Lecture 6 - mRNA Synthesis and Genome Replication

What must RNA viruses ensure when copying their genomes?

The genome must be copied end-to-end with no loss of nucleotides.

What is the key goal of viral mRNA production?

To produce mRNAs that can be efficiently translated by host ribosomes.

Is positive-sense RNA always the same as mRNA?

No, not all positive-sense RNA is mRNA.

What are the universal rules of RNA-directed RNA synthesis?

1. RNA synthesis initiates and terminates at specific sites on the template

2. RNA-dependent RNA polymerases (RdRp) may initiate synthesis de novo (from nothing) like cellular DdRp or it may require a primer A primer is a short single-stranded nucleic acid sequence that provides a starting point for replication

3. Other viral and cellular proteins may be required not just RdRp

4. RNA is synthesized by template-directed stepwise incorporation of NTPs (nucleoside triphosphates), elongated in the 5’-3’ direction

5. Some non-templated synthesis (this is rare)

What enzyme performs RNA synthesis in RNA viruses?

RNA-dependent RNA polymerase (RdRp).

What are the two types of initiation used by RdRp?

De novo initiation and primer-dependent initiation.

What is a primer in RNA synthesis?

A short nucleic acid sequence that provides a starting point for replication.

In which direction is RNA synthesized?

5' to 3' direction.

What ion does RdRp use to catalyze nucleotide addition?

Magnesium (Mg²⁺).

How do (+) strand RNA viruses like flaviviruses and picornaviruses replicate?

Their (+)ssRNA genome serves as mRNA and is copied via a (-) strand intermediate which is then used to make genomic (+)RNA

How does poliovirus do RNA synthesis?

Using a protein primer (VPg) that is uridylated to begin replication.

What is a key feature of alphavirus RNA synthesis?

They produce subgenomic mRNAs from the negative strand intermediate.

What structure at the 5' end helps poliovirus replication?

Cloverleaf RNA structure and other cis-acting elements.

Are (-)ssRNA viruses ribosome ready?

No, they require transcription into a mRNA before translation.

What is unique about rabies virus genome replication?

It uses N protein as an antiterminator to switch from mRNA to genome replication.

How to unimolecular (-)ssRNA make genomic RNA and mRNA?

By encoding an RdRp and using a nucleoprotein to prevent premature termination

How do segmented (-)ssRNA viruses (like influenza) acquire caps?

Through cap-snatching from host mRNAs and using them as primers to initiate synthesis.

Are dsRNA viruses ribosome ready?

No, they must first make (+)RNA from their genome.

How do dsRNA make mRNA and genomic dsRNA

Through synthesis of a (+)RNA or mRNA strand first which is then used to make proteins or dsRNA

What is the first biosynthetic step for dsDNA virus replication?

Must convert their viral genomes to templates for transcription

What must ssDNA viruses do first to transcribe mRNA?

Convert to dsDNA which then allows virus to make mRNA or more ssDNA

Which viruses encode an DdRp?

Only DNA viruses that replicate in the cytoplasm

What is co-transcriptional capping?

Capping that occurs during transcription, necessary for mRNA stability and export.

What are two viral alternatives to capping?

Using viral proteins (e.g., VPg) or uncapped 5’ ends (e.g., Hepatitis C).

What is cap-snatching?

A process where viruses steal 5’ caps from host mRNAs to initiate transcription.

How is the poly(A) tail added post-transcriptionally?

Cleavage of pre-mRNA followed by polyadenylation by cellular enzymes

How do some RNA viruses add a poly(A) tail during transcription?

By copying a U-rich sequence or via polymerase slippage.

What is the benefit of mRNA splicing in viruses?

It expands coding potential, marks mRNA for nuclear export, and regulates gene expression.

What are the universal rules for DNA replicaiton

1. DNA is synthesized by template-directed incorporation of dNTPs into 3’OH of DNA chain

2. DNA is always synthesized 5’-3’ by semiconservative replication (two daughter strands)

3. Replication initiates at specific sites on the template called the origin (ori)

4. Catalyzed by DdDp and accessory proteins

5. Always primer-dependent

Where do small DNA viruses get their replication machinery?

They rely mostly on host proteins.

Where do large DNA viruses get their polymerase from?

Large DNA viruses encode most of their own replication systems

What is the "5’ end problem" in DNA replication?

The lagging strand can't be fully replicated, solved by telomeres or circular genomes.

What are strategies that DNA viruses use to overcome 5’ end problem/

By using either protein priming at the end of viral genomes, bidirectional replication on circular template, DNA hairpins at the end of viral genomes, or rolling circle replication.

What are mechanisms of dsDNA synthesis

Could use replication fork when using RNA primers or strand displacement which never uses RNA primers but uses DNA or protein primers.