5. RNA Synthesis by RdRP Flashcards
what are 2 examples of (+) RNA viruses?
- polioviruses
- alphaviruses
what are 2 examples of dsRNA viruses?
- reoviruses
- rotavirus
what are 2 examples of (-) RNA viruses?
- influenza
- VSV
how did we identify RNA polymerases?
in poliovirus infected cells: measured RNA polymerase activity and poliovirus PFUs over time –> increase in RNA pol activity and poliovirus PFU –> therefore virus encodes for RNA pol
what are the 2 steps in RNA-directed RNA synthesis? how are these steps accomplished?
- replicate RNA
- make viral mRNAs to be translated by host machinery
both steps accomplished by RdRP
how is RNA synthesized? in which direction?
template-directed stepwise incorporation of complementary NTPs in the 5’ to 3’ direction
where does RNA synthesis start and stop?
RNA synthesis initiates and terminates at specific sites on the template
what are the 2 types of initiation of RNA synthesis?
- de novo initiation
- primer-dependent initiation
what is de novo initiation?
RdRP initiates RNA synthesis without a primer
what is primer-dependent initiation?
RdRP uses a primer
what are the 2 types of primers used by RdRP?
- Protein primer
- Capped primer
why do some RdRP use capped primers?
virus can steal RNA from host which is capped and some viruses may have their own machinery to cap the RNA
what is the name for the configuration of Poliovirus RdRP?
“Right handed”
what are the 3 domains of Poliovirus RdRP?
- Fingers
- Thumb
- Palm
what is the name of the special motif in Poliovirus RdRP? where is it located? what does it consist of?
DD motif
at the catalytic site
Two Mg2+ ions at two Asp resideus
what part of an enzyme is the most conserved?
the catalytic site
what is the most conserved part of the RdRP?
DD site
what is the Two Metal Mechanism of Catalysis?
- incoming NTP has three phosphates (alpha, beta, gamma)
- Mg2+ by Asp catalyze:
1. cleavage btwn alpha and beta phosphates
2. formation of phosphodiester bond btwn resulting NMP and growing RNA - beta-gamma diphosphate is discarded
are both Asp essential? what happens if you mutate each of them?
the one closer to the alpha phosphate is ESSENTIAL –> mutation = no function
the one closer to the beta and gamma phosphate is less essential –> mutation = reduced efficacy
on which face of RdRP does the template strand enter?
the front
on which face of RdRP does the dsRNA exit?
the front
on which face of RdRP do NTP enter? when are specific NTP selected?
many NTP enter in the back, specific NTP are selected during catalysis
which 3 baltimore classes of viruses use RdRP?
- (-) RNA viruses
- (+) RNA viruses
- dsRNA viruses
where do (-) RNA viruses get RdRP?
RdRP is brought in via the capsid
where do (+) RNA viruses get RdRP?
RdRP is made in the infected cell
where do dsRNA viruses get RdRP?
RdRP is brought in via the capsid along with (+) RNA genome
describe the general system for making genomic RNA for (+) strand viruses
unimolecular (unsegmented) ss (+) RNA genomes are replicated through a negative strand intermediate (i.e. to make more genome template)
how do genome RNAs compare to mRNA in flaviviruses and picornaviruses/polioviruses?
genome and full-length mRNA are identical
how do genome RNAs compare to mRNA in alphaviruses?
make full-length (+) mRNA that are identical to genome and make SUB-GENOMIC mRNAs
what is the name of the RdRP used by Poliovirus?
3Dpol
what is the primer used by Poliovirus? what type of primer?
VPg –> protein primer
how is VPg linked to RNA?
VPg is covalently linked to the 5’ end of the RNA by a phosphodiester bond
how does poliovirus RdRP recognize the viral RNA? (3)
- 5’ cloverleaf
- cis-acting RNA element (CRE)
- pseudoknot (3 strand bundle of RNA)
why is it important that RdRP recognizes the 3 structures?
allows specificity of virus to genome –> RdRP will never act on host RNA
describe the synthesis of VpG primer (3 steps)
- a molecule of VpG and 3 copies of 3Dpol bind to the CRE element
- one 3Dpol catalyzes the addition of two U nucleotides to the VpG molecule via phosphodiester linkage, using A residues in the CRE loop as a template
- VpG-U-U can then be used to prime RNA synthesis
where does poliovirus RNA synthesis occur?
on membranes
describe the 4 steps for poliovirus RNA synthesis
- cellular poly(rC) binding protein (PCbp) is required to recruit other proteins
- cellular polyA binding protein (PAbp) binds the polyA tail on the 3’ end of the viral RNA AND the cloverleaf on the 5’ end to circularize the viral genome
- the VpG-U-U primer anneals to the polyA tail and RdRP extends it to the 5’ end of the template –> making the (-) strand RNA
- then REPEAT to make more (+) RNA genome
describe the 3 steps of the transcription/replication process of Alphavirus RNA
- 5’ part of the full-length (+) RNA genome is translated to make replication proteins
- (-) complementary strand is made from the (+) template strand
- subgenomic (+) mRNA is produced from the (-) strand via an internal promoter to make structural proteins
what allows for only the 5’ part of the full-length RNA to be translated for Alphavirus?
there is a stop codon at the end of nsP4 to prevent translation of the 3’ part
what are the 2 ways to describe (-) RNA viruses? and an example of each
- unimolecular (VSV)
- segmented (influenza)
what does transcription of a unimolecular (-) RNA genome produce?
RdRP produces several subgenomic mRNAs by a mechanism of termination and re-initiation –> diff mRNAs for each protein
if small, subgenomic mRNAs are produced, how does it make a full-length genomic RNA?
- genome is transcribed immediately to make subgenomic mRNAs encoding N protein (first protein) and other viral proteins
- N is an RNA-binding protein that binds nascent RNA, acting as a transcription anti-termination factor –> inhibiting subgenomic mRNA synthesis so full-length (+) strand RNA can be made as a template for (-) RNA
describe RNA synthesis from segmented (-) RNA genomes (2 steps)
- genome has 8 (-) RNA segments
- each segment is transcribed by RdRP to a sub-genomic mRNA that encodes 1 or more viral proteins, some mRNAs are spliced
when is full-length RNA synthesis favoured over subgenomic RNA?
as concentration of N protein increases with increased segmented mRNA, full-length RNA synthesis becomes favoured
described influenza RdRP
heterotrimer of viral proteins PB1, PB2, PA
what does transcription of segmented (-) RNA genome produce? what feature do they have?
sub-genomic mRNAs that are about 20 nucleotides shorter than the segmented genome
they are polyadenylated
what allows for the production of the sub-genomic mRNAs from the segmented (-) RNA genome?
synthesis requires m7G-capped primers stolen from host cell mRNAs
describe replication of (-) RNA genome
replication makes FULL-LENGTH (+) RNA intermediate then (-) RNA genome –> occurs DE NOVO –> doesn’t require cap but requires nucleoprotein NP
what is the role of NP?
anti-termination factor to allow full-length (-) and (+) RNA to be produced
what does it mean for replication of (-) RNA to be DE NOVO?
doesn’t require primer
how does the (-) RNA virus determine whether the (-) RNA is transcribed or replicated?
initially transcribed, making mRNA which makes NP –> when high [NP], de novo synthesis of full-length RNA
what is the name for the mechanism where the virus obtains a capped primer?
cap snatching mechanism
describe the cap snatching mechanism (3 steps)
- Influenza RdRP recognizes and binds to cellular mRNAs which have 5’ caps
- RdRP cleaves the capped mRNA after ~13 nucleotides
- RdRP can then use the snatched capped RNAs primers to produce capped viral mRNAs that encode viral proteins
why are viral mRNAs considered hybrid molecules?
the viral mRNAs contain host sequences at their 5’ end
why do dsDNA viruses carry RdRP in their particles?
ribosomes cannot access dsRNA so the virus carries RdRP to make (+) RNA
describe transcription of the dsRNA genome (2 steps)
- RdRP makes (+) mRNA
- mRNA can be translated to make protein OR replicated to make dsRNA
what are the 3 options for things on the 5’ end of viral mRNA? and an example of a virus for each
- Cap acquired via CAP-SNATCHING (influenza)
- Cap acquired via VIRAL CAPPING ENZYMES (flavivirus)
- VIRAL PROTEINS (poliovirus –> VpG protein)
what are the 2 mechanisms for viruses adding a poly-A tail to its mRNA? and 2 examples of viruses for each?
- copying of long U stretch in template RNA (poliovirus, alphavirus)
- reiterative copying at stretches of U in template RNA (influenza, VSV)
describe how VSV accomplishes reiterative copying of U stretches in template RNA (3 steps)
- a stretch of 7 U’s in the (-) RNA genome causes the RdRP to slip and recopy the sequence many times
- once there are 200 A’s on the new mRNA, the RdRP terminates
- the RdRP can reinitiate on the next mRNA promoter
describe how influenza accomplishes reiterative copying of U stretches in template RNA (3 steps)
- the 5’ end of the viral RNA is anchored at the RdRP active site and RNA gets pulled thru enzyme as the polymerase is stationary
- at the end, the RdRP is physically unable to pull the last 20 nucleotides
- RdRP gets stuck on a stretch of U residues and adds stretches of A’s
