SARS-CoV-2 viroporin activity impacts viral infectivity and pathogenicity


Coronavirus disease (COVID-19), which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected more than 233 million people worldwide and claimed more than 4.7 million people. dead to date. While approved vaccines save lives, they are not permanent solutions to such a devastating pandemic.

Study of two SARS-CoV-2 viroporins – Orf3a and envelope protein (E)

Viroporins play a key role in the life cycle of SARS-CoV-2 and are one of the main determinants of viral pathogenesis. Specifically, SARS-CoV-2 viroporins are primarily responsible for ion channeling activity and participate in ion flow in the opposite direction of the host’s cellular response, as well as downstream disturbances. host cell signaling pathways.

Study: SARS-CoV-2 Orf3a viroporin activity and envelope protein impact viral pathogenicity. Image Credit: Volodymyr Dvornyk / Shutterstock.com

A team of Indian and French researchers recently studied the two main viroporins of SARS-CoV-2, namely Orf3a and the Envelope (E) protein, from a structural point of view. This study can be found on the bioRxiv* preprint server.

Orf3a shares a high level of sequence similarity with several mutation hotspots that have been identified in SARS-CoV-2 from SARS-CoV-1. While SARS-CoV-2 Orf3a is mainly localized in the endosomal-lysosomal membranes of this virus, it is rather often found in the Golgi apparatus of SARS-CoV-1.

Residue-forming SARS-CoV-2 Orf3a channel mutations promote formation of a critical inter-subunit channel which is not present in SARS-CoV-1 Orf3a. This improved structural feature correlates with higher channeling activity in SARS-CoV-2 compared to SARS-CoV-1.

The SARS-CoV-2 E protein is one of the most conserved proteins in the SARS-CoV proteome. Researchers in the present study found that water molecules formed electrostatic interaction networks with polar residues during the putative wetting condition and that there were no channels formed during the putative wetting condition. As a result of this aqueous medium, the cations move non-selectively and affect the ionic homeostasis of cell compartments. The resulting ionic imbalance induces an inflammatory response in host cells.

About the study

This study shows the mechanism of action of these two SARS-CoV-2 viroporins which could ultimately support the development of new antiviral drugs. Specifically, the researchers assessed the conservation among various Orf3a proteins of the coronavirus through the use of a
Protein-Protein Base Local Alignment Finder (BLAST). Homology modeling of the Orf3a and E proteins was also performed to obtain more information on the structural characteristics of these two viroporins.

Researchers were also interested in molecular dynamics simulations conducted through visual molecular dynamics (VMD) and nanoscale molecular dynamics (NAMD) to better understand the channeling activity of upper and inter-subunit channels. of the Orf3a protein of SARS-CoV-1. and SARS-CoV-2. A transcriptomic analysis of the proteins was also performed.

“We have elucidated the importance of the Orf3a and E proteins in the pathogenesis of COVID-19 on a structure-function association, potentially resulting in modifications of the transcriptome of the host cell. “

Study results

BLASTp search yielded 100 Orf3a sequences from various sources. Of these, 15 sequences ranging from SARS-CoV-1 and SARS-CoV-2 were analyzed. This experiment indicated that the Orf3a sequences of SARS-CoV-2 appeared to be more closely related to those from pangolins compared to human sources of SARS-CoV-1.

Compared to previous strains of SARS-CoV-1, SARS-CoV-2 has a relatively higher number of mutations in its genome and is a more contagious viral infection. In the present study, the researchers concluded that the increased pathogenicity of SARS-CoV-2 may be related to an increase in function in Orf3a compared to the characteristics observed from that of SARS-CoV-1.

Another new observation was the formation of hydrophilic pores through the inter-subunit channel of SARS-CoV-2 Orf3a. This formation could be correlated with the formation of ionic transfer media across membranes, which was not a phenomenon observed in SARS-CoV-1 Orf3a.

Overall, this finding suggests that there is a hydrophilic permeation pathway and ion channeling through SARS-CoV-2 Orf3a higher than that of the SARS-CoV-1 protein. Further analysis of the mutability of SARS-CoV-2 Orf3a demonstrated that the inter-subunit channel of this protein forms an important structure in its pore-forming regions.

The molecular dynamics simulation revealed that the protein-membrane system of the two Orf3a proteins has low structural variability in 5 nanoseconds (ns) of simulation. Comparatively, the same simulation on the SARS-CoV-2 E protein found high structural stability of the protein-membrane system during 5 ns of simulation.

The transcriptomic analysis performed by the researchers in the present study revealed that the upregulation of certain genes during SARS-CoV-2 infection is directly related to cellular processes that are impacted by the activity of channeling of ions. Specifically, significant upregulation of many immunomodulatory genes has been observed, including CD40, IFNL1, IFNL2, IFNL3, interleukin 12A (IL-12A), IL-33), IL-6 and NFkB1. This up-regulation appears to alter the host’s ability to defend itself against the virus, to participate in type II interferon (IFN) signaling, to regulate cytokine production, to respond to IFN- gamma and activate lymphocytes, to name a few.

Conclusion

The results of the present study corroborate previous findings on transcriptomic data from pulmonary alveolar cells infected with SARS-CoV-2, where inflammatory responses and molecular regulators directly affected by ion channeling were upregulated. These observations overlap with the upregulation of the transcript observed in diseases with pulmonary fibrosis, acute lung injury, and acute respiratory distress syndrome.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.


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