RNA-Directed RNA Synthesis

What is RNA-dependent RNA polymerase (RdRp)?

An enzyme used by RNA viruses to synthesize RNA from an RNA template, essential for viral genome replication and transcription.

Why do RNA viruses need RdRp?

Because host cells lack enzymes that can replicate RNA from RNA.

What types of viruses use RdRp?

+ssRNA, -ssRNA, and dsRNA viruses

What is a consequence of RdRp lacking proofreading ability?

High mutation rates in RNA viruses

Why is RdRp a good antiviral drug target?

It’s essential for viral replication and not found in host cells.

What is (-) ssRNA?

A single-stranded RNA that is complementary to mRNA and must be converted to (+) ssRNA by RdRp before translation.

Can (-) ssRNA be directly translated by host ribosomes?

No, it must first be transcribed into (+) RNA.

What enzyme converts (-) ssRNA to (+) ssRNA?

RNA-dependent RNA polymerase (RdRp)

Why do (-) RNA viruses carry RdRp in their virions?

Because host cells lack the enzyme needed to transcribe (-) ssRNA into (+) ssRNA.

Name a virus that has a (-) RNA genome

Rabies, VSV, and Influenza

What is (+) ssRNA?

A single-stranded RNA genome that can be directly translated into proteins by host ribosomes. 

How does (+) ssRNA replicate?

It uses RdRp to make a complementary (-) ssRNA, which then serves as a template to make more (+) ssRNA. 

Do (+) ssRNA viruses carry RdRp in their virions?

No, they encode RdRp in their genome and translate it after infection.

Name a virus with a (+) ssRNA genome.

Alphavirus and Polio Virus

What is subgenomic RNA?

A shorter RNA molecule transcribed from the viral genome to express specific viral proteins, especially structural or accessory proteins. 

How is subgenomic RNA produced?

By RdRp during viral replication, often through discontinuous transcription.

What is the function of subgenomic RNA?

To allow translation of downstream viral genes that wouldn’t be accessible from the full-length genome.

Name a virus that produces subgenomic RNA. 

Rabies, VSV, and Alphavirus

What is RNA editing?

A process where the nucleotide sequence of viral RNA is altered after transcription, allowing production of different proteins from the same RNA.

What enzyme is involved in RNA editing in viruses?

RdRp

Why is RNA editing useful for viruses?

It allows them to produce multiple proteins from a single gene, increasing protein diversity without expanding genome size. 

Name a virus that uses RNA editing.

Paramyxovirus and Ebolavirus

What is an intergenic junction in viral RNA?

A non-coding region between two genes that regulates transcription by RdRp.

What happens at an intergenic junction during transcription?

RdRp may pause, terminate, or reinitiate transcription, affecting gene expression levels. 

Why are genes near the 3’ end of the genome expressed more?

Because RdRp may fail to reinitiate transcription at each intergenic junction, leading to attenuation.

What types of viruses use intergenic junctions in transcription?

Nonsegmented (-)ssRNA like paramyxoviruses and rhabdoviruses.

What happens when a (+) ssRNA genome enters the cytoplasm?

It is immediately translated by host ribosomes to produce viral proteins, including RdRp.

What does RdRp do after being directly translated from (+) ssRNA?

It synthesizes a complementary (-) ssRNA strand to replicate the genome and produce more (+) ssRNA.

Can (+) ssRNA be directly translated?

Yes, it acts like mRNA and is directly translated by host ribosomes.

What types of proteins are made first from (+) ssRNA?

Non-structural proteins, including RdRp. 

What happens to newly made (+) ssRNA genomes?

They are packaged into new virions for release.

What happens when (-) ssRNA genomes enter the cytoplasm?

They are transcribed into (+) ssRNA by RdRp, which is carried in the virion, since host cells cannot perform this function.

Why must (-) RNA viruses carry RdRp in their virions?

Because host cells lack the enzyme needed to transcribe (-) RNA into (+) RNA. 

What does RdRp do with (-) ssRNA?

It transcribes it into monocistronic (+) mRNAs for translation and later replicates the genome.

How do nucleoprotein (N) levels affect RdRp activity?

Low N levels promote transcription; high N levels promote replication of full-length genomes.

What is the role of nucleoprotein in viral replication?

It binds to RNA and signals RdRp to switch from transcription to replication. It also protects the genome as it is in the cytoplasm. 

What happens when dsRNA viruses replicate?

RdRp inside the capsid transcribes (+) mRNAs from the dsRNA genome, which are then translated and used to make new genomes.

Why does transcription occur inside the capsid in dsRNA viruses?

To prevent exposure of dsRNA to the cytoplasm and avoid triggering host immune responses.

How is the dsRNA genome replicated?

(+) RNAs are packaged into new capsids, where RdRp synthesizes the complementary (-) strand to form dsRNA. 

What is an advantage of segmented genomes?

Genetic reassortment allows rapid evolution and strain diversity. Easier regulation of gene expression due to modular organizations where each segment can be transcribed independently.

What is a disadvantage of segmented genomes?

Complex packaging increases the risk of incomplete virions. Reassortment only occurs if two viruses infect the same cell, which is relatively rare.

What is an advantage of non-segmented genomes?

Simpler packaging and efficient replication. 

What is a disadvantage of non-segmented genomes?

Limited genetic diversity due to lack of reassortment as well as less flexibility in gene regulation.

Why do (+) ssRNA viruses replicate on membranes?

To protect viral RNA from host immune sensores, concentrate replication machinery, and organize replication processes.

What are replication complexes?

Membrane-bound compartments where (+) ssRNA viruses replicate their genomes. 

What host structures are used to form replication membranes?

ER, Golgi apparatus, mitochondria

What is the benefit of concentrating viral components on membranes?

It increases replication efficiency and coordination.

What is a transcription gradient in (-)ssRNA viruses?

A pattern where genes near the 3’ end of the genomes are transcribed more frequently than those near the 5’ end. 

What causes the transcription gradient?

RdRp may fail to reinitiate transcription at intergenic junctions, reducing transcription of downstream genes.

Where are highly expressed genes located in the (-) ssRNA genome?

Near the 3’ end

Why is sequential transcription important in (-) ssRNA viruses?

It determines the relative abundance of viral proteins based on gene position. 

What is cap snatching in influenza virus?

A process where the virus steals 5’ caps from host mRNAs to use as primers for viral mRNA transcription.

Why does influenza perform cap snatching?

To ensure its viral mRNAs are recognized and translated by host ribosomes.

Where does cap snatching occur in the cell?

In the nucleus

How is EBOV GP mRNA produced?

Through RNA editing, where RdRp inserts and extra adenosine, allowing translation of full-length GP instead of sGP.

How are paramyxovirus C and V mRNAs produced?

By RNA editing of the P gene transcript, where RdRp inserts extra G resides resulting in different reading frames and protein products, such as C and V.

What is a poly(A) tail?

A stretch of adenine nucleotides added to the 3’ end of mRNA to enhance stability and translation. 

How do (-) RNA viruses like VSV produce poly(A) tails?

By RdRp stuttering on a U-rich sequence, adding multiple As.

How do (+) RNA viruses like poliovirus produce poly(A) tails?

The poly(A) tail is encoded in the genome and copied during replication.

How do viruses like influenza produce poly(A) tails?

They use host nuclear machinery to polyadenylate their mRNAs. 

Which RNA viruses must package RdRp in their virions?

(-) ssRNA viruses and dsRNA viruses, because their genomes cannot be directly translated.

Why do (-) ssRNA viruses need RdRp in the virion?

To transcribe their genome into (+) RNA for translation.

Why do dsRNA viruses need RdRp in the virion?

To transcribe mRNA from the ds genome inside the capsid. 

Which RNA viruses do not need to package RdRp?

(+) ssRNA viruses, because their genomes can be directly translated.

Do (+) ssRNA viruses make one or multiple mRNAs?

One initial mRNA, plus subgenomic RNAs for additional proteins

Do (-) RNA viruses make one or multiple mRNAs?

Multiple monocistronic mRNAs, one per gene. 

Do dsRNA viruses make one or multiple mRNAs?

Multiple mRNAs, one per genome segment.

Can RNA viruses regulate how much protein they produce?

Yes, via transcription gradients, RNA editing, subgenomic RNAs, and timing.

Which of the following viruses need to package the RdRp in the viral particle?

dsRNA viruses

ssDNA viruses

(+) RNA viruses

(-) RNA viruses

dsRNA viruses and (-) RNA viruses

Subgenomic RNAs are

RNAs that are smaller than the full length genome or antigenome

What do dsRNA and (+) RNA viruses have in common?

They hide their dsRNA intermediates from cellular sensors.

Which of the following viruses do NOT produce subgenomic RNAs during replication?

Measles or Rabies

Polio or Dengue

Chikungunya or WEEV

Reovirus or Rotavirus

Polio or dengue and Reovirus or Rotavirus

Steps in Polio virus RNA replication

1. The viral genome is translated

2. Polyproteins are cleaved, forming new RdRp

3. Antigenome is produced on membranes

4. More mRNAs/genomes are produced from the antigenome

Steps in Measles Virus Replication

1. The RdRp produces mRNAs in a transcription gradient

2. N protein is made at high levels

3. Full length +RNA can be made

4. More -RNA genomes can be produced

Which of the following RNA viruses replicate in the nucleus to produce spliced mRNAs?

CHIKV

Polio

Influenza

VSV

Influenza

Which of the following uses cap snatching to make their mRNAs?

Polio

Measles

Influenza

CHIKV

Influenza

Which viruses use a transcription gradient to make their mRNAs?

Influenza

Measles and Mumps

RSV and Human metapneumovirus

Ebola and Marburg

Measles and mumps, RSV and human metapneumovirus, Ebola and Marburg