The document discusses SARS-CoV-2 and its close relative RaTG13. It aims to evaluate if the nucleocapsid proteins of SARS-CoV-2 and RaTG13 can inhibit PKR- and RNase L-mediated antiviral pathways. The SARS-CoV-2 nucleocapsid protein was found to colocalize with dsRNA and inhibit phosphorylation of PKR in infected cells. Similarly, the nucleocapsid protein of RaTG13 was able to inhibit human PKR and RNase L, indicating an ability to counteract human innate immunity.

1. LUISA FERNANDA LÓPEZ SILVA
3rd SEMESTER
UNIVERSIDAD PONTIFICIA BOLIVARIANA

2. SARS-COV-2
Betacoronavirus with a single-stranded,
positive-sense, and 30-kb RNA genome
that encodes 4 structural proteins,
membrane, envelope, and nucleocapsid,
16 nonstructural proteins (NSP1 to -16), and
9 accessory proteins.
It is the cause of acute respiratory
syndrome, which is characterized
by its symptoms resembling flu.
Infection in humans is highly
transmissible, with a wide range of
severities ranging from asymptomatic
to lethal.
INTRODUCTION

3. WHAT DO THEY WANT TO DEMONSTRATE?
We know that the SARS-CoV-2 is capable
of inhibiting the antiviral activities
mediated by PKR and RNase L.
That the SARS-CoV-2 nucleocapsid (N)
protein is capable of binding to dsRNA
and phosphorylated PKR, inhibiting
both the PKR and OAS/ RNase L
pathways.
INTRODUCTION
And that the N protein of the bat
coronavirus RaTG13, the closest
relative of SARS-CoV-2, is also
capable of inhibiting human PKR and
RNase L indicating that the it has the
evasion measures to counteract human
innate immunity.

4. GENERAL OBJETIVE
To evaluate the nucleocapsid
proteins of SARS-CoV-2 and its
close relative bat coronavirus
RaTG13 are capable of inhibiting
PKR- and RNase L-Mediated
antiviral pathways.

5. WESTERN BLOTTING INMUNOPRECIPITATION
METHODS
It is a test that is classified as an
inmunoessay, all of which
evaluate the relationship
between an antigen and an
antibody. Proteins are
separated by electrophoresis
and then identified with a
specific antibody.
It was done to be able to
evaluate the proteins involved.
It's a technique that involves
precipitating the protein
antigen into a solution using an
antibody that binds specifically
to that particular protein.
Was used for the preparation
of the SARS-CoV-2 N and
mutant protein cell lysates,
A549/dko cells were infected
with the corresponding viruses

6. RNA ELECTROPHORESIS
CONSTRUCTION OF
RECOMBINANT VACV
METHODS
It is based on the mobilization
of molecules dissolved in an
electrolyte solution through a
gel by the action of electric
current.
In this study it was used to
extract RNA molecules.
The most common method used
to produce recombinant viruses
involves the insertion of foreign
genes into the thymidine kinase
(TK) gene of the VV via
homologous recombination.
They used VACVDE3DK3 as the
parental virus to construct
recombinant viruses expressing
SARS-CoV-2 nucleocapsid (N)
and N mutants.

7. The SARS-CoV-2 N protein
colocalized with dsRNA in the
infected cells.
RESULTS
Only a trace amount of
phosphorylated PKR (p-PKR)
was detected in the SARS-
CoV-2-infected cells.
It was confirmed the expression
of the SARS-CoV-2 N protein
using a FLAG Ab.

8. VACVDE3DK3 induced
significant PKR
phosphorylation.
RESULTS
PKR was phosphorylated to
similar degrees in
A549/RNaseLko cells infected
with the DLKR and DCTD viruses.
Immunoprecipitation of SARS-CoV
N and its domain mutant proteins.
Similarly, the LKR1CTD and CTD
coprecipitated with PKR from the
lysate of VACVDE3DK3-infected
cells but not in the lysate of
uninfected cells.

9. AUTOR WHAT DID THEY SAY? COINCIDENCE
Comar CE, Otter CJ,
Pfannenstiel J, Doerger
E, Renner DM, Tan LH,
Perlman S, Cohen NA,
Fehr AR, Weiss SR
In the case of MERS-CoV, however, only the
combinational deletion of ns15 and ns4a or -4b could
abolish the inhibition of dsRNA-induced antiviral
pathways, while the single inactivation of ns15 could not.
Dutta NK, Mazumdar K,
Gordy JT
The coronavirus N protein is the most abundant and
highly antigenic structural protein.
Sa Ribero M, Jouvenet
N, Dreux M, Nisole S
We found that N is a dsRNA binding protein capable of
inhibiting PKR- and OAS/RNase L mediated antiviral
activities. In addition, previous studies have shown that
the N protein has the capacity to inhibit IFN expression.
DISCUSSION

10. It can be concluded that we now
have the mechanism of action of
the N protein and we can start
the research and creation of
antivirals associated with it. And
all thanks to molecular biology,
science without which these
findings would not be possible,
which also contribute to public
health.
CONCLUSIONS
Molecular biology can also
concentrate on modifying the N
protein as a protective antigen, and
thus be able to develop N
protein-based vaccines which may
be more effective at docking the
disease.

11. CONCEPT MAP