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MIMM 324 > Lecture 6 > Flashcards

Lecture 6 Flashcards

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1
Q

What is the 5’ cap important for?

A
  • Translation initiation
  • Also, processing, transport and stability
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2
Q

What is the structure of an mRNA?

A
  • 5’ cap and UTR: 3-1000 nt (average 50-70 in eukaryotes), often structured so must be unwound to allow passage of ribosomes, length and secondary structure influence translation efficiency
  • Start codon, open reading frame (protein), stop codon
  • 3’UTR and poly A tail: regulate translation initiation, efficiency and mRNA stability, tail (stability and translation(
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3
Q

What is the process of 5’ dependent initiation?

A
  • 40S subunit binds to eIF2 and tRNA with initiation met (ternary complex)
  • 43S pre initiation complex binds to eIF4G and then eIF4E which is cap binding protein so it is recruited to 5’ end and the eIF4A is a helices that unwinds RNA for the ribosome to find AUG
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4
Q

What is needed for translation efficiency?

A
  • Proximity of 3’ end and 5’ end which is accomplished by a PAbp that binds to eIF4G which binds to eIF4E which binds cap
  • when there is no cap, 3’ CITE (cap independent translation elements/enhancer recruit machinery and through kissing loops they can place machinery near 5’ end
  • Ex: pea nation mosaic virus, barley yellow fever dwarf virus
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5
Q

How does 60S subunit join?

A
  • When AUG start codon is found, 60S gets recruited to translate
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6
Q

True or false, 5’ cap is only present on mRNA and not pre-mRNA.

A

False, capping is cotrascriptional, so also present on pre-mRNA.

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7
Q

What is ribosome shunting?

A

The direct translocation of the 40S subunit across hairpin structures and other loops thanks to a viral structure or protein on its RNA in order to decrease the dependency to eIF4F complex that is usually needed to unwind mRNA.
Example: adenovirus late mRNAs

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8
Q

What is the mechanism of internal initiation?

A

Example of poliovirus: has VPg but also the 5’ UTR is long and structured. It has internal ribosomal entry sites (IRES) which is a structured RNA that directly recruits ribosomes and translation initiation factors. SO they found that with cap dependent ORFs, if there was no cap, there is little to no translation, but add an IRES and you get much more translation. To prove it was truly the IRES, they made circular genome, one had IRES, one did not, the one with IRES had translation, so it was clearly the IRES that recruited the ribosomes as no possibility for 5’ cap dependent translation if circle.

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9
Q

What predicts IRES element structure?

A

It is hard to do so from just the primary sequence as its ability strongly depends on the secondary structure. There is also not a lot of conservation in how these different IRES elements fold.

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10
Q

Requirements for IRES?

A
  • 5’ end dependent initiation: all eIFs
  • type 1 or 2 IRES: all eIFs except eIF4E since no cap duh
  • Hep C IRES: eIF2 and eIF3
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11
Q

How does methionine-independent initiation look like?

A
  • Cricket paralysis virus
  • Can assemble 80S ribosomes without any eIFs nor Met-tRNAi
  • the RNA mimics tRNAi
  • Turnip yellow mosaic virus has valine like molecule
  • Our prediction algorithms look for start codons, no start codons means we probably miss some ORFs
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12
Q

How do polyproteins maximize coding capacity?

A
  • Picornavirus (viral proteases) and Flaviviruses (viral and host proteases)
  • 1 mRNA that codes for multiple proteins (really just one long protein that gets cleaved by proteases)
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13
Q

Explain leaky scanning and how it maximizes coding capacity?

A
  • Multiple possible start sites but where it starts more or less often depends on context (tRNA availability, RNA structure, RNA sequence, rare tRNA)
  • Ex: Paramyxoviruses can has 5 possible start sites in different contexts for capsid proteins, there is also ribosome shunting
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14
Q

How does re-initiation help maximize coding capacity?

A

Viral structure or protein keeps the ribosome associated to the mRNA so it can reinitiate translation somewhere downstream ORF. Herpesviruses and paramyxoviruses.

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15
Q

How does suppression of termination (read through) maximize coding capacity?

A

The stop codon recruits an amino acid (like selenocysteine), so it reads through, makes new protein. Ex: retroviruses and alpha viruses

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16
Q

How does ribosomal frame shifting maximize coding capacity?

A

Ribosome suns into a slippery sequence or structure that makes it bounce back and switch reading frames. Ex: retroviruses

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17
Q

How does regulation of translation happen?

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A

Rate limiting step is the recycling of the ternary complex (GTP, eIF2, iMet) so most regulation happens here. If there are stressors like ER stress (PERK) or AA deprivation (GCN2) or dsRNA (PKR) also intermediates or infection, eIF2 alpha kinases => phosphorylated eIF2 cannot be recycled and translation is shut off

18
Q

PKR and the cellular antiviral response?

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A
  • PKR is present in an inactive form in the cell
  • PKR is induced and activated by virus infection (sensing of dsRNA)
  • Dimerizes and autophosphorylates
  • Leads to inhibition of translation and apoptosis
  • Interferon pathway activated
  • Different viral mechanisms have evolved to inactivate the PKR pathway
19
Q

How do viruses prevent activation of PKR?

Study These Flashcards
A

Ex: Adenovirus
makes VA RNA I sequesters PKR, no phosphorylation of eIF2 alpha subunit, active protein synthesis

20
Q

Viral inhibition of host cell translation

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A
  • Cell does not have opportunity to establish an antiviral response
  • Poliovirus completely turns off host translation of any proteins that are cap dependent
  • SDS PAGE at first all protein made, then as time goes by less and less proteins. By hour 5, only viral proteins are made. By hour 7 all cells are dead cuz no essential proteins made
  • Polio graph goes down (bye bye host translation) goes up (hello virus translation) goes down again cuz cells dead
21
Q

How does polio inhibit host cell translation?

Study These Flashcards
A
  • cutting eIF4G, has proteases to cleave off the viral proteins also cleave eIF4G, cutting link between eIF4G and eIF4E (cap binding protein) so now no more host proteins but viral proteins can be made because don’t need cap, they have IRES (polio and foot and mouth disease)
    Poliovirus and encephalomyocarditis virus dephosphorylate 4E-bp1 which can then sequester the cap binding protein
    Adenovirus and influenzavirus dephosphorylate eIF4E, does not bind to eIF4G anymore, no more cap binding protein
22
Q

summary of translation

Study These Flashcards
A
  • Although viruses use the host protein synthesis machinery, they often employ novel strategies to recruit and initiate protein synthesis
  • Viruses employ a number of novel mechanisms to maximize coding capacity
  • Viruses can regulate protein synthesis (and even inhibit cellular protein synthesis)
23
Q

What are the common set of assembly reactions and some supplementary steps?

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A
  • Formation of individual structural units of the protein shell from one or several viral proteins
  • Assembly of the protein shell by appropriate, and sometimes variable, interactions among structural units
  • Selective packaging of the nucleic acid genome and other essential virion components
  • Acquisiton of an envelope (suppp)
  • Release from the host cell
  • Maturation of virus particles (suppp)
24
Q

Assembly is dependent on host cell machinery like…?

Study These Flashcards
A
  • Cellular chaperones (fold well)
  • Transport systems (move within cells)
  • Secretory pathways -> virion secretion in some cases
  • Nuclear import and export machinery
25
What is the localization of viral proteins?
- Viral replication and assembly often take place in factories or inclusions (concentrate viral proteins to accelerate reactions) - Membrane targeting: signal sequences, fatty acid modifications, membrane retention signals, amphipathic helices - Nuclear localization sequences (NLS) - Nuclear export signals (to exit nucleus)
26
Why does packaging of the virus often happen in the nucleus?
- No mechanism to export DNA (why would you ever need that?) - DNA would get destroyed in cytoplasm
27
When would coral proteins localize to the plasma membrane?
- Assembly happens near there, may be an enveloped virus that acquires envelope from plasma membrane - Makes mRNA, goes into ER, into Golgi (if cell surface protein), then move with microtubules to fuse with plasma membrane, then viral RNA and proteins may go there for packaging
28
What are sub-assemblies?
- Virus particles don't get made all at once, more of an assembly line - Sub-assemblies ensure orderly formation of viral particles and virion subunits (quality control at each step and if bad, legs don't add bad legs and move on) - Formation of discrete intermediate structures - Quality control
29
What are strategies for making sub-assemblies?
- Individual protein molecules that have super high affinity for one another and just find each other in the cell - Can assemble from a poly protein precursor, already all together before getting cleaved and assembling correctly (polio) - Chaperone assisted assembly, needs to be folded properly before taking on its mature form (adenovirus type II)
30
Assembly can be sequential. Explain and give example.
Poliovirus, makes poly protein precursor before cleaves and comes together to make pentamers, that then come together and then incorporate genome (stepwise, sequential)
31
Assembly can be concerted. Explain and give example.
Influenza (-) ssRNA virus that replicates in the nucleus, brings nucleocapsid protein and matrix protein into the nucleus to assemble onto the viral genome fragments that then zip through the nuclear pore. When arrive at cell surface, interact with glycoproteins and drives budding. Virus particles only assemble in association with viral genome.
32
How do viruses distinguish viral genomes from cellular DNA or RNA molecules?
- Packaging signals in the viral genome (discrete sequences recognized by viral proteins) Examples: signals on DNA of adenovirus and SV40, recognized by viral proteins helping them pull genome into the capsid
33
How to identify a packaging signal?
If only mutated and no packaging, maybe just disrupted another part of the viral life cycle. If put in different piece of DNA and that gets packaged => probs packaging signal
34
How does Herpesvirus get packaged?
- Herpesvirus replication produces concatemers with head-to-tail copies of viral genomes from rolling circle replication - HSV-1 packaging signals (pac1 and pac2) are needed for recognition of viral RNA and cleavage once a signal is found, little motor thing that packs genome into capsid, keeps winding until capsid is full and then hits another packaging signal, when it hist second one, DNA is cleaved, and then BAM you got your packaged genome and ensures correct size of genome is packed
35
How do packaging signals work in RNA viruses?
- Often have secondary structure that are packaging signals - Retroviruses pack 2 copies of genome because the packaging signal recognized is kissing loop structure between two copies
36
How do segmented genomes get packed?
- Evidence for specific packaging sequence on each RNA segment ensuring that the right number of segments gets packaged - Influenza virus always 8 RNA segments thought was random cuz infectious particle ratio is 1/400 (ratio predicted by randomness and also reality) however they ran experiments and noticed that it was selective packaging - Serial dependence of packaging like in bacteriophage phi6, S goes in, M only goes if S in, and L only goes if both S and M in
37
How do viruses acquire an envelope?
- After assembly of internal structure (for most enveloped viruses) - Can occur at many cellular membranes (nuclear membrane, ER, Golgi, plasma membrane, etc.) I: envelope glycoproteins and capsids drive budding (alpha viruses) II: internal matrix (inner leaflet) or capsid proteins drive budding (retroviruses) III: envelope proteins drive budding (influenza virus) IV: matrix proteins drive budding, but additional components needed for efficiency and accuracy (ebolavirus)
38
How does Influenza virus acquire its envelope?
- Internal structure assembly and budding are spatially and temporally separated - matrix protein targeted to the membrane (hydrophobic region)
39
How do retroviruses acquire their envelope?
- Requires the ESCRT pathways - Late (L) domains bind cellular proteins involved in vesicle trafficking, needed for virus release (found in + and - enveloped RNA viruses) - Retroviral RNA produced in the nucleus and exported, dimerizes and bind to structural proteins which target it to the cell membrane where it buds and then maturation happens - Maturation only happens once they've left the cell by a protease cleavage event within the virion, not infectious until undergo maturation
40
Herpesvirus acquiring its envelope
- DNA viruses, replicate in the nucleus and then build their nucleocapsid in the nucleus, bud out of the nucleus into ER, then out of ER into Golgi where decorated by envelope glycoproteins, then bud out of Golgi into secretory vesicle and then exocytosed from the cell => envelope from Golgi not plasma membrane
41
Maturation of dengue virus
- creates iron in ER then passes through the trans Golgi network where change in pH and exposure to the furin molecule causes a cleavage event which cleaves the PR protein and then when viral particles are released, PR portion of PPRM protein are released from the particle and this protein is mature
42
What is a way to prevent reinfection of a previously infected cell?
- often down regulate the receptor from the virus, so no other virus gets in - Vaccinia virus propulsion on actin tails away from the cell

MIMM 324 (25 decks)

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