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COVID-19 Research - ORF3a Is Not A Viroporin  Jan 27, 2023  1 year, 3 weeks, 4 days, 1 hour, 33 minutes ago

BREAKING! COVID-19 Research: U.S. NIH Funded Study Discovers That SARS-CoV-2 ORF3A Is Not An Ion Channel But Does Interact With Trafficking Proteins.

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BREAKING! COVID-19 Research: U.S. NIH Funded Study Discovers That SARS-CoV-2 ORF3A Is Not An Ion Channel But Does Interact With Trafficking Proteins.
COVID-19 Research - ORF3a Is Not A Viroporin  Jan 27, 2023  1 year, 3 weeks, 4 days, 1 hour, 33 minutes ago
COVID-19 Research: A new study that was funded by the U.S. NIH has found that despite earlier claims by many, the SARS-CoV-2 accessory protein ORF3A is not an ion channel but does interact with trafficking proteins.


 
Typically, the SARS-CoV-2 and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus.
 
However certain past COVID-19 Research reports have led to annotation of both Orf3a proteins as viroporins.

https://www.thailandmedical.news/news/covid-19-immunology-study-shows-that-sars-cov-2-viral-protein-orf3a-activates-nlrp3-inflammasome-causing-severe-inflammatory-responses
 
https://www.thailandmedical.news/news/covid-19-mutations-study-that-tracks-mutation-patterns-of-sars-cov-2-warns-of-highly-possible-changes-on-nucleocapsid-and-3a-viroporin-proteins
 
The study team showed that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductance measured are common contaminants in overexpression and with high levels of protein in reconstitution studies.
 
Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively-charged aqueous vestibule, which would not favor cation permeation.
 
The study team observed enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39.
 
Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39.
 
In order to explain this difference, the study team found that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes.
 
The study findings suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.
 
The study findings were published in the peer reviewed journal: ELife.
https://elifesciences.org/articles/84477
 
The study team comprised of scientists from Janelia Research Campus, Ashburn-USA, Universidad Austral de Chile, Valdivia-Chile, University of Pennsylvania-USA and the United States National Institute of Child Health and Human Development, Bethesda-USA.
 
The study findings have numerous implications as it now also invalids certain claims about possible drugs for COVID-19 treatments etc.
ors-including-amlodipine,-nifedipine,-felodipine-and-the-phytochemical-neferine-can-treat-covid-19-infecti">https://www.thailandmedical.news/news/austrian-study-shows-calcium-channel-inhibitors-including-amlodipine,-nifedipine,-felodipine-and-the-phytochemical-neferine-can-treat-covid-19-infecti
 
Typically, viroporins are small viral membrane proteins that often form weakly‐ or non‐selective pores. They are widely distributed among different viral families and, accordingly, their contributions to viral survival range widely from assisting with viral propagation to participating in host immune evasion.
 
Four proteins encoded by SARS‐CoV‐1 have been proposed to function as viroporins ‐ E protein, Orf3a, Orf8a and Orf10.
 
In order to demonstrate that a novel protein unequivocally forms a viroporin requires the identification of its pore‐lining residues. This is ideally done by introducing a pore‐lining cysteine that can be modified by a methanethiosulfonate reagent, which when added, partially blocks the pore or alters ion channel selectivity.
 
However, this has yet to be demonstrated for SARS‐CoV‐1 or SARS‐CoV‐2 Orf3a.
 
The study team asked if Orf3a from SARS‐CoV‐2 is a bona fide viroporin and took a comprehensive approach to investigate its function in several mammalian cell lines.
 
The study team first assessed sub‐cellular localization of SARS‐CoV‐2 Orf3a to determine where it may be functioning in mammalian cells and observed its enrichment at the PM and in the endocytic pathway.
 
They then performed whole‐cell patch‐clamp in HEK293 and A549 cells, and endo‐lysosomal patch‐clamp from HEK293 cells, in numerous cationic conditions. 
 
They also tested SARS‐CoV‐2 Orf3a at the PM of Xenopus oocytes and in reconstituted systems using purified SARS‐CoV‐2 Orf3a.
 
None of the findings suggested that SARS‐CoV‐2 Orf3a was indeed a viroporin.
 
The study team observed an enrichment of Rab7 puncta in cells overexpressing SARS‐CoV‐2 Orf3aHALO, but not SARS‐CoV‐1 Orf3aHALO.
 
Consistent with this, VSP39 selectively interacts with SARS‐CoV‐2 Orf3a, and not SARS‐CoV‐1 Orf3a. 
 
By generating Orf3a chimeras (LC) the study team showed that an unstructured loop of SARS‐CoV‐2 Orf3a mediates binding to VPS39.
 
Though SARS‐CoV‐1 Orf3a LC displays enhanced interaction with VPS39, it is not fully restored, and overexpression of the chimera is not sufficient to generate the Rab7 enrichment phenotype. 
 
The study findings suggests that the VPS39 interaction with Orf3a was acquired in SARS‐CoV‐2 and, by sequence and structural comparison between SARS‐CoV‐1 and SARS‐CoV‐2 Orf3a, and that the study findings have likely identified SARS‐CoV‐2 Orf3a’s novel region of interaction.
 
The study findings propose that the unstructured loop, W193 substitution, and the neutral charge in this region contributes to promoting the SARS‐CoV‐2 Orf3a and VPS39 interaction. 
 
However, the question remains, how does the acquired interaction between SARS‐CoV‐2 Orf3a and VPS39 contribute to viral pathogenesis?
 
Numerous studies have implicated the endocytic pathway as the primary mode of viral egress for several beta‐coronaviruses, including SARS‐CoV‐2, and that Orf3a may mediate this process.
https://pubmed.ncbi.nlm.nih.gov/33157038/
 
https://pubmed.ncbi.nlm.nih.gov/34706264/
 
A possibility is that the SARS‐CoV‐2 Orf3a and VPS39 interaction promotes viral exit by perturbing forward endocytic trafficking through disrupting the HOPS complex‐mediated fusion of LE with Lyso.
 
This could be due to 1) Orf3a sequestering VPS39 from the HOPS complex or, 2) Orf3a interacting with the HOPS complex through VPS39, an important distinction that has yet to be elucidated.
 
A second, related possibility is that the SARS‐CoV‐2 Orf3a and VPS39 interaction promotes viral egress by interacting with known membrane tethering, fusion and trafficking complexes that facilitate lysosome movement to the cell periphery.
 
In particular, VPS39 knockdown in SARS‐CoV‐2 Orf3a overexpressing cells reduces the localization of LAMP1 vesicles near the PM, and concomitantly diminishes recruitment of protein complexes involved in Lyso‐PM trafficking.
 
The study findings suggest that VPS39 is necessary for SARS‐CoV‐2 Orf3a‐mediated Lyso‐PM trafficking and implicates the recruitment of the HOPS complex by Orf3a in this process.
 
Another possibility is that the SARS‐CoV‐2 Orf3a and VPS39 interaction prevents HOPS‐mediated AP‐Lyso fusion, which the study team and other researchers have observed.
https://pubmed.ncbi.nlm.nih.gov/33422265/
 
https://pubmed.ncbi.nlm.nih.gov/34386498/
 
Significantly, autophagy is an intracellular surveillance process that targets damaged cellular or foreign materials, such as viruses, for lysosomal degradation. Many viruses hijack host cell autophagy to prevent its degradation and promote survival.
 
Overall, the acquired SARS‐CoV‐2 Orf3a and VPS39 interaction may function to assist with SARS‐CoV‐2 exit and host intracellular immune evasion. The molecular details of the host cell response to SARS‐CoV‐2 Orf3a need to be examined during viral infection to fully elucidate the contributions of Orf3a to SARS‐CoV‐2 cell physiology and pathogenesis.
 
For the latest COVID-19 Research, keep on logging to Thailand Medical News.

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