Lecture 5

Question 1

True or false: RNA polymerase activity increases when PFU (viral particles) are synthesized

Answer 1

True

Question 2

How can RdRP synthesize RNA

Answer 2

De novo or requiring a primer

Question 3

What are the two types of primers used by RdRP?

Answer 3

Protein primer
Capped primer

Question 4

What is special about the poliovirus RdRP?

Answer 4

Two Mg2+ ions coordinated by two Asp residues at the catalytic DD motif

Right handed configuration

Question 5

What do Mg2+ ions do in RdRP?

Answer 5

Catalyze cleavage between alpha and beta phosphates of incoming NTP

Forms phosphodiester bond between resulting NMP and growing RNA chain

Beta-gamma disphosphate discarded

Question 6

What can RdRP synthesize in viruses?

Answer 6

+ mRNA and sub-genomic mRNA

Question 7

For poliovirus what is the name of the RdRP and primer used for RNA synthesis?

Answer 7

3Dpol RdRP
VPg protein used as primer that is covanlently linked to the 5’ end of the RNA by a phosphodiester bond

Question 8

What does the RdRP recognize in the genome?

Answer 8

5’ clover leaf
Cis-acting RNA element (CRE)
Pseudoknot

Question 9

How is VpG-U-U primer created

Answer 9

1 molecule of VpG and 3 copies of viral 3Dpol RdRP binds to CRE element

One of the polymerases then catalyze the addition of two U nucleotides to the VpG molecule. A stretch of A residues in the CRE loop is used as template

Question 10

What is the role of PCbp in poliovirus?

Answer 10

Cellular poly(rC) binding protein (PCbp): required for poliovirus replication

Question 11

What is the role of PAbp in poliovirus?

Answer 11

Binds simultaneously to the polyA tail at the 3’ end oft he viral RNA and to the cloverleaf structure at the 5’ end

Circularizes the viral genome

Question 12

How is poliovirus RNA synthesized?

Answer 12

PAbp binds to PolyA tail at the 3’ end fo the viral RNA and to the cloverleaf structure at the 5’ end.

VpG-U-U anneals to polyA tail and is extended by RdRP all teh way to the 5’ end fo the +RNA. This generates -strand

Whole procedure repeated using the newly synthesized - strand RNA as the template to create + strand RNA

Question 13

Describe the difference between the mRNA and subgenomic mRNA of the alphavirus that are created from - strand RNA.

Answer 13

mRNA:
- Only the 5’ part of the full length RNA is translated
- Makes the replication proteins
- stop codon at the end of the nsP4 prevents translation of the 3’ part

Subgenomic mRNA:
- 3’ part of the RNA
- synthesized from an internal promoter
- Makes structural proteins

Question 14

How does RNA synthesis work for VSV?

Answer 14

Has - RNA
Transcription of this genome by RdRP produces several subgenomic mRNAs by a mechanism of termination and re-initiation

Virus can switch between making mRNAs to full length + RNA

Question 15

What promotes the switch between subgenomic to full length RNA synthesis?

Answer 15

Nucleocapsid (N) Protein
Acts as a transcription anti-termination factor and inhibits subgenomic RNA synthesis

Question 16

How does RNA synthesis work for Influenza?

Answer 16

Has segmented - RNA that gets transcribed by RdRP into sub-genomic RNA. Some mRNAs are spliced

Review lecture 5 slide 26

Question 17

The influenza RdRP is a heterotrimer of which viral proteins?

Answer 17

PB1, PB2, PA

Question 18

Why do dsRNA viruses have RdRP?

Answer 18

Ribosomes cannot access ds RNA so these viruses need RdRP to make +RNA in the cell that can be sued as mRNA or a template for synthesis of -RNA strand

Question 19

What are alternative capping strategies at the 5’ end of mRNAs?

Answer 19

Cap snatching
Viral capping enzymes

(Alternative but not capping: viral proteins)

Question 20

How to add polyA tails to mRNA in viruses?

Answer 20

Copying of long U stretch in template RNA

Reiterative copying at stretches of U in template RNA

Question 21

Describe the mechanism of reiterative copying of U stretches in template RNA during mRNA synthesis in VSV

Answer 21

Slippery stretch of 7 U residues in the intervenir region

Causes the RdRP to slip and recoup this sequence several times, resulting in the addition of 200 A nucleotides that function as the poly A tail

At this point the polymerase terminates, and can reinitiate on the next mRNA promoter

Question 22

How does reiterative copying of U stretches in template RNA during mRNA synthesis work for Influenza?

Answer 22

The 5’ end of the viral RNA is anchored to the RdRP active site

The polymerase is stationary and RNA gets pulled through the enzyme

At the end, RdRP cannot pull the last 20 nucleotides and gets stuck on a stretch of U residues

The RdRP adds stretches of complementary A residues that make up the PolyA tail