BIOL 512 Exam 2 Pt. 1

What direction on the template strand does RNA polymerase travel, and in what direction are nucleotides added?

* RNA polymerase travels 3’ - 5’
* New nucleotides are added to the 3’ end (written 5’-3’)

What is unique about eukaryotic transcripts from most RNA virus transcripts?

Eukaryotic transcripts have introns and exons. Introns are removed, exons are expressed.

What features are needed at the 5’ and 3’ end of eukaryotic mRNA for translation to occur?

A 5’ cap and 3’ poly-A tail are needed.

What is leaky scanning? What is ribosomal frameshifting? What are the similarities and differences between the two?

Leaky scanning→ ribosomes can bypass the first AUG codon and start translating at the second.

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Frameshifting→ ribosomes can intentionally “slip” into a different reading frame

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Similarities→ Both result in the translation of a different protein/protein sequence being made.

What are the 3 types of genomes that RNA viruses can have?

Negative (-), positive (+), and dsRNA

What are the size ranges of RNA viruses?

6-41 kb

Are dsRNA viruses segmented, non-segmented, or both?

(+) RNA Viruses?

(-) RNA viruses?

dsRNA viruses→ exclusively segmented

(+) RNA viruses → non-segmented

(-) RNA viruses → either segmented or non-segmented

Why must an RNA virus encode for an RNA-dependent RNA polymerase?

There are no cellular enzymes that can copy RNA from RNA, and transcription/replication must proceed from the 3’ end of the genome.

What is the difference between replication and transcription? Why can these processes overlap in RNA viruses? What class of RNA viruses do these processes overlap?

Replication → making exact copies of the genome to be packaged into a new virion.

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Transcription → making an RNA to be translated into a functional protein.

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In some RNA viruses, RNA is made for transcription/replication, while in others, genomic RNA can be used as mRNA.

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These processes overlap in (+) sense RNA viruses.

Do RNA viruses use the same enzyme to replicate and transcribe DNA? Do they use the same complex of proteins?

The same set of enzymes and protein complexes are used to replicate and transcribe DNA.

What are the signals in the genome that help initiate replication/transcription?

In dsRNA, the genome termini must have a poly-A tail and a cis-acting element.

Do RNA viruses require a cap and a poly-A tail? Of the RNA viruses discussed in class, which require caps/poly-A tails and which do not?

RNA virus genomic RNA do not require a 5’ cap and a poly-A tail, but their mRNA contains both.

What are the basic properties of (-) RNA viruses? Are they single- or multi-segmented?

All (-) RNA viruses are enveloped, form helical capsids, do not make polyproteins, and replicate in the cytoplasm (except for the flu)

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They encode a similar set of genes

→ nucleocapsid genes

→ glycoprotein genes

→ RNA Polymerase genes

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(-) RNA viruses can have one or more segments

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What non-structural protein must be in the virion for (-) RNA viruses?

RNA Polymerase

What are some common (-) RNA viruses?

Measles, RSV, rabies, Ebola, Marburg

What are the five proteins in all monopartite (-) RNA viruses? Are they made of one or multiple transcripts? In what order are these proteins made? Which proteins comprise the RNP complex?

Nucleoprotein, Phosphoprotein, Matrix protein, Glycoprotein, and Large protein (polymerase)

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They are made from 5 separate mRNAs, 1 for each protein.

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N→P→M→G→L

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Nucleo-, phospho- and large proteins make up the CIRCULAR RNP complex.

Describe the process of rhabdovirus transcription.

* 5 separate mRNAs are cleaved, one for each protein.
* Made in decreasing proportions as RNA polymerase moves towards the 5’ end of the genome
* Transcription is carried out by the ribonucleoprotein complex

What is the leader RNA in Rhabdoviruses?

* 49 nucleotide RNA strand
* No cap or poly-A tail
* RNP moves along the genome **without transcription** until it encounters a **transcription start site.**

When Rhabdovirus RNA polymerase encounters a termination sequence, what are its two choices?

1. Dissociate from RNA, go back to the beginning, and start over.
2. Move along the genome until it encounters another initiation sequence (stuttering).

What is the stuttering mechanism? What is EIS?

The end domain sequence is 3’-AUACU-5’ (E)

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RNP copies U7 region (poly-A), skips dinucleotide (intergenic region, I)

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Reinitiates at start sequence 3’-UUGUC-5’ (S)

How is rhabdovirus transcription regulated? How is this regulation achieved?

Regulated by switching between transcription and translation

What must happen for Rhabdovirus (-) RNA to be replicated vs. transcribed?

RNP switches from transcription to replication of the viral genome and produces the **antigenome.** The replication process is asymmetric, creating more (-) RNA than (+) antigenome.

Do Rhabdovirus mRNAs encode for one or multiple proteins?

Rhabdovirus mRNAs encode for 5 highly conserved proteins.

Is Rhabdovirus RNA replication symmetric or asymmetric? What does this mean? Which strand is made in abundance?

Rhabdovirus RNA replication is asymmetric.

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Polymerase ignores the termination sequence that normally stops during transcription.

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(-) RNA >>>> (+) antigenome

What type of disease is caused by rotaviruses? Who does it affect? How does it transmit?

Rotaviruses commonly cause **diarrhea** in **children**.

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It infects cells of the small intestine.

Spread by the **fecal-oral route.**

→What are the basic properties of rotaviruses?

→How many segments does it have and what is the total amount of genetic material?

→How many proteins does it encode?

→How does one segment make different proteins?

→Where does it replicate?

→ Rotaviruses are non-enveloped dsRNA viruses

→ 11 segments of helical dsRNA; 1 segment: 1 protein (except for one)

→ 12 proteins total.

→ Total of 18.5 kb

→ Occurs in the cytoplasm.

How do rotaviruses prevent transcription of their genome before the virus has entered a cell?

RdRP is inactive in the virion, but becomes active after it loses some structural proteins.

Where is rotavirus transcribed? How does it exit this location?

(+) RNA is transcribed/replicated **in the core** and leaves the core through channels through the external spike proteins.

How do rotaviruses promote translation without a poly-A tail?

NSP3 acts as a translational enhancer to make up for the lack of a poly-A tail.

Though mRNA levels are similar at late times after rotavirus infection, describe how protein abundances could be different.

* mRNAs are translated with different efficiencies
* The sequence surrounding AUG start codon is important
* Consensus Kozak sequence

Where does RNA replication occur for rotaviruses?

Inside the virion.

What families of viruses are “simple” (+) RNA viruses?

Picornaviridae and Flaviviridae

What are the basic features of a “simple” (+) RNA virus?

→ Monopartite genome

→ No difference between genome RNA and mRNA

What are some medically relevant picornaviruses?

Poliovirus, Rhinoviruses, Enteroviruses, and Hepatitis A virus.

What are the basic features of picornaviruses?

→ Pico means small in size, 7.5 kb genome

→ non-enveloped virus with icosahedral capsid.

What is an IRES? How does it allow for translation in the absence of a cap?

An IRES is an **Internal Ribosome Entry Site** that adopts a specific 3D conformation that binds to ribosomes and directs translation. It is an RNA sequence between VPg and AUG initiation codon.

How do picornaviruses prevent host translation?

The genome is protected by protein VPg at the 5’ end. The virus targets eIf44G1 and eIf4G2 for degradation which halts protein synthesis.

Describe the process of picornavirus translation.

Where are structural/non-structural genes located?

How is the abundance of protein regulated?

* The genome is translated as a polyprotein which is cleaved into individual proteins by viral proteases.
* The structural proteins @ the 5’ end
* The non-structural proteins @ the 3’ end of the genome.

PV replication occurs on-----membrane vesicles derived from ---- or ---- membranes.

PV replication occurs on __**double-membrane**__ vesicles derived from the __**ER or autophagic membranes.**__

Why must the viral genome be translated prior to any RNA replication?

The virus must translate **RNA-dependent RNA polymerase** since it is not in the virion like (-) RNA viruses. The virus must make the (-) RNA next.

How is the picornavirus polymerase activated?

Poly-A and poly-C binding proteins are brought together by the 3CD protein and is the precursor to polymerase 3D. The polymerase is activated by proteolytic cleavage.

What is a replicative intermediate? How many polymerase complexes can be on the genome at one time?

* All the complexes are simultaneously present on the strand.
* Up to 5 complexes can be formed.

Picornavirus RNA virus replication is asymmetric. Which strand is produced in abundance?

(+) RNA >> (-) RNA

What are some medically relevant flaviviruses?

West Nile Virus, Zika virus, Dengue virus

What are the basic characteristics of the Flaviviruses?

* The genome is 10-11 kb
* (+) RNA and the virion is spherical with an envelope.

What are two similarities between Flaviviruses and PVs in their replication?

1. Both genomes are translated into a single polyprotein to be cleaved by proteases.

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2. The structural genes are at the 5’ end and the non-structural proteins are at the 3’ end.

What are three differences between Flaviviruses and PVs in their replication?

1. The polyprotein is cleaved by both viral and host proteases in FVs
2. FVs have a cap at the 5’ end of their genome.
3. Do not have a poly-A tail.

What is sfRNA and how is it created?

sfRNA is **sub-genomic flavivirus RNA**.

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It is the incomplete digestion of genomic RNA by cellular exoribonucleases.

What Flavivirus proteins are NOT membrane-bound proteins?

The capsid, NS1, N3 protease, NSJ polymerase, or protein.

What is the major difference between complex and simple (+) RNA viruses?

Complex (+) RNA viruses make >1 strand of (+) sense RNA and are generally larger than simple (+) RNA viruses.

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Examples: Caliciviridae and astroviridae such as togaviridae and coronaviridae.

What are the basic characteristics of alphaviruses and what alphavirus was mentioned in class?

Alphaviruses are arthropod-borne viruses that generally cause encephalitis or severe arthritis.

Icosahedral capsid and enveloped genomes

Genome is 10-13 kb

Example: Chikungunya virus

Be able to describe how alphavirus non-structural proteins are created compared to structural proteins. Which are created from genomic RNA?

Non-structural proteins are located at the 5’ end of the genome; structural proteins are created from sgRNA at the 3’ end.

Does the genome have a 5’ cap and poly-A tail?

Yes

Understand the process of transcription/replication for alphaviruses. How is it different from simple (+) RNA viruses? How many unique (-) RNA molecules are created?

Full-lengthh (-) gRNA/mRNA is produced by polymerase

gRNA is translated into a polyprotein which is cleaved to produce structural proteins.

What is the protease/polymerase for alphaviruses?

nsP2 → protease

nsP4 → polymerase

What nsP complex directs (-)-strand synthesis? Which nsP complexes direct sub-genomic RNA synthesis?

P1234 → (-) strand synthesis

P123 and P4 → (+) strand, sgRNA synthesis

Which RNAs (sg or gRNAs) are made in greatest abundance?

sgRNA >> gRNA

What are the basic characteristics of coronaviruses?

Large genome (\~30 kb)

Enveloped, helical nucleocapsid ((+) sense)

Which end of the genome contains the non-structural proteins and which end contains the structural proteins?

nsPs at 5’ end

structural proteins at 3’ end

Same as alphaviruses

Besides the structural proteins, what other types of proteins are encoded throughout the 3’ end of the CoV genome?

Accessory proteins

What is unique about the polyprotein encoding the CoV non-structural proteins compared to alphaviruses?

All structural and accessory proteins are separately transcribed and are NOT created as polyproteins.

How are the two polyproteins created and what are they called?

The 5’ end is translated into two long polyproteins


1. pp1A
2. pp1AB

Translation depends on the slippery sequence where the ribosome slips back 1 nucleotide causing a ribosomal frameshift.

What is ribosomal frameshifting? How does it direct the production of new proteins? How often does ribosomal frameshifting of the polyprotein occur?

→ Ribosome slips back 1 nucleotide, bypasses the stop codon to produce nsPs 12-16

→ Produces nsPs in different quantities, nsPs 12-16 << nsPs 1-10

→ Occurs 25% of the time

What two non-structural CoV proteins have protease activity? Which one makes most of the cleavages in the polyprotein?

Papain-like protease makes the first cleavages

3CL protease (main protease) makes all other cleavages

What are 2 advantages of the RTCs for coronavirus replication?

1. Provides a scaffold to anchor the replication complex/confine process to a specific location in the cytoplasm
2. Helps prevent activation of innate immune system sensing of dsRNA

What additional enzymatic activity are encoded by CoV?

→ polymerase/helicase

→ proteases

→ capping enzymes

→ endoribonuclease

→ exoribonuclease

→ ADP ribosyl hydrolase

What property does the CoV exoribonuclease give to CoVs that no other RNA virus has?

Proofreading

Describe the process of sgRNA production during coronavirus infection. How does this differ from alphaviruses?

sgRNAs act as mRNAs for all the structural and accessory proteins and both types of RNAs are copied through (-) strand intermediates.

Each sgRNA generally encodes for 1 protein, no polyprotein.

What are the components of every sgRNA?

→ Leader

→ TRS

→ Body

→ 3’ UTR

→ Poly-A tail

How many proteins are generally produced from each sgRNA?

1 protein per 1 sgRNA

Do all sub-genomic RNAs have the same 5’ and 3’ end?

All sgRNA's have the same 3’ end, but translation stops once it reaches a TRS (transcription regulatory sequence)

Be able to describe the process of how CoVs produce (-) sgRNAs. How are the leader/TRS sequences joined?

sgRNAs are transcribed until it reaches a TRS sequence. When RdRP reaches a TRS-B, it either continues to make (-) gRNA or stalls and “jumps ship” to TRS-L (more common), looping around and coding another section further down.