➡️ Cell reprogramming expressed through RNA-induced mutagenicity of #SARSCoV2 in infected cells confirmed.👇

1) SARS-CoV-2 is an enveloped beta-Coronavirus RNA belonging to the Coronaviridae family.

The genome is packaged inside a helical capsid formed by the
2) nucleocapsid (N) protein.

Three other structural proteins are associated with the viral envelope: membrane (M), envelope (E) and glycoprotein peak (S).

Cellular entry of SARS-CoV-2 depends on the binding of protein S to the angiotensin converting enzyme 2 (ACE2) which is a
3) specific cell receptor located on the surface of the host cell.

This receptor facilitates zoonotic transfer because these viruses can involve ACE2 from different animal species.

Beta-coronaviruses replicate in the cytoplasm; cellular compartments such as the
4)👉 endoplasmic reticulum (ER) and the endoplasmic reticulum-intermediate compartment of the 👉Golgi apparatus (ERGIC) undergo intense remodeling.

This implies the contribution of host membranes and organelles for viral replication.

Therefore, remodeling of
5) intracellular membranes due to coronavirus infection is also observed for many 👉RNA viruses.

➡️ After internalization and release of RNA, into the cytoplasm, a set of proteins is synthesized triggering the formation of vesicles which become a viral platform ensured
6) efficient 👉RNA replication and transcription.

The new coronavirus particles are assembled in the 👉endoplasmic reticulum and 👉Golgi complex.

Membrane budding between these compartments has been reported in association with 👉protein N and 👉genomic RNA
7) along with 👉proteins M, 👉E and 👉S.

Complete virions are sent to the extracellular environment following a conventional 👉"secretory pathway".

In this work, isolated SARS-CoV-2 was used (GenBank MT126808.1)

The virus was cultured in Vero cells👉 (Monkey African Green
8) kidney cell line) in the 👉Molecular Virology Laboratory, at the Federal University, of Rio de Janeiro, Brazil.

➡️ Vero cells were maintained in DMEM supplemented with 5% fetal bovine serum at 37 ° C and 5% CO 2 (all work involving infectious SARS-CoV2 was
9) performed in a biosafety level containment laboratory BSL3).

➡️Fig 1)
cell surface infected by mock at 48 h. (F) The adhesion of the virus to the cell surface and SP (arrowheads) became more evident with the MOI of 0.1 (F). Bars 200 nm.👇
➡️ Fig. 2)
Structure similar to a joint(B). Other viral particles were enveloped with thin (≃70nm) cell projections that resemble nanotubes(C). Membrane bridges connecting 2 cells showed the presence of viral particles,on their surface,that induce shape/direction (D, E).👇
11) Magnification of the "membrane bridges" connecting two cells.👇
12) Magnification increased by 100 times
👇
13) The abundance of SARS-CoV-2 particles retained on SP has recently been demonstrated and may facilitate the rate of viral propagation in the airway epithelium leading from the lumen of the upper respiratory tract because this environment is colonized by hair cells.
14) Coronavirus infection leads to massive remodeling of cell membranes; the proposed mechanism for virus export into the extracellular space is via fusion of the transport compartment membrane with the cell plasma membrane.
15) ➡️ Cell bridges containing viral particles👇
16) The viral particles adhered to cell surface protrusions which were shown to connect two cells.

This observation suggests viral 👉"cell surfing" previously described by other enveloped viruses such as HIV and human metapneumovirus.

It is assumed that this
17) mechanism allows the virus to penetrate in vivo into 👉mucosal surfaces showing cells rich in 👉microvilli.

👉Actin filaments play a vital role in 👉viral extrusion by the cell for both RNA and DNA viruses.

Actin offers the strength to discharge offspring virus particles
18) into the extracellular medium, as do some viruses that leave the cell and germinate, including the 👉Fowlpox and 👉West Nile viruses.

Previous studies have shown that the cytoskeleton network plays an important role in the maturation and possibly replication process
19) of SARS-CoV 2.

👉The communication between the two cells, in Fig. 2 (CD), suggests the presence of a thin filament (<0,7 µm) of F-actin containing👉 tunnel nanotube.

These 👉intercellular membranous connections can provide the 👉transfer of
20) molecular information, particularly viruses.

Similarly, viral cell navigation was shown on SARS-CoV-2 infection, which offers new insights into 👉cell-to-cell propagation and virus transmission.

➡️ Author: Lucio Ayres Caldas👇

nature.com/articles/s4159…
21) ➡️ The CK2, and N, co-localize virus-induced joint protrusions Phosphoproteomics data indicated the regulation of several kinases and effector proteins related to cytoskeletal organization following SARS-CoV-2 infection.

👉Kinases downstream of Rho /
22) Rac / Cdc42 GTPases (PAK1 / 2 and ROCK1 / 2) and several well characterized phosphorylation site targets of PAK1 / 2 kinase in vimentin (VIM S39 and S56) and stathmina (STMN1 S16 and S25) be downregulated during infection.

The interaction of Nsp7 with
23) RHOA (Gordon et al., 2020) may contribute to this downregulation. In contrast, signaling via CK2 is strongly upregulated, as determined by the increase in phosphorylation of well characterized target sites.

Among the many roles of this kinase, we noted increased
24) phosphorylation of cytoskeletal protein targets such as the ɑ-catenin (CTNNA1 S641) and the heavy chain of the motor protein Myosin IIa (MYH9 S1943). In addition to these kinase-mediated effects, the SARS-CoV-2 protein 👉Nsp2 also interacts directly with strumpellin
25) (WASHC5), a subunit of the WASH complex that induces actin assembly (Gordon et al., 2020), implying further regulation of the cytoskeleton during infection.

To investigate the relevance of these observations in a human infection model, high-resolution
26) immunofluorescence imaging of fixed Caco-2 human colonic epithelial cells was performed 24 hours after infection👉 (STAR methods).

2 infected with SARS-CoV-2 were imaged for filamentous actin and SARS-CoV-2 M protein, revealing prominent M protein clusters,
27) possibly marking assembled SARS-CoV-2 viral particles, located along trees and on the tips of actin-rich filopods.

👉SARS-CoV-2 infection induced a dramatic increase in filopo-protrusions, which were significantly longer and more branched than in uninfected cells.
28) The uninfected cells also exhibited filopodial protrusions, but their frequency and shape were markedly different.

The reorganization of the actin cytoskeleton is a common feature of many viral infections and is associated with different stages of the viral life cycle.
29) Containing viruses, could be important for SARS-CoV-2 release and / or cell-to-cell spread within epithelial monolayers.

Since Rho / PAK / ROCK signaling is downregulated, we then wondered if CK2 could play a role in this process.

At 24 hours, the infected cells
30) showed expression of CK2 along the thin filopodial protrusions, partially co-localized with the SARS-CoV-2 N protein.

Scanning and transmission electron microscopy was used to visualize the cell protrusions at higher resolution.

The assembled viral particles are
31) clearly visible along these filopods, with cases where the viral particles appear to sprout from the protrusions.

Finally, a global phosphoproteomic analysis of Vero E6 cells that overexpress the N protein was performed and we observed that CK2 activity was
32) significantly upregulated, since👉 CK2 activity can promote actin polymerization (D'Amore et al., 2019), it is consequential that 👉protein N can control 👉CK2 👉activity and regulate the organization of the cytoskeleton.👇
33) Scientists found that the pharmacological inhibitors of CK2, MAPK signaling p38, PIKFYVE and CDK, possess strong antiviral efficacy.

The cells were pretreated with inhibitory molecules, followed by SARS-CoV-2 infection (STAR methods) and the amount of virus
34) (anti-NP antibody against SARS-CoV-2) and cell viability were quantified 48 hours later. 'infection.

➡️ Silmitasertib, an inhibitor of CSNK2A1 and CSNK2A2, was found to possess antiviral activity (IC 50 = 2.34 μM.)

Along with data supporting
35) physical interaction (Gordon et al., 2020) and co-localization with the N protein, as well as a potential role in extracellular matrix remodeling in case of infection, CK2 signaling appears to be an important pathway hijacked by SARS-CoV-2.

Additionally, 👉silmitasertib is
36) currently being studied for human testing as a potential treatment for COVID-19.
37) Important link Paper:👇
doi.org/10.1016/j.cell…
38) Robert Grosse: 👇

Research continues in several directions to try to keep CK2 as small as possible, Grosse and his team are currently testing different methods.

Furthermore, the researchers want to inhibit other virus-activated enzymes that polymerize actin
39) fibers in the cell skeleton, i.e. separate them.

For years the protein actin, which constitutes an important part of the cell skeleton, has been researched.

He has also occasionally studied how viruses, particularly HIV and vaccinia, modify the cellular skeleton.
40) With SARS-CoV-2, Grosse suddenly had new research material of him, together with his doctoral student Svenja Ulferts, they started working, in March 2020, in the 👉BSL3 laboratory, at the University of Freiburg Medical Center.
41) ➡️ CONCLUSION: 👇

Scientists found that the on-off switches changed significantly in 40 of the 332 proteins interacting with the novel coronavirus.

The changes occur because the virus makes or composes 49 enzymes called kinases.

The upward or downward
42) composition of the kinases causes them to alter 40 of the proteins that interact with the virus. Imagine kinases as "guards" who protect our health until the new coronavirus turns them against us.

In any case, however, the new study has identified treatments
43) that can prevent the virus from turning guards into attackers.

The virus most potently hijacks a kinase called CK2, which plays a key role in the basic structure of the cell as well as in its growth, proliferation and death, leading scientists to study a drug called
44) Silmitasertib. Tests found that this drug inhibits CK2 and eliminates the novel coronavirus.
45)➡️ FINAL: 👇

It has been shown, from molecular microscopy studies, that RNA, expressed by SARS CoV 2 in the cell, induces 👉overexpression of the👉 N protein, which upregulates the activity of 👉CK2; since the activity of CK2 can promote the 👉polymerization of actin and
regulate both the👉 filopodic protusion (👉overwriting for the remodeling of the extracellular matrix), and the 👉organization of the cytoskeleton itself.
END.
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