Source: SARS-CoV-2 Viral Proteins   Nov 12, 2020  2 years ago
Canadian Study Shows SARS-CoV-2 Viral Proteins Disrupt Processing Bodies And Reshape Production Of Interferons And Proinflammatory Cytokines
Canadian Study Shows SARS-CoV-2 Viral Proteins Disrupt Processing Bodies And Reshape Production Of Interferons And Proinflammatory Cytokines
Source: SARS-CoV-2 Viral Proteins   Nov 12, 2020  2 years ago
SARS-CoV-2 Viral Proteins: Canadian scientist from the University of Calgary, University of Ottawa, University of British Columbia and Dalhousie University have in a new study found that coronaviruses’ viral proteins including those of the SARS-CoV-2 virus is able to disrupt so called processing bodies (PBs) in the human host resulting in the modifications of interferon and proinflammatory cytokine production.

The Coronaviridae are a family of viruses with large RNA genomes. Seven coronaviruses (CoVs) have been shown to infect humans, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease of 2019 (COVID-19). The host response to CoV infection is complex and regulated, in part, by intracellular antiviral signaling pathways triggered in the first cells that are infected.
New emerging evidence suggests that CoVs hijack these antiviral responses to reshape the production of interferons and proinflammatory cytokines.
Processing bodies (PBs) are membraneless ribonucleoprotein granules that mediate decay or translational suppression of cellular mRNAs; this is particularly relevant for proinflammatory cytokine mRNA which normally reside in PBs and are repressed. Recent evidence also suggests that PBs or their components play important direct-acting antiviral roles, providing a compelling reason for their frequent disassembly by many viruses. No information is known about how human CoVs impact PBs.
The study findings show that infection with the human CoV, OC43, causes PB disassembly. Moreover, the study teamed showed that several SARS-CoV-2 gene products also mediate PB loss and virus-induced PB loss correlates with elevated levels of proinflammatory cytokine mRNAs that would normally be repressed in PBs.
The study team also demonstrated that stimulating PB formation prior to OC43 infection restricts viral replication. These data suggest that SARS-CoV-2 and other CoVs disassemble PBs during infection to support viral replication and evade innate immune responses. As an unintended side effect, the disassembly of PBs enhances translation of proinflammatory cytokine mRNAs which normally reside in PBs, thereby reshaping the subsequent immune response.
The study findings were published on a preprint server but are currently being peer reviewed.
This new study describes a vital mechanism whereby the novel SARS-CoV-2 pathogen can restrict the human host response. In doing so, the study explores how this novel feature of SARS-CoV-2 works and how it results in a reshaping of the human host's immune response.
The study team describes processing bodies (PBs), which are aggregations of biomolecules that occur throughout the body. These are formed via a liquid-liquid phase separation of proteins that have regions of intrinsic disorder, as well as from interactions between RNA and proteins or between different RNA molecules. They are composed of enzymes that act on messenger RNA (mRNA), producing RNA turnover by various processes.
 < br /> Importantly the RNA in PBs is delivered there by RNA-binding proteins (RBPs). PBs are actually granules of ribonucleoprotein (RNP) and undergo constant fluctuations in size and number as they respond to various stimuli. For instance, anything that activates the p38/MK2 MAPK pathway causes disassembly of the PBs, as do viral infections.
Significantly, the RNA within PBs makes up about a third of all coding but imperfectly translated RNA, as well as noncoding RNA. Transcribed mRNA within the PBs consists mostly of regulatory mRNAs in groups related by function.
One such group includes growth factors, inflammatory cytokines and vascular growth factors. This group carries destabilizing AU-rich elements (AREs) at the 3'-untranslated region (UTR). This group was subject to a higher rate of degradation and suppression when PBs were visible, while constitutive expression of ARE-mRNA was restored with the loss of PBs. This is important in view of their physiological function.

Processing bodies are a nexus point for virus-host conflict. In part one of our model, we hypothesize that PBs are direct-acting antiviral granules that can restrict virus infection when present as visible condensates; for this reason, they are targeted for disassembly by most viruses. In part two of our model, we propose that viral PB disassembly is perceived by the cell as a danger signal and relieves suppressed cytokine transcripts to produce proinflammatory cytokines that recruit and activate immune cells. In this way, PB disassembly may also contribute to the pathogenic cytokine responses that underly many viral illnesses including COVID.
Hence the ARE-mRNA mechanism allows cells to respond to stimuli like viral infections that result in the PBs being lost, with the secretion of ARE-containing inflammatory cytokines at a high level, including IL-6, IL-8, IL-1β, and TNF.
Effect of viral proteins on PBs
It has been found that disassembly of PBs may be through the direct effect of a viral protein on a PB component, which then moves towards viral replication and transcription pathways within the cell, or is broken down by viral proteases. However, indirect disassembly can occur in response to viruses via p38/MK2 signals.
However the assembly of PBs by viral gene products is much less common. This may indicate that PBs are directly antiviral and that the tearing down of these particles is virus replication-friendly. This is in contrast to other RNPs like stress granules, which have a more indirect antiviral activity, mediated by the enzymatic activity of the PBs.
Also another indirect antiviral effect of PBs is by their stockpiling of proteins involved in innate immune signaling pathways. More studies will be needed to identify the importance of the organization of proteins within the PBs or the higher-order condensation of multiple proteins within a PB in modulating antiviral PB effects.
Although all coronaviruses are known for their ability to take over host interferon and antiviral responses, SARS-CoV-2 is exceptionally skillful at doing this. The interferon response's misdirection is turning out to be one of the most important processes that determine the clinical outcome of COVID-19.
Emerging evidence is piling up to show how this virus defeats the host's antiviral measures, such as four nonstructural proteins (NSPs) that have been recently reported to act upon specific cellular RNAs to limit IFN-β secretion and thus favor viral spread steeply.
This research is the first to show that human coronaviruses can disassemble PBs specifically. The viral RNA can translate to several independent proteins that can cause PB loss. The formation of PBs limits later infection with the seasonal coronavirus, OC43, in many ways, by hindering nucleocapsid protein production and preventing the production of infectious progeny. Thus, PBs are a significant component of antiviral cellular responses in coronavirus infection.
The study team screened SARS-CoV-2 genes for those which mediate the loss of PBs and found six genes that might cause PB loss. These included the nucleocapsid (N) protein, NSP1, which shuts off host immunity, NSP6 and NSP11, and the accessory proteins ORF7b and ORF3b.
The team found that two of these also markedly enhanced PB disassembly, namely, NSP1 and ORF7b. They then looked at the expression of these PB-disassembling genes in endothelial cells and found N, (an RBP that induces the assembly of viral particles) and NSP14 were associated with PB loss. With its multiple and indiscriminate RNA-binding sites, it could well pull RNA from the cytoplasm and thus prevent phase separation of PBs by physically preventing RNA-protein interaction.
The study team also found NSP14, an enzyme that has two different functionalities. Though NSP14 caused PB loss in endothelial cells, it failed to do so in the laboratory cell line (called HeLa cells), perhaps because of competing PB-stabilizing factors in the latter. Another explanation may be that all endothelial cells studied here were selected for NSP14 positivity, while HeLa cells were not selected. Both cell lines showed some mysterious dots, which were not PBs, and these remain to be identified. However, the researchers suggest that this protein may be a stabilizer or translation promoting factor for cytokine mRNA found within PBs and containing AREs and could thus enhance inflammatory cytokines such as TNF and IFN-β.
Importantly  the NSP1 protein is the earliest viral protein to be expressed after viral entry and is also known to restrict interferon expression as well as interferon-stimulated genes (ISGs). To achieve this, it binds to the ribosomal 18S subunit to block the mRNA entry channel within the 40S subunit.
Also the ORF7b and ORF3b are known to oppose interferon responses to viral infection or to activate the kinase p38, which elicits PB disassembly. The scientists are working on ways to examine further the roles played by NSP6 and NSP11.
Interestingly when endothelial cells were infected with human coronaviruses, PB loss occurred, with much lower levels of PBs at 12 and 24 hours post-infection. This was accompanied by a 20-fold rise in the levels of IL-6 and IL-8 mRNA.
Most important, the presence of PBs was also found to restrict infection by coronaviruses. Cells to which PBs were delivered prior to viral infection with OC43 showing limited viral replication.
Corresponding author Dr Jennifer A. Corcoran from the Microbiology, Immunology and Infectious Diseases Department and Charbonneau Cancer Research Institute at University of Calgary-Canada told Thailand Medical News, “This provides a compelling reason why SARS-CoV-2 would coordinate an attack on cellular PBs using multiple viral proteins."
However the mechanism of restriction by the PBs remains to be clarified. The study team moreover wishes to determine if the technique used to increase PB levels within the treated cells itself produced antiviral effects or caused the formation of larger PBs. Moreover, it is a moot question whether the proteins localized to the PB have antiviral activity irrespective of whether they are within a formed PB or not.
Dr Corcoran added, "We consider that the antiviral restriction promoted by PB localized enzymes requires the granule formation for optimal function. By that definition, factors that promote PB condensation may also be antiviral."
Hence, the formation of PBs could be targeted by the virus, since only when they are present in visible condensate form are they capable of restricting viral infections.
Lastly, the breakdown of PBs prevents the normal suppression of cytokine ARE-containing mRNAs. This means that viral infection stimulates PB loss signals to the immune system as well, indicating danger. In this case, the loss of PBs could be crucial for the cellular response of the innate immune system and an automatic part of the immune activation-signaling pathway set off by viral infection.
Most importantly when this signal is combined with the inability to mount an interferon response, as seen with SARS-CoV-2 infection, this could cause dangerously high and dysregulated cytokine secretion. Thus, the study concludes, PBs form a meeting point for both direct antiviral responses and proinflammatory cytokine induction, making them "a central player in the antiviral response that coordinates the immune reshaping that occurs after CoV infection."
The study team concluded, “We now propose a model for how PBs may regulate cellular innate responses to CoV infection. Our model places PBs at a nexus point, a connection between intracellular direct-acting antiviral responses and proinflammatory cytokine responses. We propose that PBs function as direct-acting antiviral granules that can restrict virus infection if robustly induced into phase separated molecular condensates; for this reason, they are targeted by disassembly by most viruses. Second, when viral invaders cause PB loss, the cell responds to the inactivation of PBs with a call for reinforcements. This occurs because PBs house suppressed cytokine transcripts which are relieved of their suppression by viral PB disassembly. This model places the PB as a central player in the antiviral response that coordinates the immune reshaping that occurs after CoV infection.”
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