Chapter 6 RNA directed RNA synthesis

Viruses with (-) ss RNA genomes

RdRp

RNA coated with protein (nucleocapsid)

ex: VSV (unimolecular); Influenza (segmented)

Viruses with dsRNA genomes

RdRp

Naked RNA

mRNAs and genomic RNAs are produced from distinct templates in different viral particles

ex: Reoviridae, reovirus, rotavirus

Reovirus

(+) strand is not accessible by ribosome because it is double stranded

Rotavirus

Each dsRNA segment is attached to RdRp via the 5’ cap

Viruses with (+) ssRNA genomes

No RdRp

Naked RNA (exceptions include retroviruses and coronavirus)

ex: Polio virus

RNA structure

Rules for Viral RNA Synthesis

1. RNA genome must be copied end to end with no loss of nucleotide sequence

2. Viral mRNAs must be produced that can efficiently translated by cellular protein synthesis machinery

Rules for RNA-directed RNA synthesis

1. RNA synthesis initiates and terminates at specific sites

2. RdRp may initiate synthesis w/ or w/out a primer

3. Other viral and cell proteins may be required

4. RNA is synthesized by adding NTPs one by one from the 5’ end to the 3’ end.

5. There is some non-templated synthesis

De novo

Without a primer

NTP added to 3’ end

Primer-dependent

Protein linked primer (polio)

or nucleic acid primer (influenza)

Two-metal mechanism of polymerase catalysis

Polymerases synthesize nucleic acids

Polymerase enzyme contains two Asp residues which coordinate two Mg2+ ions.

A phosphodiester bond forms, extending the sugar-phosphate backbone

Template sequence determines next NTP

UTP

Are bound to polio virus RdRp

D328 & D329 are 2 aspartates in active site of RdRp that coordinate metal ions

Hydrogen bond between 2’-OH of ribose and D238 (only UTP of RNA works)

Poliovirus Replication: VPg

Linked to first base in RNA at 5’ end by phosphodiester bond

Acts as a primer for RNA synthesis

Why copies of viral RNAs are made

Cloverleaf, cre stemloop, and pseudoknot structures

(-) strand RNA Virus Replication

Viral genome is not mRNA

There must be a switch from mRNA to genome RNA synthesis

A specific protein acts as that molecular switch (N protein)

N protein

With this viral nucleocapsid bound to the (-) strand genome RNA, the RdRp reads through the intergenic juctions where termination occurs during mRNA synthesis

Activation of influenza virus RNA Polymerase

Influenza has a (-) strand RNA genome

PB2 binds to capped primer, activating binding of(-) strand genome RNA at conserved sequence

Endonuclease cleaves host cell RNAs, producing primers for viral mRNA synthesis

Diversity among RNA viruses

Lack of proofreading in RdRp

Exchange of nucleotide sequences among different genomic RNA molecules (Shapes RNA virus world by rearranging genomes and creating new ones)

Nidovirales

exoN protein is 3’ to 5’ exonuclease that corrects RNA polymerase errors and allows replication of large RNA genomes