Lecture 10 & 11: RNA Virus Replication

Question 1

+ strand RNA genomes

Answer 1

-Naked
-Ready to be translated upon entry (Not in retroviral genomes)
-Genome by itself is infectious

Question 2

• strand RNA genomes

Answer 2

-Coated with protein
-Ready to begin mRNA synthesis upon entry
-Virions contain polymerase
-Genome is non-infectious by itself
-Must firt be transcribed into + sense RNA by RDRP

Question 3

dsRNA genome

Answer 3

-Cannot be copied into mRNA by the cell
-Virions contain RNA polymerase

Question 4

Replication strategy for + sense RNA viruses

Answer 4

-mRNA/Genomic RNA is translated to produce a polyprotein. Cleavage of polyprotein can make structure and non-structural viral proteins
-Non-structural proteins (RDRP) catalyze RNA replication using (+) sense genome to synthesize - antigenomic RNA
-Antigenomic RNA is template for nascent (+) strand genomes

Question 5

Replication strategy for ambisense genome

Answer 5

-Genome having both positive and negative characters. Part can be directly translated and part cannot be.
-Both genome and complement contain coding information
-Classified as “Negative sense” because they carry their polymerase
-During infection virus first makes mRNA from the (-) part of the genome which is translated into protein which then replicates the genome and make mRNA from

Question 6

(+) sense RNA replication in alphaviruses

Answer 6

-1st Open reading fram translated to produce non structural proteins (an unprocessed Rep & processed Rep)
-Unprocessed Rep uses (+)-sense genome to make (-)-sense genome
-Processed Rep uses (-)-sense genome to make more (+)-sense genome and also recognize a subgenomic promoter in (-)-genome from which a subgenomic RNA of positive polarity is made and translated to produce a stuctural polyprotein

Question 7

Replication strategy for - sense RNA viruses

Answer 7

-(-) sense must be converted to (+) sense by RdRP before proteins can be made, therefore, they must package the RdRp in the virion so they can make mRNA upon infecting cell.
-Genome is a template for mRNAs
-Viral nonstructural proteins join with full length (+) sense complementary RNA to make new (-) sense genomes

Question 8

Replication strategies for dsRNA

Answer 8

-DOES NOT FUNCTION AS AN MRNA
-Bring their own RdRp into the particle
-(-) sense strands are used as templates for (+) sense strands
-These (+) sense strands serve as mRNA or as templates for (-) sense strand synthesis

Question 9

RNA-dependent RNA Polymerases

Answer 9

-Unique process with no cellular parellel
-Hallmark: Resistant to actinomycin D, an inhibitor of DNA-directed RNA synthesis
-Show functional similarities to cellular polymerases (DdRp and DdDp)
-RdRp’s catalyze template directed RNA synthesis from 5’ to 3’
-Viral RNA synthesis is highly efficient but highly error prone

Question 10

Recognition of secondary RNA structures

Answer 10

-First order information content is contained int he sequence of an RNA
-Second order information content is contained in the structure
-Ability to form G-U base pairs and non-watson crick base pairs gives RNA the ability to produce a variety of structures
-Wide variety of structural possibilities provides for specificity of interaction with other biomolecules

Question 11

Roles of viral accessory proteins in assisting RdRp’s during replication

Answer 11

-Used to direct RDRP to the correct interacellular site
-Can target RDRP to correct initiation site on RNA template
-Helicases unwind RNA secondary structures

Question 12

Processive and Distributive Helicases

Answer 12

-Processive: Unwind along an mRNA
-Distributive: Unwind at one particular spot

Question 13

Role of cellular proteins in bacteriophage QB RNA synthesis

Answer 13

RdRp hijacks a host ribosomal protein and two host Translation Elongation Proteins for their RNA binding properties

Question 14

Role of cellular proteins in poliovirus RNA synthesis

Answer 14

-Host Poly(rC)-binding protein 2 helps target viral proteins to an RNA secondary structure that is the site of initiation for genome replication
-Host Poly-A binding protein 1 (PABP-1) used in both initiation of replication and translation

Question 15

Initiation mechanisms of RdRp

Answer 15

-Most initiation occurs de-novo (From new) with no primer requirement

Question 16

Exception to RdRp mechanism by capped RNA fragments

Answer 16

-Influenza steals methyl-Gppp caps from cellular mRNAs to prime viral mRNA synthesis

Question 17

Synthesis of polyA tracts

Answer 17

-3’ polyA tails are required for translation of most mRNA’s
-PolyA is attached to the 3’ in the nucleus and RNA viruses are replicated in cytoplasm

Question 18

Exception to RdRp mechanism by protein priming

Answer 18

-In poliovirus VPg a protein covalently linked to 5’ end of genome
-VPg becomes polyuridylated (PolyU). Base pairs with polyA 3’ end of genome are made. Interaction with RdRP serves to target replicase to primed 3’ end of genome

Question 19

Evolutionary mechanisms to acquire polyA tracts

Answer 19

-Encode a 3’ polyA sequence on the (+) strand and/or 5’ polyU on the (-) strand
-Reiterative copying “Stuttering” on short 3’ U-sequences on the (-) strand: polymerase pauses between genes and adds a polyA tail by reiterative copying of U residues

Question 20

Stuttering

Answer 20

After each A is added the polymerase moves back one nucleotide along with the mRNA, this repeats over and over until hundreds of A’s have been added.

Question 21

Stuttering mechanism of flu

Answer 21

1. PB1 subunit of RdRp binds the 5’ end of a viral (-) sense RNA segment
2. Causes a confirmational change that triggers PB2 binding to host mRNA
3. Binding of the 3’ end of the viral RNA segment to a second site in PB1 activates the endonuclease activity of RdRp which allows it to cleave the host mRNA 10-13 nucleotides downstream of cap.
4. Elongation begins using the cleaved host mRNA 5’ end as a primer
5. RdRp continues to hold the 5’ end of the viral RNA while the 3’ end is threaded through as synthesis of the mRNA continues
6. Polyadenylation occurs by RdRp stuttering near but not at the 5’ end of the segment over a run U’s.

Question 22

Ribosome/RDRP clash problem

Answer 22

-In (+) sense RNA viruses, RNA is both template for translation and replication
-Translation moves in the 5’ to 3’ direction along the (+) strand
-Replication moves in the 3’ to 5’ direction along the (+) strand
-Problem: At some point in the middle they will collide so virus must evolve around this

Question 23

Switching from mRNA production to genome RNA synthesis

Answer 23

-No switch required when mRNA and gRNA are identical
-mRNAs of most RNA viruses are not complete copies of the viral RNA so a switching mechanism is needed

Question 24

Switch mechanism 1: Different polymerases for different functions

Answer 24

-Viral (-) strand genomes are template for the production of either mRNAs or full length (+) strand RNAs
-L+P polymerase=mRNA synthesis
-L+P+N polymerase=genome replication

Question 25

Switching mechanism 2: Antiterminator activity

Answer 25

-To produce full length (+) strand RNA the stop-start reactions at the intergenic region must be suppressed.
-As more and more N protein is made, it coats the genome, preventing termination of synthesis and allowing read-through of the polyA signals and production of full length (+) sense antigenomes

Question 26

Switching mechanism 3: Different templates used for RNA synthesis and genome replication

Answer 26

-in dsRNA viruses replication of gRNA occurs only after packing RNAs inside of capsids
-All unpackaged viral RNAs are mRNA by default.