There have been many outbreaks of major zoonotic CoV diseases in the 21st century: SARS-CoV in 2002/3, the ongoing MERS-CoV that begun in 2012, and the current worldwide SARS-CoV-2 pandemic. Currently there are no approved drugs for these viruses. CoVs has a nsp14, bifunctional enzyme with an amino terminal domain coding for proofreading exoribonuclease and N7-Methyltransferase domain able to methylate the viral RNA cap . The exoribonuclease function of ExoN 3' to 5' is a central player in a variety of essential life-cycle processes for coronaviruses and has made many previously active antiviral compounds ineffective against CoVs. the most recent SARSr-CoV outbreak, has emerged at a time when post-genomics technologies are increasingly evolving in molecular biology in terms of sophistication and target specificity. ExoN is a logical therapeutic target for new technologies in genomics
2. Outline:
1- Introduction
2- the coronavirus genome organization
3- Coronavirus Proofreading Mechanism
4- The Bifunctional nonstructural Protein 14
5- The role of nsp14 in RNA recombination
6- Nsp 14 potential as a drug target
7- Conclusion
3. Introduction:
• Coronaviruses are enveloped, single-stranded
positive-sense RNA viruses with some of the
largest identified RNA viral genomes about 30 kb
• Severe Acute Respiratory Syndrome (SARS‐CoV),
Middle East Respiratory Syndrome (MERS‐CoV),
Severe Acute Respiratory Syndrome -2 (SARS-
2‐CoV). belongs to the genus Betacoronavirus .
• All three of these viruses considered to be
transmitted from animals to humans (zoonotic
viruses)
• coronaviruse can cause respiratory infection in
human
4. coronavirus genome organization:
• The polycistronic RNA genome of SARS-CoV is
approximately 30 kb and encodes 14 open reading
frames (ORFs), some of which overlap.
• It is capped at the 5′ end and has a 3′ poly(A) tail.
• Two-thirds of the genome encodes for 16 non-
structural proteins.
• The last one-third of the genome encodes structural
and accessory proteins (spike, envelope, membrane,
• and nucleocapsid)
6. Coronavirus Proofreading Mechanism
• Usually, replication of RNA virus has a high error rate, due
to it polymerase lacks the proofreading ability found in
DNA virus polymerase.
• The distinctive 3′ to 5′ exoribonuclease (ExoN)
proofreading feature which provided by non-structural
protein 14 is obstacle to the production of nucleoside
analogs (NAs) as antivirals against CoVs.
• ExoN function is important for preservation of broad
genome of coronaviruses
7. The Bifunctional nsp14 Protein
• Nsp14 is a bifunctional enzyme with
an N-terminal ExoN domain
implicated in RNA proofreading and a
C-terminal N7-methyltransferase
domain involved in mRNA capping.
• Nsp14 is also involved in several
other processes of the virus life cycle
and pathogenicity including :
innate immune responses
viral genome recombination
Figure 2. The Overall Structure Nonstructural
protein 14
8. Exoribonuclease Domain
• The nsp14 exoribonuclease activity resides in
the N terminus of this bifunctional protein
• The Exon domain , correct errors made by
the RNA polymerase by removing
mismatched nucleotides from the 3′ end of
the growing RNA strand
• Exon has two zinc fingers (ZF1 and ZF2) that
are essential for Exon activity and structural
stability. Also Exon activity depend on metal,
namely (Mg+2 )
• Nsp10 as cofactor for nsp14, improve Exon
activity and stability
Figure 3. Structure model of SARS-CoV
nsp10/nsp14 complex
9. The role of nsp14 in RNA recombination
• Recombination in RNA viruses involves the exchange of genetic
information between two RNA genomes.
• Recombination is proposed to be critical for coronavirus (CoV)
diversity and emergence of SARS-CoV-2 and other zoonotic CoVs
• Studies show that SARS-CoV-2 has acquired the capacity to infect
human cells by recombining within the spike protein series.
• CoV recombination, resulting in multiple CoVs vaccine failure.
• Nsp14-ExoN is required for recombination, and that inactivation of
ExoN results in decreased recombination frequency.
10. Nsp 14 potential as a drug target
• the multiple critical functions of nsp14, further defines nsp14 as
highly selective target for antiviral treatment inhibition.
• While inhibitors of ExoN alone might be effective , Combining
nsp 14 inhibitor with an RNA mutagen would become more
effective and drive high-level mutagenesis.
• since beginning of the COVID-19 t, the class of antivirals that
are RNA polymerase inhibitors such as (Remdesivir), have
been of high importance
• These drugs, which are nucleoside analogs, had a strong effect
on viruses that do not have proofreading enzymes
11. Nsp 14 potential as a drug target
• In SARS-CoV-2 Due to its 3'-5 'exoribonuclease proofreading operation,
ExoN is capable of excising integrated nucleoside analogs
• This results in the negation of the function of these medicines.
• There is currently no available drug for inhibiting nsp14.
• molecular docking studies to find small molecules/peptides/natural
molecules that have the potential to inhibit nsp14 ExoN, are required
urgently.
12. Figure 3. Schematic describing ExoN proofreading activity and mode of
action of inhibitors.
13. Conclusion
• There have been many outbreaks of major zoonotic CoV diseases in the 21st century:
SARS-CoV in 2002/3 ,MERS-CoV that begun in 2012, and the current worldwide SARS-
CoV-2 pandemic.
• Currently there are no approved drugs for these viruses.
• The exoribonuclease function of ExoN 3' to 5' is a central player in a variety of essential
life-cycle processes
• nsp14, bifunctional enzyme with an amino terminal domain coding for proofreading
exoribonuclease.
• has made many previously active antiviral compounds ineffective against CoVs.
• . ExoN is a logical therapeutic target for new technologies in genomics.
14.
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