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MIMM 324 > 5. RNA Synthesis by RdRP > Flashcards

5. RNA Synthesis by RdRP Flashcards

1
Q

what are 2 examples of (+) RNA viruses?

A
  1. polioviruses
  2. alphaviruses
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2
Q

what are 2 examples of dsRNA viruses?

A
  1. reoviruses
  2. rotavirus
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3
Q

what are 2 examples of (-) RNA viruses?

A
  1. influenza
  2. VSV
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4
Q

how did we identify RNA polymerases?

A

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

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5
Q

what are the 2 steps in RNA-directed RNA synthesis? how are these steps accomplished?

A
  1. replicate RNA
  2. make viral mRNAs to be translated by host machinery

both steps accomplished by RdRP

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6
Q

how is RNA synthesized? in which direction?

A

template-directed stepwise incorporation of complementary NTPs in the 5’ to 3’ direction

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7
Q

where does RNA synthesis start and stop?

A

RNA synthesis initiates and terminates at specific sites on the template

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8
Q

what are the 2 types of initiation of RNA synthesis?

A
  1. de novo initiation
  2. primer-dependent initiation
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9
Q

what is de novo initiation?

A

RdRP initiates RNA synthesis without a primer

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10
Q

what is primer-dependent initiation?

A

RdRP uses a primer

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11
Q

what are the 2 types of primers used by RdRP?

A
  1. Protein primer
  2. Capped primer
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12
Q

why do some RdRP use capped primers?

A

virus can steal RNA from host which is capped and some viruses may have their own machinery to cap the RNA

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13
Q

what is the name for the configuration of Poliovirus RdRP?

A

“Right handed”

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14
Q

what are the 3 domains of Poliovirus RdRP?

A
  1. Fingers
  2. Thumb
  3. Palm
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15
Q

what is the name of the special motif in Poliovirus RdRP? where is it located? what does it consist of?

A

DD motif

at the catalytic site

Two Mg2+ ions at two Asp resideus

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16
Q

what part of an enzyme is the most conserved?

A

the catalytic site

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17
Q

what is the most conserved part of the RdRP?

A

DD site

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18
Q

what is the Two Metal Mechanism of Catalysis?

A
  • 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
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19
Q

are both Asp essential? what happens if you mutate each of them?

A

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

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20
Q

on which face of RdRP does the template strand enter?

A

the front

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21
Q

on which face of RdRP does the dsRNA exit?

A

the front

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22
Q

on which face of RdRP do NTP enter? when are specific NTP selected?

A

many NTP enter in the back, specific NTP are selected during catalysis

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23
Q

which 3 baltimore classes of viruses use RdRP?

A
  1. (-) RNA viruses
  2. (+) RNA viruses
  3. dsRNA viruses
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24
Q

where do (-) RNA viruses get RdRP?

A

RdRP is brought in via the capsid

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25
Q

where do (+) RNA viruses get RdRP?

A

RdRP is made in the infected cell

26
Q

where do dsRNA viruses get RdRP?

A

RdRP is brought in via the capsid along with (+) RNA genome

27
Q

describe the general system for making genomic RNA for (+) strand viruses

A

unimolecular (unsegmented) ss (+) RNA genomes are replicated through a negative strand intermediate (i.e. to make more genome template)

28
Q

how do genome RNAs compare to mRNA in flaviviruses and picornaviruses/polioviruses?

A

genome and full-length mRNA are identical

29
Q

how do genome RNAs compare to mRNA in alphaviruses?

A

make full-length (+) mRNA that are identical to genome and make SUB-GENOMIC mRNAs

30
Q

what is the name of the RdRP used by Poliovirus?

A

3Dpol

31
Q

what is the primer used by Poliovirus? what type of primer?

A

VPg –> protein primer

32
Q

how is VPg linked to RNA?

A

VPg is covalently linked to the 5’ end of the RNA by a phosphodiester bond

33
Q

how does poliovirus RdRP recognize the viral RNA? (3)

A
  1. 5’ cloverleaf
  2. cis-acting RNA element (CRE)
  3. pseudoknot (3 strand bundle of RNA)
34
Q

why is it important that RdRP recognizes the 3 structures?

A

allows specificity of virus to genome –> RdRP will never act on host RNA

35
Q

describe the synthesis of VpG primer (3 steps)

A
  1. a molecule of VpG and 3 copies of 3Dpol bind to the CRE element
  2. 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
  3. VpG-U-U can then be used to prime RNA synthesis
36
Q

where does poliovirus RNA synthesis occur?

A

on membranes

37
Q

describe the 4 steps for poliovirus RNA synthesis

A
  1. cellular poly(rC) binding protein (PCbp) is required to recruit other proteins
  2. 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
  3. 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
  4. then REPEAT to make more (+) RNA genome
38
Q

describe the 3 steps of the transcription/replication process of Alphavirus RNA

A
  1. 5’ part of the full-length (+) RNA genome is translated to make replication proteins
  2. (-) complementary strand is made from the (+) template strand
  3. subgenomic (+) mRNA is produced from the (-) strand via an internal promoter to make structural proteins
39
Q

what allows for only the 5’ part of the full-length RNA to be translated for Alphavirus?

A

there is a stop codon at the end of nsP4 to prevent translation of the 3’ part

40
Q

what are the 2 ways to describe (-) RNA viruses? and an example of each

A
  1. unimolecular (VSV)
  2. segmented (influenza)
41
Q

what does transcription of a unimolecular (-) RNA genome produce?

A

RdRP produces several subgenomic mRNAs by a mechanism of termination and re-initiation –> diff mRNAs for each protein

42
Q

if small, subgenomic mRNAs are produced, how does it make a full-length genomic RNA?

A
  1. genome is transcribed immediately to make subgenomic mRNAs encoding N protein (first protein) and other viral proteins
  2. 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
43
Q

describe RNA synthesis from segmented (-) RNA genomes (2 steps)

A
  1. genome has 8 (-) RNA segments
  2. each segment is transcribed by RdRP to a sub-genomic mRNA that encodes 1 or more viral proteins, some mRNAs are spliced
43
Q

when is full-length RNA synthesis favoured over subgenomic RNA?

A

as concentration of N protein increases with increased segmented mRNA, full-length RNA synthesis becomes favoured

44
Q

described influenza RdRP

A

heterotrimer of viral proteins PB1, PB2, PA

44
Q

what does transcription of segmented (-) RNA genome produce? what feature do they have?

A

sub-genomic mRNAs that are about 20 nucleotides shorter than the segmented genome

they are polyadenylated

45
Q

what allows for the production of the sub-genomic mRNAs from the segmented (-) RNA genome?

A

synthesis requires m7G-capped primers stolen from host cell mRNAs

46
Q

describe replication of (-) RNA genome

A

replication makes FULL-LENGTH (+) RNA intermediate then (-) RNA genome –> occurs DE NOVO –> doesn’t require cap but requires nucleoprotein NP

47
Q

what is the role of NP?

A

anti-termination factor to allow full-length (-) and (+) RNA to be produced

48
Q

what does it mean for replication of (-) RNA to be DE NOVO?

A

doesn’t require primer

49
Q

how does the (-) RNA virus determine whether the (-) RNA is transcribed or replicated?

A

initially transcribed, making mRNA which makes NP –> when high [NP], de novo synthesis of full-length RNA

50
Q

what is the name for the mechanism where the virus obtains a capped primer?

A

cap snatching mechanism

51
Q

describe the cap snatching mechanism (3 steps)

A
  1. Influenza RdRP recognizes and binds to cellular mRNAs which have 5’ caps
  2. RdRP cleaves the capped mRNA after ~13 nucleotides
  3. RdRP can then use the snatched capped RNAs primers to produce capped viral mRNAs that encode viral proteins
52
Q

why are viral mRNAs considered hybrid molecules?

A

the viral mRNAs contain host sequences at their 5’ end

53
Q

why do dsDNA viruses carry RdRP in their particles?

A

ribosomes cannot access dsRNA so the virus carries RdRP to make (+) RNA

54
Q

describe transcription of the dsRNA genome (2 steps)

A
  1. RdRP makes (+) mRNA
  2. mRNA can be translated to make protein OR replicated to make dsRNA
55
Q

what are the 3 options for things on the 5’ end of viral mRNA? and an example of a virus for each

A
  1. Cap acquired via CAP-SNATCHING (influenza)
  2. Cap acquired via VIRAL CAPPING ENZYMES (flavivirus)
  3. VIRAL PROTEINS (poliovirus –> VpG protein)
56
Q

what are the 2 mechanisms for viruses adding a poly-A tail to its mRNA? and 2 examples of viruses for each?

A
  1. copying of long U stretch in template RNA (poliovirus, alphavirus)
  2. reiterative copying at stretches of U in template RNA (influenza, VSV)
57
Q

describe how VSV accomplishes reiterative copying of U stretches in template RNA (3 steps)

A
  1. a stretch of 7 U’s in the (-) RNA genome causes the RdRP to slip and recopy the sequence many times
  2. once there are 200 A’s on the new mRNA, the RdRP terminates
  3. the RdRP can reinitiate on the next mRNA promoter
58
Q

describe how influenza accomplishes reiterative copying of U stretches in template RNA (3 steps)

A
  1. 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
  2. at the end, the RdRP is physically unable to pull the last 20 nucleotides
  3. RdRP gets stuck on a stretch of U residues and adds stretches of A’s

MIMM 324 (37 decks)

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