Canadian Study Shows That SARS-CoV-2 And Coronavirus RaTG13 Nucleocapsid Proteins Inhibit PKR and RNase L-Mediated Antiviral Pathways.

COVID-19 News: The novel coronavirus, SARS-CoV-2, and its close relative, the bat coronavirus RaTG13, have demonstrated a powerful ability to evade key antiviral pathways in the human body. This unique capability likely contributes to the high transmissibility and widespread infection rate of the COVID-19 pandemic.


 
A new research led by scientists from the National Microbiology Laboratory, Public Health Agency of Canada delves into the molecular intricacies that allow these viruses to manipulate and inhibit host immune responses, with a focus on the nucleocapsid (N) proteins of SARS-CoV-2 and bat-CoV RaTG13.
 
Innate immunity serves as the frontline defense against viral infections. Viruses must be able to evade this system to establish a successful infection. Throughout their replication cycle, viruses produce double-stranded RNA (dsRNA), a pathogen-associated molecular pattern (PAMP) recognized by the host's pattern recognition receptors (PRRs), such as RIG-I/MDA5. The interaction between a PRR and PAMP leads to a cascade of signaling pathways resulting in the production and secretion of interferon (IFN) and other cytokines/chemokines, effectively launching the body's antiviral defense.
 
Two crucial antiviral proteins that can be induced by type I IFNs and directly activated by dsRNA are PKR and OAS/RNase L. When activated, PKR phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in the arrest of protein translation, while RNase L degrades RNAs, including viral mRNAs. Both PKR and OAS/RNase L act as inhibitors of viral replication by shutting down viral protein synthesis.
 
Coronaviruses, including SARS-CoV-2, produce dsRNA and are therefore subject to the inhibitory action of PKR and OAS/RNase L. To counteract these defenses and facilitate their replication, coronaviruses must have an antagonist that interferes with the antiviral activities mediated by PKR and OAS/RNase L.
 
The SARS-CoV-2 N protein, the most abundant viral structural protein, has been identified as a potential antagonist. It has demonstrated its ability to bind to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. This capability was also seen in the N protein of bat-CoV RaTG13, the closest known relative of SARS-CoV-2.
 
To better understand how the SARS-CoV-2 N protein functions, a mutagenic analysis was performed. The results showed that the C-terminal domain (CTD) of the N protein was sufficient for binding dsRNA and inhibiting RNase L activity. However, inhibition of PKR antiviral activity required not just the CTD but also the central linker region (LKR) of the N protein. This finding indicates that the SARS-CoV-2 N protein can antagonize two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities necessitates more than just dsRNA binding.
 
Beyond its ability to antagonize innate immune responses, the SARS-CoV-2 N protein also plays a crucial role in the virus's life cycle as demonstrated in previous studies and COVID-19 News reports. It helps in packaging the viral genomic RNA into the ribonucleoprotein complex and facilitates virus replication by modulating the cel lular environment.
 
Experiments using an ectopic-overexpression system showed that the SARS-CoV-2 N protein could inhibit PKR phosphorylation and the formation of stress granules (SGs) induced synthetic dsRNA poly(I·C).
 
The study also revealed that this inhibition was not solely due to the N protein's ability to bind dsRNA but involved a more complex mechanism, including the interaction with the phosphorylated form of PKR.
 
Stress granules (SGs) are a type of RNA granule that forms in response to cellular stress, such as viral infection. They serve to protect the cell by stalling the translation of a majority of cellular mRNAs, thus saving resources and energy during stress conditions. PKR is one of the key proteins that promote the formation of SGs by phosphorylating the eukaryotic initiation factor 2α (eIF2α), leading to a general inhibition of translation. It's clear that the ability of SARS-CoV-2 N protein to inhibit the formation of SGs would provide a significant advantage to the virus, allowing it to hijack the host's cellular machinery for its own replication.
 
Importantly, the study experiments showed that ectopic overexpression of SARS-CoV-2 N protein can inhibit not only PKR phosphorylation but also the downstream formation of stress granules induced by poly(I:C). This implies that the N protein can interfere with the antiviral response at multiple levels, from the initial sensing of viral dsRNA to the formation of SGs.
 
Moreover, the study revealed that the N protein's ability to inhibit PKR phosphorylation and SG formation is not entirely due to its dsRNA binding capability. Instead, the study data suggest that the N protein might also interact directly with PKR, potentially blocking its activation. The central linker region (LKR) and C-terminal domain (CTD) of the N protein appear to be crucial for this interaction, as mutants lacking these regions failed to inhibit PKR activation.
 
The study findings also suggest that the N protein might interfere with the function of activated PKR, which is an entirely novel discovery. However, further research is required to understand the exact molecular mechanism behind this interaction and its implications on the viral lifecycle.
 
This newly discovered ability of the SARS-CoV-2 N protein to inhibit PKR activation and stress granule formation provides valuable insights into the strategies used by the virus to evade the host's innate immune response. Understanding these mechanisms is crucial for the development of more effective antiviral therapies and vaccines. The N protein, with its multiple functions in the viral lifecycle and host interaction, represents an attractive target for therapeutic intervention.
 
The study findings clearly show that the N protein of coronaviruses, including SARS-CoV-2 and bat-CoV RaTG13, appears to be a key player in undermining the host’s innate immune response. The ability of these viruses to inhibit the antiviral activities of PKR and OAS/RNase L raises the question of whether this capability contributed to the high transmission rates and pathogenicity witnessed during the COVID-19 pandemic.
 
Another noteworthy discovery from the study is that despite being capable of inhibiting PKR and OAS/RNase L, some truncated N mutants such as ΔN/IDR and ΔC/IDR lost their ability to bind to G3BP1, a key component of stress granules.
Similarly, the N domain mutant LKR+CTD, while capable of inhibiting PKR and OAS/RNase L, did not bind to G3BP1. This observation suggests that G3BP1 binding by the N protein does not relate to the inhibition of PKR and OAS/RNase L, highlighting the multi-faceted nature of the N protein's functions.
 
Given the high sequence identity between SARS-CoV-2 and bat-CoV RaTG13, it's unsurprising that the N protein of bat-CoV RaTG13 also possesses the ability to inhibit human innate antiviral immunity mediated by PKR and OAS/RNase L. This raises concerns about the potential for bat-CoV RaTG13 to cause infections in humans.
 
The study findings provide valuable insights into the mechanisms by which coronaviruses, including SARS-CoV-2 and bat-CoV RaTG13, evade the host’s innate immune response. The inhibition of the PKR and OAS/RNase L pathways by the N protein, a process that requires more than dsRNA binding, highlights the complexity of virus-host interactions. The implications of these findings are twofold: they deepen our understanding of viral pathogenesis and provide a strong foundation for developing novel antivirals and vaccines against SARS-CoV-2 and its close relatives.
 
The study findings were published in the peer reviewed journal: Microbiology Spectrum.
https://journals.asm.org/doi/10.1128/spectrum.00994-23
 
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