Lecture 5 Flashcards
(37 cards)
1
Q
What are some barriers and conditions for RNA-directed RNA synthesis?
A
- RNA genome must be copied from end to ed with no loss of nucleotide sequence (to continue propagation)
- Production of viral mRNAs that can be efficiently translated but the cellular protein synthesis machinery since (+) RNA does not necessarily = mRNA (needs cap, not ribosome ready) although sometimes the genome is the mRNA like in the case of poliovirus
2
Q
The universal rules for RNA-directed RNA synthesis are…
A
- RNA synthesis initiates and terminates at specific sites on the template
- RdRp may initiate synthesis de novo (from nothing) like DdRp or require a primer
- Other viral and cellular proteins may be required (cofactors)
- RNA is synthesized by template-directed stepwise incorporation of NTPs, elongated in the 5’-3’ direction so to starts reading at 3’
- Some non-templated synthesis although this is rare
3
Q
How does RNA-dependent RNA synthesis initiate for flaviviruses?
A
- De novo initiation, 1 nucleotide and then another
- Internal initiatiation: triphosphate G initiation, slips back and then starts elongation at the end of the viral genome
4
Q
How does RdRsynthesis initiate for poliovirus?
A
- Has a termina protein primer
5
Q
How does RdRsynthesis initiate for influenza?
A
It has a capped primer
6
Q
How does RdRp do its thang?
A
It has a two-metal mechanism of catalysis a which is coordinated with the help of aspartic acid residues.
7
Q
What are the different active sites for RdRp and different RNA viruses?
A
- First of all, typically has right hand topology
- (+) ssRNA => gly-asp-asp (GDD motif)
- (-) ssRNA-RT, segmented (-) ssRNA => Asp-Asp (DD motif)
- Non segmented (-) ssRNA => Gly-asp-asn (GDN motif)
8
Q
How does making mRNA from (+) ssRNA viruses work?
A
- Picornavirus + flavi: genome is mRNA, makes (-) ssRNA to continue making copies of the (+) strand
- Alphaviruses: strand is mRNA but only codes for some genes, then it replicates to make (-) strand, then this is used to make full length mRNA as well as subgenomic mRNAs which usually translate to structural proteins like the capsid proteins
9
Q
Poliovirus mRNA production
A
- Has a VPg (viral protein genome linked) linked to the genome, it also has an encoded polyA tail, and it gets translated into one poly protein which then gets cleaved by viral proteases to make mature viral proteins
- Viral polymerases only copy viral RNA’s thanks to specific signals like in polio there is a cloverleaf, a cis-acting RNA element and pseudo knot (specific features in the 3’UTR region)
- SO the viral 3CD binds to the cis acting RNA element (3D is polymerase) and then the VPg binds to them and can get uridylated, and it is the U residues which are used to prime viral RNA synthesis
- 3CD also binds to a Poly A binding protein that binds to the poly A tail and leads to the circularization of the genome
- uridylated VPg can then interact with the polyA tail to make a primer for RNA synthesis and polymerase is right there thanks to circularization
10
Q
Alphavirus RNA synthesis
A
- Has a cap so it’s ribosome ready but only translates to a stop codon so only 4 viral proteins are made which are proteolytically processed to make mature viral proteins which include the RNA polymerase and other replication proteins so they can make a full length (-) ssRNA which can make more (+) full length ssRNA or internally initiate elsewhere and make subgenomic mRNAs that encode the structural proteins
- This is a way in which a virus can control the time of gene expression so you can actually have a genome to incorporate into the capsid that is made
11
Q
Making mRNA and genomic RNA from (-) ssRNA viruses
A
- Can be unimolecular or segmented
- Either way use (-) template to make mRNAs to make proteins which can THEN do replication to make full length (+) RNA to make more genome
12
Q
Vesicular Stomatitis Virus RNA synthesis
A
- Unimolecular
- Makes mRNA which make all viral proteins, but none are full length so none can serve as template make more genome
- To fix this the genome is coated in N protein, the first protein translated is N protein which coats the nascent positive strand mRNAs as well as the RdRp and acts as an anti-terminator, so the RdRp pushes forward and makes the full-length (+) RNA to serve as the template to make viral genome
13
Q
Influenza RNA synthesis
A
- Genome is segmented, only segments of mRNA, no full length template to make more genome
- Cap-snatching from host mRNA to prime (-) genome which is coated in NP protein and acts as an anti-terminator so full length template is made, will also bind to (+) mRNA to act as an anti-terminator to make more genome segments
- Different 5’ end since longer sequence since it takes some host mRNA when it snatches cap
14
Q
How does making dsRNA viruses RNA work?
A
- Must carry RdRp
- Reoviruses and rotaviruses
- Strands are separated and (+) strand is ribosome ready and can also serve as template to complete replication of genome
15
Q
What is transcription?
A
Making mRNA from dsDNA (making mRNA from RNA is NOT transcription by definition)
16
Q
Making mRNA from DNA
A
- First biosynthetic activity IS transcription
- when it is ssDNA, they first make dsDNA and then transcription happens
=> Hepadnavirus: gapped dsDNA so DNA repair and then dsDNA and then transcription
=> Parvovirus: ssDNA so makes dsDNA
=> Retrovirus: RT, makes dsDNA, integrates into genome and then transcription
17
Q
What are the three eukaryotic DdRp?
A
- Pol I: pre-rRNA (no touching viruses)
- Pol II: pre-mRNA, pre-miRNA, snRNA (viruses: pre-mRNA, pre-miRNA, HDV genome and mRNA)
- Pol III: pre-tRNA, 5S rRNA, U6 snoRNA (viruses: Ad-2 VA RNAs, EBV EBER RNAs, MHV68 pre-mRNA)
18
Q
DO viruses ever encode a DdRp?
A
Only DNA viruses that replicate in the cytoplasm do like poxviruses and giant viruses.
19
Q
How is transcription regulated?
A
- All gene sequences encode an upstream promoter which includes
=> core promoter: TATA box for ribosome + initiator sequence for initiation
=> Promotore: core promoter + local regulatory sequences where TFs bind
=> Transcriptional control region: promoter + distant regulatory sequences like enhancers and silencers
20
Q
What types of proteins are involved in transcription regulation?
A
- Host and/or viral sequence-specific DNA binding proteins
- Viral co-activating molecules (do not bind DNA but CAN modulate transcription) like modulating structure of nucleosomal templates
- Many viruses temporarily control mRNA expression like with subgenomic transcription (early and late gene expression, latency and lysogeny, etc.)
21
Q
How does regulation happen?
A
- Can be a positive or negative auto-regulatory loop: gene makes protein that either enhances same gene or suppresses same gene
- Positive or negative cascade regulation: gene makes protein that either enhances or suppresses expression of ANOTHER gene
- Direct (binds DNA like TFs or promoters) or indirect (modulators that bind nucleosomes or other proteins involved in transcription)
22
Q
What constitutes mRNA processing?
A
- 5’ cap structure
- Poly(A) tail
- Splicing
- Other modifications like deamination
23
Q
Co-transcriptional capping
A
- Most eukaryotic mRNAs habe a cap
- 5’7-methylguanosine (m7G) joined by a 5’5 phosphodiester linkage
- Directs premRNAs to processing and transport pathways (exit out of nucleus)
- Regulates stability of RNA (protects mRNA from degradation)
- Required for efficient end-dependent translation mechanism (ribosome docks onto 5’cap)
- Process: initiation and addition of 20-30 nucleotides, CTD phosphorylation to get RNA pol II, capping enzyme can attach there and cap can be synthesized
24
Q
Eukaryotes have caps but what happens at the 5’ end of viral mRNAs?
A
- Alternative capping strategies: cap snatching like Influenza or own viral capping enzyme like flavivirus (may or may not be the same as our caps)
- Viral proteins that can lead initiation of transcription (poliovirus, VpG protein) or no cap like HCV 5’triphosphate that slips back and stuff
25
Addition of poly A tails to mRNA mechanism, enzyme and examples...
- Post transcriptionally: cleavage of pre-mRNA followed by polyadenylation, enzyme is cellular, adenovirus, HBV, herpesvirus, polyomavirus, retrovirus
- During mRNA synthesis by copying of long U stretch in template RNA: enzyme is viral, poliovirus, alphavirus
- During mRNA synthesis by reiterative copying at stretches of U in template RNA: viral enzyme, influenza virus and VSV
26
What exactly is the process of copying U stretches in template RNA?
- VSV: slip and slide due to all the Us making poly A tail, then end of one mRNA, starts the next at initiation
- Influenza: mRNA synthesis until reaches lots of Us, elastic band like retention and makes polyA tail that way
27
What are the benefits of splicing?
- Marks mRNAs for nuclear transport
- Alternative splicing creates different mRNAs
- Coding information of small DNA genomes is expanded
- Regulation of gene expression
28
What are some DNA viruses?
- Adenovirus, herpes simplex virus, polyoma and papillomaviruses
29
What are the universal rules of DNA replication?
- DNA is synthesized by template directed incorporation of dNTPs into 3'OH of DNA chain
- DNA is always synthesized 5'-3' in a semiconservative fashion (two daughter strands)
- Replication initiates at specific sites on the template called the origin (ori)
- Catalyzed by DdDp and accessory proteins
- Always primer dependent
30
What is the chemistry of the active site of a DdDp?
- Two metal mechanism of catalysis with aspartic acid residues but more open since it must scan a double strand not just sing strand like with RdRp
31
Why do DNA viruses need the host?
- Viral DNA replication always requires the synthesis of at least one viral protein, sometimes many
- Simple viruses require more host proteins (hello genetic economy)
- Complex viruses encode many but not all proteins required for replication (so many in the roster including DNA polymerases, accessory proteins, origin binding proteins, helices, exonuclease, enzymes for nucleic acid metabolism, etc.)
32
Where does the DNA polymerase come from for viral replication?
- Small DNA viruses do not encode an entire replication system just proteins that orchestrate the host (example: polyomaviruses, papillomaviruses and parvoviruses)
- Large DNA viruses encode most of their own replication system like herpesviruses, adenoviruses and poxviruses
33
What are viral origins of replication like?
- AT-rich segments recognized by viral origin recognition proteins since only 2 H bonds to break, easier to melt
- Assembly points for multi-protein DNA replication machines (bound by origin-recognition binding proteins and transcriptional regulator binding sites in close proximity)
- Some viral genomes have 1 ori others can have up to 3
34
What are the two mechanisms of dsDNA synthesis?
- Replication fork, RNA primers, one leading, one lagging strand: papillomaviruses, polyomaviruses, herpesviruses and retroviral proviruses
- Strand displacement (other molecule serves as primer), never RNA primed since strand is simply displaced and can be replicated: adenoviruses (protein), parvoviruses (DNA hairpin) and poxviruses (DNA hairpin)
35
Eukaryotic cells use telomerase and telomeres to solve the 5' end problem, how do viruses do it?
- Circularization of replication system, no end, bidirectional replication and whatever is left can be put in during lagging strand synthesis (poliovirus)
- DNA priming (parvoviruses): hairpin serves as primer, bottom half is nicked after synthesis begins, replication of bottom strand, hairpin unfolds, bottom and top hairpin again, reinitiation of synthesis, displacement of top strand which becomes genomic DNA, process starts over
Protein priming (adenoviruses): origins at both ends, DdDp will bind to pre terminal protein (primer) DNA pol links dCMP to OH of serine residue of pTP, strand displacement synthesis but ptp stays at 3'end of template, other strand is covered by DNA binding protein to keep it separate (no interference), then new ptp goes to displaced strand and recruits DdDp and kicks of DBP to make other genome, no end problem thanks to protein and DdDp specific binding so all of the genome gets replicated
36
What is rolling circle replication?
- Herpesviruses: DNA enters linear but converts to circle thanks to host proteins, replicates as rolling circle so there is never an end problem, there is a continuous strand and discontinuous strand but gets fixed by ligases
37
Poxvirus DNA replication
- Replication in cytoplasm
- Mechanism has a nickase and a resolvase to mediate a mechanism that is similar to parvoviruses with the hairpins
