4 - RNA viral replication (MICROM 450)

What are the different kinds of RNA viruses?

(+), (-) and dsRNA

• RNA dependent RNA polymerase

What are the different kinds of DNA viruses?

dsDNA, ssDNA

• DNA dependent DNA polymerase

What are the different kinds of retroviruses?

ssRNA-retro & DNA-retro

• RNA dependent DNA polymerases

What is necessary to do RNA virus replication

• must encode RNA-dependent RNA polymerase (there is no RdRp in host cells as it breaks central dogma)

  • RdRp stills add NTPs to 3’ OH end so new strand are still made 5’ → 3’

• Initiation of new strands needs: primer OR de novo w/o primer

How does + RNA make a virus?

Naked viral RNA can let virus progeny out

Why can’t - viruses make a virus

Naked viral RNA cannot be directly translated into proteins without first being converted into a positive-sense RNA. This conversion process involves the action of an RdRd

How are recombinant viruses made for genetic systems?

1. (+): will make viral mutant when mRNA from plasmid introduced

2. (-): must introduce genome first & necessary mRNAs & encoded proteins to get system started

What are key structural features of all polymerases?

• right hand palm, fingers, thumb domains: all polymerases

• motifs A, B, C, D (GDD catalytic triad found in domain C of most +ssRNA viruses)

• Domain F unique to RdRp (forms NTP channel)

Why must RNA polymerase only incorporate NTP no dNTP’s

NTP is a base with both OH groups while dNTP’s only have one OH

Why do RNA viruses no have proof reading capabilities?

No help from host sine there is no RdRp (higher error rate)

• 1 mistake after every copy of a full length genome

• RNA viruses = poor fidelity, DNA = higher fidelity since their polymerases correctly proofread and repair errors

**reverse transcriptase = poor fidelity

How have RNA viruses mutated/evolved?

poor fidelity creates mutations per genome, leading to various sequences for each round of replication

• some viruses have multiple serotypes/strains due to higher mutational space

• some viruses have limited strains bc they cannot tolerate mutations

**twice as many RNA families than DNA

What is Ribavirin?

Antiviral compound for many RNA viruses

• binds to RdRp & causes increased mutations level to levels that make viral genomes untenable/viral failure

How does RdRp find viral genome & not cellular RNA’s around the cell?

Recognizing specific, highly conserved structural within signal sequences found on on viral RNA

• polymerase proteins recognize specific motifs on viral genome (specific sequences/structures)

**CELLULAR RNA’S NEVER COPIED BY VIRAL POLYMERASE

How does RdRp initiate of replication?

Two Modes-

1. De novo: no primer & similar DdRp (transcription)

2. Primer dependent initiation: similar to DdDp (replication), terminal protein primer, stolen cap primer

How can we ensure no loss of information passed to next generation during genome copying?

Must initiate at the end of the genome to maintain sequence integrity

(+) strand RNA virus replication

Replication must copy entire genome from first to last base of genome to make (-) strand

and subsequently use the (-) strand as a template to synthesize new (+) strand RNA genomes for complementary strand

Why do some + strand RNA viruses not encode mRNAs for full length replication

Alphaviruses encode both full length & sub genomic RNA

-full length only makes non structural proteins

-once there is enough of that, subgenomic RNA is synthesized to make structural proteins

Why are structural proteins important?

They are higher level proteins compared to enzymatic proteins. They allow for temporal expression & virus assembly

(-) strand RNA replication

Polymerase comes in w capsid to synthesize + strand to be translated into proteins and replicated back into the original (-) strand RNA genomes

** Process ensures the virus can produce more infectious particles

How is full length & subgenomic synthesis regulated?

Extensive regulatory mechanisms

Ex-

1. viral proteins bind & cause polymerase to read through polyA signals to make full length

2. Rhabdoviridae have series of stops & restarts along RNA polymerase at polyU tracts

GENERAL: build up of viral protein which binds to genome & favors full length RNA production over mRNA

What dictates if subgenomic or genomic RNAs are produced?

Level of N protein dictates if mRNAs or full length genomic RNA is made

• low levels: polymerase stutters at UUU sequence

• high: template coated w proteins & this blocks stuttering so full length can be made

What is cap snatching?

Orthomyxoviruses steal caps for Cap-primer intitiated mRNA building

Uncapped when primer independent RNA initiation begins & full genome completed

Why do double-stranded RNA viruses require RdRp in their capsid?

dsRNA cannot be translated (cannot act as mRNA)

-polymerase will come in to make + strand which can then act as mRNA

Why must RNA viruses replicated in cytoplasm of host cell?

1. + strand needs to be where ribosomes are to get translated upon entry

2. barrier to get into nucleus & viral protein don’t need nuclear localization

3. they have their own RNA polymerase for gene expression, don’t require host enzyme

EXCEPTION: orthomyxoviruses - RNA transported to nucleus