Lecture 12. Coronavirus Replication

Question 1

What are coronaviruses?

Answer 1

Baltimore class 4: +ve ssRNA genome
Order: Nidovirales
Family: Coronaviridae
Genera: Alpha-, Beta-, Gamma-, Delta-coronaviruses

Question 2

Where do alpha and betacoronaviruses originate from and what are examples of these coronaviruses?

Answer 2

Bats, include human viruses and mouse hepatitis virus (MHV)

Question 3

Where do gamma and deltacoronaviruses originate from?

Answer 3

Avian origin

Question 4

What are well known betacoronaviruses?

Answer 4

SARS-CoV, SARS-CoV-2, MERS-CoV

Question 5

Before the 21st century, what were coronaviruses known to cause?

Answer 5

Mild upper respiratory tract infections: HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU

Question 6

In the 21st century, what were coronaviruses known to cause?

Answer 6

Severe lower respiratory tract infections: SARS-CoV, MERS-CoV, SARS-CoV-2

Question 7

When did SARS-CoV emerge?

Answer 7

2002, outbreak controlled in 2003
High case mortality rate but lower transmissibility

Question 8

When did MERS-CoV emerge?

Answer 8

Emerged in 2012 from camels, now only sporadic cases

Question 9

When did SARS-CoV-2 emerge?

Answer 9

2019, became global pandemic

Question 10

What is the structure of coronavirus?

Answer 10

Enveloped spherical particle (125 nm diameter) with crown-like surface proteins

Question 11

How long is the coronavirus genome and how many open reading frames are there?

Answer 11

30kb +ve sense ssRNA (longest viral RNA genome)
13 ORFs

Question 12

What is one of the features that allows coronaviruses to adapt so well?

Answer 12

They are less error prone
Coronaviruses are the one example where the replicase complex has a proof reading enzyme - introduces less error (SARS-CoV-2 is an exception with how much variability there is)

Question 13

What can the coronavirus genome acts as mRNA for?

Answer 13

The translation of ORF1a and ORF1b (but not the ones at the 3’ end)

Question 14

What happens in the coronavirus replication cycle?

Answer 14

Attachment by S (spike) protein to receptor (S2)
Entry by endocytosis into cytoplasm (taken into endosome)
Uncoating of +ve sense ssRNA genome (released into cytoplasm)
Translation of genome to produce transcriptase/replicase complex
Replication and transcription of viral RNAs
Translation of structural proteins
Assembly of viral particles in ER/Golgi vesicles
Exit by exocytosis

Question 15

How does SARS-CoV attach and enter an endosome?

Answer 15

Trimer of S protein binds to angiotensin-converting enzyme 2 (ACE2) on human epithelial cells
Binding ACE2 triggers endocytosis and viral particle enters cell in an endosome

Question 16

Where are angiotensin-converting enzyme 2 (ACE2) receptors present?

Answer 16

In a lot of organs (resulting in the widespread issue caused by coronavirus infections)

Question 17

What are the two domains of the spike protein?

Answer 17

S1 interacts with the receptor
S2 contains a fusion peptide
Fusion requires protease cleavage between S1 and S2 domains to release fusion peptide

Question 18

What are the S proteins that make up the spike protein?

Answer 18

S protein is class I viral membrane fusion protein (like influenza HA)

Question 19

What is the difference between coronavirus spike protein cleavage and influenza HA?

Answer 19

What causes the cleavage and when it happens
Protease cleavage in spike protein of CoV is carried out by proteases in the cell membrane
Cleavage is a late stage process when compared to influenza where the fusion peptide is released much earlier (when viral particle being made)

Question 20

What proteases release the fusion peptide from the S2 domain?

Answer 20

Proteases in the cell membrane (e.g. TMPRSS2) and within the endosome (e.g. cathepsins)

Question 21

What does insertion of the fusion peptide into the endosomal membrane lead to?

Answer 21

Fusion of viral and endosome membrane
Coronavirus genome (+ve sense ssRNA), wrapped up in N protein, is released into the cytoplasm
Similar mechanism to influenza HA and HIV Env

Question 22

In SARS-CoV-2, what contributes to its high infectivity?

Answer 22

Second cleavage site (for furin) that allows pre-activation step

Question 23

In SARS-CoV-2, what does cleavage of the spike protein by furin do?

Answer 23

Makes the spike protein a lot more accessible for the cleavage that releases the fusion peptide (makes S2 more accessible)

Question 24

What is the receptor binding domain (RBD) of S1 and what does it do?

Answer 24

Has two conformations
Lying down for immune evasion
Standing up for receptor binding

Question 25

How is SARS-CoV-2 more infective than SARS-CoV?

Answer 25

SARS-CoV 2’s RBD (receptor-binding domain) stood up less so invaded the immune system more, and when it did it had a higher binding affinity for ACE2 receptor than SARS-CoV

Question 26

How is the genomic RNA of coronaviruses translated?

Answer 26

+ve sense ssRNA has a 5’ cap and poly(A) tail and can act directly as mRNA
Ribosomes translate the genomic RNA to produce polyproteins pp1a and pp1ab (by ribosomal frameshifting)
Pp1a and pp1ab encode components of the transcriptase/replicase complex

Question 27

How do coronaviruses utilise -1 ribosome frameshifting?

Answer 27

A pseudoknot structure in the RNA (folds back in on itself) causes the ribosome to pause on a slippery sequence (Us and As), then slip back 1 nucleotide (Efficiency ≈20%, high efficiency)

Question 28

What happens to Pp1a and pp1ab after replication?

Answer 28

Cleaved by viral proteases within pp1a
The RNA-dependent RNA polymerase is a component of pp1ab
After cleavage into individual proteins, the components of pp1a and pp1ab assemble into
a transcriptase/replicase complex on the ER membrane

Question 29

What is included in the transcriptase/replicase complex in coronaviruses?

Answer 29

The polymerase, RNA helicase, capping enzyme (can make capped mRNAs), exoribonuclease (proofreading)

Question 30

What is genomic RNA of coronaviruses used a template for?

Answer 30

Making either full length -ve sense RNA (template for producing more genomic +ve sense RNA)
Or subgenomic -ve sense RNA (templates for producing subgenomic mRNAs that encode viral structural proteins) by discontinuous transcription

Question 31

What is discontinuous transcription?

Answer 31

Transcriptase/replicase complexes pause at transcription-regulating sequence (TRS) and either continue creating full length -ve antigenome RNA (-gRNA)
Or dissociate and re-associate at complementary leader sequence resulting in shorter -ve subgenomic RNAs (-sgRNAs)

Question 32

What are -ve sense gRNA (genomic RNA) and -ve sense sgRNAs (sub-genomic RNA) templates for?

Answer 32

Many copies of new positive sense +ve sense gRNA and +ve sense sgRNAs

Question 33

What do all subgenomic RNAs contain?

Answer 33

The same leader region and the same 3’ end (poly A tail) - known as “nested” since only the bit between the 5’ and 3’ ends are different

Question 34

What do the structural proteins encoded by sgRNAs include?

Answer 34

S (spike), M (matrix), E (envelope) and N (nucleocapsid)

Question 35

What are the S, M and E proteins co-translationally inserted into?

Answer 35

ER membranes

Question 36

Where are the S and M proteins highly glycosylated?

Answer 36

In the ER and then the Golgi

Question 37

How is the nucleocapsid (N) protein translated?

Answer 37

N is translated on free ribosomes and binds to new genomic RNA as it is synthesised

Question 38

Where does coronavirus particle assembly take place?

Answer 38

Occurs at ER-Golgi intermediate compartment (ERGIC) membranes

Question 39

What drives coronavirus particle assembly?

Answer 39

Driven by M protein, but also requires E
The way the M protein is embedded causes the membrane to curve and encloses copy of the virus genome

Question 40

How are newly formed coronavirus particles released?

Answer 40

New particles bud into the lumen of the ER-Golgi intermediate compartment (ERGIC)
Transported out of cell by exocytosis in cargo vesicles