Nidovirales Flashcards
Order Nidovirales
-> Nidus (latin) = nest
Arteriviridae:
13-16 kb RNA genome
e.g. Equine Arteritis Virus
Coronaviridae:
26-32 kb RNA genome
- Torovirus
- Coronavirus
e.g. - SARS Coronavirus
- Human CV 229E
- Mouse Hepatitis Virus
Virus particles of Coronaviridae members
- 100 to 120 nm diameter
- Lipid envelope
- Envelope proteins:
- S (spike): receptor binding, membrane fusion
- HE: Hemagglutinin-Acetyl-Esterase (not SARS-CoV)
- E (small envelope protein): critical for budding
- M (membrane): interacts with N-protein -> contact to nucleocapsid
- Nukleocapsid: Genome + N-protein = more like RNP of negative strand RNA viruses?
- No ikosahedral internal structure in the virion
Receptor usage of Coronaviruses
Mouse Hepatitis Virus (MHV), uses CEACAM1a.
Deletion of the gene makes mice resistant to infection!
SARS CoV-1 und -2: Angiotensin Converting Enzyme 2 (ACE2)
HCoV-229E: Amino Peptidase N (APN)
Receptor can determine host tropism
(comp. poliovirus)
Corona Virus Genome
- (+) strand RNA genome with 5 ́cap and 3 ́poly A
- 5 ́ and 3 ́untranslated regions (UTRs)
- 9-14 open reading frames (ORFs)
- ORF1 encodes a polyprotein which is proteolytically processed
- translation of ORF1a stops at leaky stop codon; read through in 25% of cases
‘slippery’ heptanucleotide sequence and pseudoknot; ribosome makes −1 frameshift.
(Exact balance: deregulation severely hampers virus replication)
Corona Virus Replication
- first step: translation of incoming genome; assembly of replicase
- synthesis of a complete (-)-strand, complementary to the (+)-strand genome
- followed by the synthesis of many (+)-strand genomes on (-)-strand template
Corona Virus Transcription
- a „nested set“ of 3 ́co-terminal mRNAs with identical 5 ́sequence is generated
- capping by viral enzymes
Special mechanism:
Discontinous transcription (leader primed) mechanism:
1. negative strand synthesis starts at 3 ́end of genome
2. stop at internal genomic sequence, the transcription-regulating sequence (TRS)
transfer of this (-)-strand RNA to the TRS at the 5 ́end of the (+)-genome RNA via base pairing between TRS and leader TRS sequence
4. restart of RNA synthesis and completion of (-)-strand synthesis; 3 ́ends of all negative strands identical = leader sequence
5. subgenomic (sg) negative strand RNA serves as template for synthesis of mRNA
One single negative strand RNA serves as template for synthesis of many mRNA molecules!
Corona Virus Translation
- from each mRNA only the 5 ́proximal ORF is translated
ORF 1ab region: processing by virus-encoded proteases = protease cleavage site
3CLpro (also called Mpro or main protease)
Four structural proteins: S, E, M and nucleocapsid (N) proteins
Subset of group 2 coronaviruses encode hemagglutinin-esterase (HE) - HE is structurally similar to HA of influenza virus! Ancient recombination event?
Structural Basis for Helices-Polymerase-Coupling in the SARS-CoV-2 Replication-Transcription Complex
SARS-CoV-2 is the causative agent of the 2019–2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp8/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain (NiRAN*), detailing a new pocket for anti-viral therapy development.
SARS-CoV-2
The genome of SARS-CoV-2 encodes 16 non-structural proteins (nsp1-nsp16)
to assemble the Replication-Transcription Complex (RTC) that plays key roles in the replication of genome-length viral RNAs and in the transcription of viral mRNAs.
CoV mRNAs bear a 5’ cap structure (7MeGpppA2’OMe).
In SARS-CoV-2 RTC, co-transcriptional capping of mRNA occurs after elongation initiation by four sequential actions in the Co-transcriptional capping complex(s) (CCC).
The first and the second capping actions that sequentially generate the 5’-diphosphate end (ppA) and the cap core (GpppA) at the 5’ end of the nascent pre-mRNA are mediated by nsp13 and the nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain in nsp12.
In the subsequent capping actions, an N7-methyltransferase (N7-MTase) in nsp14 methylates the first guanine of GpppA at the N7-position to produce the
cap(0) (7MeGpppA), being the substrate for the final capping action
facilitated by a 2’-O-methyltransferase (2’-O-MTase) in nsp16 to yield the mature mRNA.
The mechanism of RNA capping by SARS-CoV-2
-> Compare: Canonical eukaryotic capping mechanism
1) RNA Triphosphatase (RTPase)
2) Guanyltransferase (GTase)
3) N7-MTase
4) 2’-O-MTase
The mechanism of RNA capping by SARS-CoV-2
First step: RNAylation of nsp9 with viral RNA by NiRAN domain of nsp12 (RdRp)
Second step: nsp9 covalently bound to viral RNA is replaced by GDP (Comparison: Capping by Vesicular stomatitis virus)
PRNTase: GDP-polyribonucleotidyltransferase (removes one gP from P-P-P-RNA)
A proofreading function for RNA virus replication?
Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing
- Nsp14 has 3 ́ -> 5 ́exonuclease activity (ExoN)
- inactivation by mutation still allows viral replication (not essential) .
- MHV: 15-fold decrease in replication fidelity (15x more mutations)
- SARS: 21-fold increase in mutation rate
Method: -10 independent cell cultures infected with wt virus or mutant
- passage and continuous sequencing of whole genomes
Deep sequencing technique
- “454 Sequencing”: primers with biotin tag are used to generate cDNA/ or DNA fragm.
- fixation of each DNA to one streptavidin bead; each sorted into a well of a plate
- PCR amplification; addition of a nucleotide by PCR leads to ligth emission
(pyrosequencing); detection for each well; millions of sequences in parallel - ability to sequence 400-600 million bp per run with 1000 bp read lengths
A live, impaired-fidelity coronavirus vaccine protects in an aged immunocompromised mouse model of lethal disease
“Coronavirus (CoV) replication fidelity is approximately 20-fold greater than that of other RNA viruses and is mediated by a 3’→5’ exonuclease (ExoN) activity that probably functions in RNA proofreading. In this study we demonstrate that engineered inactivation of severe acute respiratory syndrome (SARS)-CoV ExoN activity results in a stable mutator phenotype with profoundly decreased fidelity in vivo and attenuation of pathogenesis in young, aged and immunocompromised mice. The ExoN inactivation genotype and mutator phenotype are stable and do not revert to virulence, even after serial passage or long-term persistent infection in vivo. ExoN inactivation has potential for broad applications in the stable attenuation of CoVs…”
Anti-host function of CoV nsp1
nsp1 of mouse hepatitis virus (MHV):
- is critical for blocking the IFN response of the host
nsp1 of SARS coronavirus:
- binds to 40S ribosomal subunit
- stops translation of cellular mRNA (non-specific, includes IFN mRNAs)
- modifies 5 ́end of mRNA to render it translationally incompetent
- later discovered: induces endonucleolytic cleavage of mRNA near to 5 ́end nsp1 is most likely no nuclease itself -> host nuclease
- leader sequence in SARS coronavirus mRNAs protects against nsp1 mediated degradation (mechanism?)
Compare: nonsense mediated RNA decay detects stalled ribosomes at mRNAs with premature stop codons; degradation by Xrn1
