replicated there
- infects people who are living in closed quarter housing (military recruits, boarding schools, Korea war)
- developed a vaccine for it and military recruits have been vaccinated ever since
- 10 years after virus discovery, easy to work with, injected into rodents and almost all of them developed tutors (first pathogen/virus shown to cause cancer into model like rodent) But does not cause cancer in humans (mice don’t have the most robust tumour suppressor mechanisms since they don’t have very long life spans anyway, kinda useless to suppress cancer in such a sophisticated way when doesn’t need to be suppressed for long) + virus does not replicate well in rodent cells so it deliver genes (growth promoting), sits there and causes cancer => in humans, viral replication and cell death => no cancer
- Cancer is never the goal of a virus, just a byproduct
What scientific use has Adenovirus had for us?
- Very used vector for vaccines such as AstraZeneca
Adenovirus family
adenoviridae
Adenovirus morphology
- non-enveloped
- icosahedral
- 90 nm in diameter
- genome: dsDNA 35-45 kb, linear, mid size genome
- vertices are receptors for cell surface
Although the different genera of adenoviruses are not needed to be known (except for that ONE), what is something interesting about adenovirus when it comes to their classification?
- been around for eons, co-evolved with vertebrates, so each vertebrate has an adenovirus associated to it
Know that mastadenovirus is for mammals (included all humans adenoviruses)
Different pathologies associated with adenovirus and at risk populations (from most common to least common)
Acute respiratory illness => military recruits, boarding schools, closed quarters, etc. (cam become viral pneumonia which can be life-threatening especially if secondary infections)
Pharyngitis => infants
Gastroenteritis => infants
(most of us infected at young age and quickly collected antibodies against it, most common serotype is Ad5)
Conjunctivitis (very common cause of viral pink eye) => all
Pneumonia => infants, military recruits
Keratoconjunctivitis (if conjunctivitis penetrates deeper into skin / more serious)
Acute Hemorrhagic cystitis (kidneys, usually immunocompromised infants, quite rare) => infants
Hepatitis => infants, liver transplant patients (new cases of unknown origin, half of them have an adenovirus, serotype 41, very surprising, but only half, 90-95% of these cases have parvoviruses, specifically adeno associated viruses)
What are some common adenovirus serotypes?
Over 100 adenovirus types identified in which about 50 are known to infect humans, don’t need to know them actually just that Dif types have Dif genes and they have different impacts on human health (different pathology) serotype means different antigenically, now use more sequence similarity, Ad5 def most common though (5 and 2 most worked with in lab)
What are inclusion bodies?
viral replication centers/factories
In adenovirus, inclusion bodies in nucleus (more expected in DNA viruses) => forms a crystalline lattice of different viral particles assembling
Clinical progression of adenovirus
After about 16-24 hours, has made over 20k copies of the virus.Virus has mechanism to break open the cell and release the viral particles and start over again
Working with adenovirus in the lab
- very easy to work with, replicates in almost any type of human epithelial cell even if it prefers surface airway epithelial cells, replicate well in immortalized cancer cell line from epithelial cells and human kidney cell line
Kidney cell => after 16-24 hours, CPE, cells round up, are full of virus and start to shed the virus
How do adenoviruses transmit so quickly and well in closed quarters housing?
- very tough virus, survives for long on pillows, developed vaccine, may have been more efficient to just switch out pillows
How do we purify the virus in the lab?
- Using caesium gradients (CsCl), density gradients and genome floats somewhere in it (where it has neutral buoyancy), 80,000 rpm ultracentrifuge, may see a band higher up, lower density => probably viral particles that are not infectious since probs missing genome
What kinds of proteins make up the adenovirus genome?
Most proteins are structural (genome also in structure lmao) and relies on the host for everything else, smallish genome
Capsid structure
Each facet is made up of herons (each has 6 neighbours) but corners are pentons (5 neighbours), each penton is a pentamer with finer protein (beta spiral) sticking out of centre, interacts with host cell receptors. Also protein 5, core protein, forms core that surrounds the genome. Interacts with proteins bound to linear genome. Protein six, on inside of the capsid, links core to the outer capsid, holds everything together, so is one of the main triggers to cause disassembly. Protein 8 helps cement the capsid, holds everything together. Genome is decorated with protein VII, analogous to histones (acidic proteins that bind to DNA, condenses and tightly packs). Terminal protein covalently bound to genome (phosphodiester bond between DNA and serine residue)
Genome of Adenovirus type 2
- 40k basepairs, about 4o different proteins, regulated temporally. There’s E1A and E1B, made first, early promoters then E2,E3, E4 also early promoters. We also have intermediate gene IVa2. Then we have lates genes L1 to L5, made by major late promoter. Lots of functionality packed into very small space. A lot of splicing and polyAs to get all the different proteins. Tripartite leader is spliced onto each one of the late genes. Splicing discovered by studying adenovirus.
Who won the Nobel prize in phgy or medicine in 1993?
Richard J. Roberts and Phillip A. Sharp for their discovery of split genes, tripartite leader in adenovirus
How does splicing work in adenovirus?
Observed hybridization of mRNA of hexon gene to genomic DNA making an echo fragment of the adenovirus genome. When looked at closely, start to see looping out (introns) (A to C, smallest to biggest)
Adenoviruses also have teminal repeats reminiscent of poxviruses, but how do they work?
- Inverted terminal repeats (ITR) about 100 bases at each of the two ends come into play in replication with terminal protein
What is the most minimal region of adenovirus genome needed to cause cancer in rodents?
Only the leftmost 10% region here was necessary to cause cancer, the rest was not needed. Basically just E1A and E1B are enough to turn rodent cells into cancer cells. Learn a lot about cancer from studying these viruses.
What is the receptor for adenovirus?
Known as CAR (Coxsackie B virus and adenovirus receptor), below tight junction so virus relies on micro lesions to actually enter and recognize receptor. Binding helps for viral release too because lots of fibre protein made that can bind with CAR to prevent it from binding with itself and blocking viral release.
How does entry happen for adenovirus?
- binds to CAR receptor, then gets slightly internalized by receptor mediated endocytosis. Each Denton has an RGD loop, amino acid sequence present on the Penton base. This is a sequence that integrins, on surface of cells for cell-cell interaction, can interact with to have some interactions, close proximity to bind receptor and then recepetor-mediated endocytosis (RGD can interact with clathrin pit). Once internalized, goes in endosome, which gets acidified, important for viral replication, activation of protease, cleaves protein 6, the one that triggers disassembly, outer capsid is shed off. Then they must escape endosome, endosome escape, probably through some phosphorus lipase or lipase activity (not much known). Free core that can move on microtubules with dynein to go into nucleus through nuclear pore complex although not known how as it does not have NLS. Then transcription starts almost immediately.
Early genes transcribed in adenovirus
- Set the cell for viral replication
- E1A (very first) has constitutively active promoter, turns on S phase (responsible for tutors in rodents) and acts as transcription factor (binds to other Tfs not directly DNA) that turns on all the other promoters (E1B, E2A, E2B, E3, E4), different splice products, 2 main ones are 289R and 243R (number of residues).
289R: N term domain, CR1, CR2, (these three domains important to push into S phase, bind to Rb family proteins, most important being Rb CR1 and CR2) CR3 (transactivation domain, really important for activation of transcription of all other proteins, has TBP binding site (TATA box binding protein) and ATF binding site (canonical type of transcription factor that binds in a sequence specific way to different promoters) forms bridge with E3, E1B and E4) without CR3, virus replication very defective, but interestingly can still form tumors
243R: Nterm domain, CR1, CR2, no CR3 (one difference)
CR (conserved regions, are very very conserved across adenovirus serotypes)
Mechanism of action of E1A with Rb
Rb represses F phase by repressing E2F important TF for S phase activation (DNA polymerases, DNA synthesis, nucleotide synthesis (thymidylate synthétase, reductase, thymidine kinase, etc.) and cyclins required for progression through cell cycle (importance discovered thanks to adenovirus). Usually Rb inhibited by Cdk phosphorylation, but in adenovirus, inhibited by E1A binding in lieu of E2F, making it constitutively active. Rb is main gatekeeper of G1/S, committing step to cell division, sensitive to growth factors.
Function of N term domain in E1A
Bind P300 and CBP (Dif family of proteins) large TFs and inhibit cell differentiation. They are related cAMP binding proteins which have histone-acetyltransferase activity (HAT) required for activating transcription, loosens of helix, chromatin modification, relaxing of histone/DNA complex formation resulting in expressions of genes involved in various cellular functions including cell differentiation. Differentiation mediates cell cycle arrest. E1A therefore inhibits cell differentiation through binding p300/CBP therefore keeps cells capable of proliferation. Mutation in this domain makes it more difficult but it doesn’t completely lose ability to trigger S-phase.
