Lecture 5
• Coronaviruses (+ sense RNA viruses)
• Genome
• Genes and proteins
• Entry by fusion
• Synthesis of viral proteins
• Genome replication
• Assembly and release
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Coronaviruses
• Spherical enveloped particles studded with trimeric spikes (diameter ~120-160 nm) (corona or
crown)
• Mostly cause respiratory illnesses in humans (e.g. SARS-CoV2) but in rare cases can cause
gastroenteritis (inflammation of GI tract) and encephalitis (inflammation of the brain)
• Also cause important veterinary diseases such as feline peritonitis (inflammation of abdominal linings),
infectious bronchitis (inflammation of bronchi), murine hepatitis etc.
SARS-CoV2 structure
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SARS-CoV2 EM image
Coronaviruses
• Family Coronaviridae: from Latin (crown)
• Three genera based on genome homologies: Alpha-, beta- and gammacoronaviruses
• Betacoronaviruses were causative
agents for 3 deadly outbreaks in
humans (within last 20 years)
• SARS-CoV in 2002 → SARS
disease
• MERS-CoV in 2012 → MERS
disease
• SARS-CoV-2 in 2019 → COVID19 disease
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Coronavirus Genome
• Linear, single-stranded positive sense RNA genome
• Largest known RNA genomes (27-32 kb) of any known virus families
• Introduction of purified genome RNA into a cell is enough to initiate infection and result in
production of new virus particles → infectious genomes!
• The order of genes is highly conserved among all coronaviruses
• Genome ends have a 5’ terminal cap (methylguanosine) and a 3’ poly(A) tail
31.5 kb murine
hepatitis virus
genome (a type of
coronavirus)
What is this similar to?
Fig 14.1
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Coronavirus Genome
• First gene from 5’ end occupies 2/3rd of the entire genome → translated into polyprotein –
precursor protein that is cleaved to form mature functional protein
• Cleaved by viral proteinases into multiple (~14-16 or more) different functional proteins
→ involved in genome replication, subgenomic mRNA synthesis
• Final 1/3rd generally encodes for virion structural and non-structural proteins (nsps)
• Hemagglutinin-esterase (HE; not present in all coronaviruses)
• Spike (S), small envelope protein (E), membrane protein (M), nucleocapsid protein (N)
1-16 indicate nsps
Fig 14.1
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Coronavirus Genome
• Example: Genome features of SARS-CoV2
See: https://viralzone.expasy.org/9076
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Coronaviruses – Genes and Proteins
•
Coronaviruses have enveloped virions containing
helical nucleocapsids
•
Nucleocapsids formed from viral N protein bound to
viral RNA in a helical fashion
EM of murine hepatitis virus
Fig 14.2
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Coronaviruses – Genes and Proteins
•
Nucleocapsids formed from viral N protein bound to
viral RNA in a helical fashion
•
Most other ‘+’ strand RNA viruses
nucleocapsids with icosahedral symmetry
•
Coronaviruses resemble ‘–’ strand RNA viruses
with helical nucleocapsids
have
•
Some coronaviruses may have a spherical core structure
(shell) formed via the M protein → spherical core with
internal helical nucleocapsid
•
Coronavirus virions
proteins (E)
contain
multiple
EM of murine hepatitis virus
envelope
Fig 14.2
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Coronaviruses – Spike Protein
• Coronavirus virions contain multiple envelope proteins:
spike (S), membrane (M), envelope (E), nucleocapsid (N)
and in some cases HE (hemagglutinin-esterase) proteins
• Spike Protein (S):
• Major surface transmembrane
protruding from the surface
glycoprotein
Fig 14.2
• Responsible for viral entry and tropism
• Targeted by neutralizing antibodies and T-Cells in
infection → possible vaccine / therapeutic target
• Generally forms trimers
3D model of SARS-CoV2
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Coronaviruses – Spike Protein
• Spike Protein (S):
• Synthesized as a single polypeptide chain → cleaved by a
cellular proteinase to yield an N-terminal S1 domain (or
subunit) and a C-terminal S2 domain (subunit)
RBD
• S1 recognizes specific cellular receptors and initiates
attachment
• Receptor Binding Domain (RBD) is present within
S1 for most coronaviruses
• S2 forms the stalk with a short C-terminal tail, a
hydrophobic transmembrane domain and exterior domain
of interacting alpha-helices
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Coronaviruses – Spike Protein
• SARS-CoV-2 Spike protein structure:
RBD
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Coronaviruses – Spike Protein
• Spike proteins bind to a variety of cellular receptors:
• Alphacoronaviruses bind to aminopeptidase-N (aka Alanyl
aminopeptidase),
a
family
of
zinc-binding
metalloproteinases (use metal ions in catalytic mechanisms)
present on cell surface:
• Broadly distributed on epithelial and fibroblast cells in
small intestine, kidneys (renal epithelium), CNS etc.
• Species-specific
• CoVs that have HE can bind to sialic acid (9 carbon sugar),
found on a variety of glycoproteins and glycolipids
Sialic acid structure
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Coronaviruses – Spike Protein
• Spike proteins bind to a variety of cellular receptors:
• Betacoronaviruses use a variety of receptors:
• SARS-CoV binds to metalloproteinase Angiotensinconverting enzyme (ACE2) and a co-receptor, LSIGN (a lectin – carbohydrate binding proteins that
recognize specific sugar motifs)
• Transmembrane Serine Protease 2 (TMPRSS2), a
cellular serine protease for host-cell entry → cleaves at
S2 to activate viral fusion
• SARS-CoV-2 also binds ACE2 and possibly other
receptors
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Coronaviruses – Entry by Fusion
• Spike protein generally mediates entry via fusion
• External S1 subunit mediates attachment
• Stalk subunit S2 (a class I fusion protein) facilitates fusion
• Series of conformational changes → insertion of S2 into target cell membrane → brings
cell membrane and viral envelope into close contact
• Some CoV S proteins can also cause formation of syncytia
• In some cases, fusion can be pH dependent (e.g., fusion induced at low pH →
indicates entry via endosomes, followed by fusion with endosomal membranes)
• Fusion at plasma membrane is also required for egress from cells
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Coronaviruses – SARS-CoV2 Entry
• Example: SARS-CoV2 can enter via endosomal or non-endosomal (fusion) pathway
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Coronaviruses – SARS-CoV2 Entry
• In SARS-CoV2, spike protein cleavage by TMPRSS2 (tempress 2; on cell surface) or
cathepsin L (endosomal route) is required:
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Coronaviruses – Synthesis of Viral Proteins
• First step after entry is synthesis of viral proteins that organize and catalyze viral RNA
synthesis
• The replicase gene (Gene 1) is translated from genomic RNA into a polyprotein that is
processed by viral proteinases
• Gene 1 is comprised of ORF1a and ORF1b (partially overlapping reading frames)
• Translation starts at the start codon (AUG) → progresses 5’ – 3’
Fig 14.1
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Coronaviruses – Synthesis of Viral Proteins
• Ribosomes translating ORF1a pause at a
pseudoknot and frameshift (ribosomal
frameshift) allowing translation of ORF1b →
yields two polyproteins, pp1a and pp1ab
• Pseudoknot: Secondary structures formed
in RNA via hydrogen bonds
• Ribosomal
frameshift:
Ribosome
‘slipping’ by one nucleotide in 5’ (-1 nt) or
3’ (+1 nt)
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Coronaviruses – Synthesis of Viral Proteins
• Example: Ribosomal frameshift in SARS-CoV2
Papain-like cysteine
proteinase
See: https://viralzone.expasy.org/9076
Chymotrypsin-like
cysteine proteinase
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Coronaviruses – Viral Proteins
• pp1a yields 10-11
mature
proteins
(including
viral
proteinases)
• pp1ab yields up to 16
mature proteins
• ORF1b is the most
conserved part of the
genome – What does
this tell you?
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Coronaviruses – Viral Proteins
• Virus proteinases carry
out proteolysis of the
pp1a and pp1ab.
• Papain-like cysteine
proteinases (PLP1
and PLP2 from
nsp3)
• Chymotrypsin-like
cysteine proteinase
(3CL
proteinase
from nsp5)
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Coronaviruses – Viral Proteins
• Various proteins needed
for RNA replication are all
encoded by the replicase
gene (Gene1, nsp 1-16):
• RNA-dependent RNA
polymerase (ORF1b,
nsp12)
• Another
noncanonical
RNA
polymerase
is
encoded by nsp8
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Coronaviruses – Viral Proteins
• RNA
helicase
and
nucleoside triphosphatase
activities
assist
with
replication and packaging
of the viral genome
(ORF1b, nsp 13)
• RNA
exonuclease
(ORF1b,
nsp14)
has
proofreading activity →
rare for RNA viruses
• Various nsps may be
involved in various roles /
activities (see next slide)
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Coronaviruses – Viral Proteins
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Coronaviruses – Viral Proteins
• Coronavirus proteins and functions:
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Coronaviruses – Viral Proteins
• Coronavirus proteins and functions:
Methylates the 5’ cap
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Coronaviruses – Replication Complexes
• Membrane association of viral RNA synthesis is common among ‘+’ strand RNA viruses of
eukaryotes (i.e. can be found in membrane-associated complexes in cytoplasm)
• Replication complexes are the site of viral RNA synthesis (RNA factories) → consists of viral
and cellular proteins associated with membranes in the cytoplasm of the infected cell
• Rearrangement of subcellular architecture (cytoplasmic membranes) induced upon CoV
infection
• Replication complexes commonly observed on double membrane vesicles by EM
Membrane structures induced by
SARS-CoV1
(a) 2 hours p.i. (b) 8h p.i.
Fig 14.3
M = mitochondria
N = nucleus
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Coronaviruses – Replication Complexes
• Replication complexes are the site of viral RNA synthesis (RNA factories) → consists of viral
and cellular proteins associated with membranes in the cytoplasm of the infected cell
Advantage?
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Coronaviruses – Replication Complexes
• The virus-induced membrane alterations have been termed a ‘reticulovesicular network’ of interacting
structures (e.g.; double-membrane vesicles (DMVs); convoluted membranes (CMs) and doublemembrane spherules (DMSs))
• Networks are formed by a combination of cell stimuli to produce new membrane and modification of
existing membranes by virus proteins (e.g., nsps 3,4,6 are integral membrane proteins → proposed to
initiate formation of replication complexes and induce membrane modifications)
• Nucleocapsid protein can also be found in abundance at these sites → encapsidation of newly
synthesized genome RNA can also occur at these sites
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Coronaviruses – Genome Replication
• Genome
replication
proceeds via a full-length
negative-strand
intermediate (antigenome)
→ used to direct synthesis
of full-lengths ‘+’ strand
genome
• Negative-strand
RNAs
account for 1-2% of total
viral RNA
• Most negative-strands are
associated with one or
more growing positivestrands in a replicative
intermediate
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Coronaviruses – Genome Replication
• Replicative intermediate: RNA molecule
on which one or several growing RNA
strands are being synthesized
• The growing strand typically forms
base-pairing to template RNA only near
their growing 3’ end.
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Coronaviruses – Subgenomic mRNAs
• The expression of all genes downstream of the replicase gene occurs from a series of
subgenomic mRNAs
• The subgenomic mRNAs all have identical “untranslated leader” (UTR) sequences at the 5’
end, poly(A) tails, and represent a nested set of mRNAs and all contain overlapping sequences
on their 3’ end.
• UTR is attached to a unique mRNA ‘body’ sequence with one or more ORFs e.g. genes 2
through 7 in murine hepatitis virus
Fig. 14.4 Nested CoV mRNAs
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Coronaviruses – Nested mRNAs in SARS-CoV2
• Example: Subgenomic RNAs in SARS-CoV2
See: https://viralzone.expasy.org/9076
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Coronaviruses – Discontinuous Transcription
• Subgenomic (sg) mRNAs are
transcribed from subgenomic
negative strand mRNA templates
• Negative strand mRNA
templates are made by
discontinuous
transcription
• Polymerase begins at 3’ end
and stops at the end of a
transcription-regulating
sequence (TRS)
L = leader, B = body
Template switching from body TRS to leader TRS
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Coronaviruses – Discontinuous Transcription
• RNA polymerase begins at 3’ end
and stops at the end of a
transcription-regulating sequence
(TRS)
Fig. 14.5 modified
• E.g., in murine hepatitis virus,
repeated short TRS are located
at 5’ ends of the genes 2-7 and at
the 3’ end of the leader sequence
in genome RNA
• Polymerase then pauses and
disassociates the nascent RNA chain
from TRS → jumps to TRS located
at the end of the leader sequence
(template switching)
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Coronaviruses – Discontinuous Transcription
• Dissociated nascent RNA chain
forms RNA-RNA hybrids of
complementary sequences at 5’
TRS
Fig. 14.5 modified
• RNA Pol can pause and
dissociate at any of the TRSs
• Each of the subgenomic ‘-’
strands are used to make a ‘+’
strand mRNA
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Coronaviruses – Discontinuous Transcription
• The discontinuous model could explain recombination between viral genomes
• Viral RNA polymerase can switch between two different positive-strand genome RNAs if
they are both in the same cell
• Could be two different virus strains infecting the same cell, or mutations in the virus
genome during replication
• Template switching may help with genome repair and/or generate new viral strains /
variants
• After dissociation, polymerase has to find another template or transcription will abort
→ can lead to template switching
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Coronaviruses – Discontinuous Transcription
• After dissociation, polymerase has to find another template or transcription will abort → can
lead to template switching
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Coronaviruses – Assembly and Release of Virions
• Assembly of virions takes place at intracellular membrane structures –
the endoplasmic reticulum-Golgi intermediate compartment (ERGIC):
interconnected system of membranes that lies between ER and Golgi
and is involved in transport, processing and modification of proteins.
Generally located in the perinuclear (around or near the nucleus) region
of the cell.
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1. Helical nucleocapsids (curved blue lines) containing genome RNA are
delivered from site of synthesis to these membranes for packaging
3
2. Virus particles are formed by budding into the lumen of these membranes
(virions acquire donut-shaped cores)
3. Progress to smaller and more uniformly dense cores as transit through
Golgi membranes, envelope proteins also undergo glycosylation
4. Secretory vesicles transport virions to cell surface, for fusion with plasma
membrane and release
2
1
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Coronaviruses – Assembly and Release of Virions
• M and E proteins play important roles in the formation of the virus envelops by budding
• Enveloped virus-like particles can be formed in ERGIC when only M and E are
expressed → What does this indicate?
• C-terminal cytoplasmic tail of M is thought to interact with packaging signals in N → ensures
only full-length viral RNA gets packaged into virions
• HE (if present) and S are incorporated into the membrane through interactions with the M
protein
• As they transverse through Golgi, envelope proteins are glycosylated
• Mature virions are packaged into vesicles → targeted to plasma membrane for release
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Coronaviruses – Assembly and Release of Virions
• Example: Replication cycle of SARS-CoV2.
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Coronaviruses – Evolution
• Bats are evolutionary pools (reservoirs) for new CoVs in mammals
• Subtle changes can alter tissue tropism, host range and pathogenicity
• Changes can occur by:
• Relatively high theoretical error rate of viral RNA polymerase (1 mutation per 10,000
nucleotides)
• RNA-RNA recombination
• Some mutations may enhance virulence or cross species barriers, others may make the virus
less effective
These kind of changes have a potential to cause future pandemics!
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Coming up….
• Positive strand RNA viruses (continued…)
• Picornaviruses
• Flaviviruses
• Togaviruses
• Quiz 4 – Available from Oct 1 – Oct 3 (10 PM)
• Covers lecture 3 (slide 31 onwards and lecture 4)
• Format similar to other quizzes
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