Lecture 12. Coronavirus Replication Flashcards
What are coronaviruses?
Baltimore class 4: +ve ssRNA genome
Order: Nidovirales
Family: Coronaviridae
Genera: Alpha-, Beta-, Gamma-, Delta-coronaviruses
Where do alpha and betacoronaviruses originate from and what are examples of these coronaviruses?
Bats, include human viruses and mouse hepatitis virus (MHV)
Where do gamma and deltacoronaviruses originate from?
Avian origin
What are well known betacoronaviruses?
SARS-CoV, SARS-CoV-2, MERS-CoV
Before the 21st century, what were coronaviruses known to cause?
Mild upper respiratory tract infections: HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU
In the 21st century, what were coronaviruses known to cause?
Severe lower respiratory tract infections: SARS-CoV, MERS-CoV, SARS-CoV-2
When did SARS-CoV emerge?
2002, outbreak controlled in 2003
High case mortality rate but lower transmissibility
When did MERS-CoV emerge?
Emerged in 2012 from camels, now only sporadic cases
When did SARS-CoV-2 emerge?
2019, became global pandemic
What is the structure of coronavirus?
Enveloped spherical particle (125 nm diameter) with crown-like surface proteins
How long is the coronavirus genome and how many open reading frames are there?
30kb +ve sense ssRNA (longest viral RNA genome)
13 ORFs
What is one of the features that allows coronaviruses to adapt so well?
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)
What can the coronavirus genome acts as mRNA for?
The translation of ORF1a and ORF1b (but not the ones at the 3’ end)
What happens in the coronavirus replication cycle?
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
How does SARS-CoV attach and enter an endosome?
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
Where are angiotensin-converting enzyme 2 (ACE2) receptors present?
In a lot of organs (resulting in the widespread issue caused by coronavirus infections)
What are the two domains of the spike protein?
S1 interacts with the receptor
S2 contains a fusion peptide
Fusion requires protease cleavage between S1 and S2 domains to release fusion peptide
What are the S proteins that make up the spike protein?
S protein is class I viral membrane fusion protein (like influenza HA)
What is the difference between coronavirus spike protein cleavage and influenza HA?
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)
What proteases release the fusion peptide from the S2 domain?
Proteases in the cell membrane (e.g. TMPRSS2) and within the endosome (e.g. cathepsins)
What does insertion of the fusion peptide into the endosomal membrane lead to?
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
In SARS-CoV-2, what contributes to its high infectivity?
Second cleavage site (for furin) that allows pre-activation step
In SARS-CoV-2, what does cleavage of the spike protein by furin do?
Makes the spike protein a lot more accessible for the cleavage that releases the fusion peptide (makes S2 more accessible)
What is the receptor binding domain (RBD) of S1 and what does it do?
Has two conformations
Lying down for immune evasion
Standing up for receptor binding
How is SARS-CoV-2 more infective than SARS-CoV?
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
How is the genomic RNA of coronaviruses translated?
+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
How do coronaviruses utilise -1 ribosome frameshifting?
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)
What happens to Pp1a and pp1ab after replication?
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
What is included in the transcriptase/replicase complex in coronaviruses?
The polymerase, RNA helicase, capping enzyme (can make capped mRNAs), exoribonuclease (proofreading)
What is genomic RNA of coronaviruses used a template for?
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
What is discontinuous transcription?
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)
What are -ve sense gRNA (genomic RNA) and -ve sense sgRNAs (sub-genomic RNA) templates for?
Many copies of new positive sense +ve sense gRNA and +ve sense sgRNAs
What do all subgenomic RNAs contain?
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
What do the structural proteins encoded by sgRNAs include?
S (spike), M (matrix), E (envelope) and N (nucleocapsid)
What are the S, M and E proteins co-translationally inserted into?
ER membranes
Where are the S and M proteins highly glycosylated?
In the ER and then the Golgi
How is the nucleocapsid (N) protein translated?
N is translated on free ribosomes and binds to new genomic RNA as it is synthesised
Where does coronavirus particle assembly take place?
Occurs at ER-Golgi intermediate compartment (ERGIC) membranes
What drives coronavirus particle assembly?
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
How are newly formed coronavirus particles released?
New particles bud into the lumen of the ER-Golgi intermediate compartment (ERGIC)
Transported out of cell by exocytosis in cargo vesicles
