Lecture 5 Flashcards
True or false: RNA polymerase activity increases when PFU (viral particles) are synthesized
True
How can RdRP synthesize RNA
De novo or requiring a primer
What are the two types of primers used by RdRP?
Protein primer
Capped primer
What is special about the poliovirus RdRP?
Two Mg2+ ions coordinated by two Asp residues at the catalytic DD motif
Right handed configuration
What do Mg2+ ions do in RdRP?
Catalyze cleavage between alpha and beta phosphates of incoming NTP
Forms phosphodiester bond between resulting NMP and growing RNA chain
Beta-gamma disphosphate discarded
What can RdRP synthesize in viruses?
+ mRNA and sub-genomic mRNA
For poliovirus what is the name of the RdRP and primer used for RNA synthesis?
3Dpol RdRP
VPg protein used as primer that is covanlently linked to the 5’ end of the RNA by a phosphodiester bond
What does the RdRP recognize in the genome?
5’ clover leaf
Cis-acting RNA element (CRE)
Pseudoknot
How is VpG-U-U primer created
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
What is the role of PCbp in poliovirus?
Cellular poly(rC) binding protein (PCbp): required for poliovirus replication
What is the role of PAbp in poliovirus?
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
How is poliovirus RNA synthesized?
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
Describe the difference between the mRNA and subgenomic mRNA of the alphavirus that are created from - strand RNA.
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
How does RNA synthesis work for VSV?
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
What promotes the switch between subgenomic to full length RNA synthesis?
Nucleocapsid (N) Protein
Acts as a transcription anti-termination factor and inhibits subgenomic RNA synthesis
How does RNA synthesis work for Influenza?
Has segmented - RNA that gets transcribed by RdRP into sub-genomic RNA. Some mRNAs are spliced
Review lecture 5 slide 26
The influenza RdRP is a heterotrimer of which viral proteins?
PB1, PB2, PA
Why do dsRNA viruses have RdRP?
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
What are alternative capping strategies at the 5’ end of mRNAs?
Cap snatching
Viral capping enzymes
(Alternative but not capping: viral proteins)
How to add polyA tails to mRNA in viruses?
Copying of long U stretch in template RNA
Reiterative copying at stretches of U in template RNA
Describe the mechanism of reiterative copying of U stretches in template RNA during mRNA synthesis in VSV
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
How does reiterative copying of U stretches in template RNA during mRNA synthesis work for Influenza?
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
