These amyloidogenic peptides are capable of causing a variety of neurological issues in the human host.
Even after so called deemed ‘recovery’, circulating amounts of these protein fragments are still being found in individuals and are able to keep on causing these neurological damages.
The study findings are alarming as it implies a variety of scenarios in terms of what we can expect to see months and years down the road in those that had been infected with the SARS-Cov-2 virus. Ever growing and deteriorating neurological issues are going to be a strain on the public healthcare infrastructures in countries across the world.
Despite COVID-19 primarily known as being a respiratory disease caused by the virus SARS-CoV-2, neurological symptoms such as memory loss, sensory confusion, cognitive and psychiatric issues, severe headaches, and even stroke are reported in as many as 30% of cases and can persist even after the infection is over (so-called ‘long COVID’ or PASC).
These neurological symptoms are thought to be caused by brain inflammation, triggered by the virus infecting the central nervous system of COVID-19 patients, however the mechanisms for these symptoms have never been fully understood.
The neurological effects of COVID-19 share many similarities to neurodegenerative diseases such as Alzheimer’s and Parkinson’s in which the presence of cytotoxic protein-based amyloid aggregates is a common etiological feature.
Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology the study team performed a bioinformatic scan of the SARS-CoV-2 proteome, detecting peptide fragments that were predicted to be highly amyloidogenic.
The study team selected two of these peptides and discovered that they do rapidly self-assemble into amyloid.
Alarmingly, these amyloid assemblies were shown to be highly toxic to a neuronal cell line.
The study findings introduce and support the idea that cytotoxic amyloid aggregates of SARS-CoV-2 proteins are causing some of the neurological symptoms commonly found in COVID-19 and contributing to long COVID, especially those symptoms which are novel to long COVID in contrast to other post-viral syndromes.
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.11.24.469537v1
The disease caused by viral infection with SARS-COV-2 is known as COVID-19 and whilst predominantly a respiratory disease affecting the lungs it has been shown to display a remarkably diverse array of symptoms. These include a range of moderate to severe neurological symptoms that persist for up to 6 months after infection reported in as many as 30% of patients.1
These symptoms include memory loss, sensory confusion (such that previously pleasant smells become fixed as unpleasant, for example) cognitive and psychiatric issues, severe headaches, brain inflammation and haemorrhagic stroke.1–5 The molecular origin of these neurological symptoms is as yet unknown but they are similar to hallmarks of amyloid related neurodegenerative diseases such as Alzheimer’s (AD),6, 7 and Parkinson’s.8
Impaired olfactory identification ability in particular has been reported in AD and prodromal AD ailments, such as mild cognitive impairment (MCI).9 COVID-19 related anosmia and phantosmia has been shown to correlate with a persistence of virus transcripts in the olfactory mucosa and in the olfactory bulb of the brain, and with persistent inflammation, however negative evidence for continuing viral replication has also been shown for long-term anosmia.10
Proteins from the Zika virus11 and also the coronavirus responsible for the Severe Acute Respiratory Syndrome (SARS) outbreak in 2012 (Sars-CoV-1)12 have been shown to contain sequences that have a strong tendency to form amyloid nanofibrils.
As the proteome of SARS-COV-1 and SARS-COV-2 possess many similarities13, 14 we propose amyloid nanofibrils formed from proteins in SARS-COV-2 may be responsible for the neurological symptoms in COVID-19. Further, there is evidence that SARS-COV-2 is neuroinvasive with either the full virus15, 16 or viral proteins16 being found in the CNS of mouse models and the post-mortem brain tissue of COVID-19 patients.
Therefore, amyloid forming proteins from the SARS-COV-2 virus in the CNS of COVID-19 infected patients could have similar cytotoxic and inflammatory functions to amyloid assemblies that are the molecular hallmarks of amyloid related neurodegenerative diseases such as Alzheimer’s (Aβ, Tau) and Parkinson’s (α-synuclein).
Viral genomes evolve rapidly and are highly constrained by size, therefore every component is typically functional either to help the virus replicate or to impede the host immune system, allowing also some constraints to genome physical structure. If the proteome of SARS-COV-2 does contain amyloid forming sequences, this raises the question “what is their function?”
To this end, there are a number of potential roles for amyloid assemblies in pathogens generally17 and specifically in coronaviruses such as SARS-COV-2. The simplest is that amyloid is an inflammatory stimulus18, and pro-inflammatory cytokines can up-regulate expression of the spike protein receptor ACE-2 such that intercellular transmissibility of SARS-COV-2 is increased19.
Eisenberg and co-workers found that the Nucleocapsid protein (NCAP) in SARS-COV-2, which is responsible for packaging RNA into the virion, contains a number of highly amyloidogenic short peptide sequences within its intrinsically disordered regions (IDRs).20 It has also been shown that the self-assembly of these peptides is enhanced in the presence of viral RNA, during liquid-liquid phase separation (LLPS is an important stage in the viral replication cycle).21, 22 These findings suggest amyloids may play an important role in RNA binding and packaging during the viral replication cycle.
It is also possible that amyloid assemblies in coronaviruses might have a role in inhibiting the action of the host antiviral response. Mitochondrial antiviral-signalling proteins (MAVS) are activated during viral infections, this activation is a binary effect where nearly all the MAVS in a cell are activated in quick succession, this triggers the cell to process antiviral molecules such as type I interferons and pro-inflammatory cytokines.23
It is believed that this rapid activation occurs by a catalytic prion-like process where the MAVS self-assemble into fibrillar structures on the surface of the mitochondria. Amyloids formed by the SARS-COV-2 proteome may interact with the prion-like MAVS, binding to and inhibiting their self-assembly. This would prevent the binary activation of the MAVS and inhibit the antiviral signalling effects.
Such an inhibitory mechanism based on co-assembly or amyloid self-recognition would closely resemble action of peptide inhibitors which have been developed as potential anti-amyloid therapies for Alzheimer’s and Parkinson’s diseases.24 Similar co-assembly mechanisms between murine cytomegaloviruses and human RIPK3 kinase have been seen by Pham et. al.25 These hybrid amyloids are also proposed to inhibit the signalling capabilities of the host protein.
In a related mechanism to the inhibition of the prion-like activity of the MAVS it is possible that amyloids within SARS-COV-2 might sabotage the antiviral action of lipid droplets. Such lipid droplets have been found to play vital roles in controlling the antiviral immune response through modulation of type-1 interferon and viral replication rates in a number of different viral infections including COVID-19.26–29
Furthermore, the importance of lipidic structures (extracellular vesicles, lipid droplets etc) in the antiviral response is becoming more apparent.30, 31 Therefore, as amyloids are well known to be efficient binders and disrupters of lipid membranes32–35, it is entirely possible that amyloids in viral proteomes may have evolved to disrupt the membrane of antiviral lipid bodies either destroying them totally, or reducing their antiviral properties.
In this study we choose to focus on a selection of proteins from the SARS-COV-2 proteome known as the open reading frames (ORFs). These ORF proteins were chosen as they have no obvious roles in viral replication36, perhaps freeing them up to have yet uncharacterised roles in disrupting the host antiviral responses. By sequence and length they appear to be largely unstructured, making them good candidates for amyloid formation in vivo.
We performed a bioinformatic screening of the ORF proteins to look for potential amyloidogenic peptide sequences. This analysis was used to select two subsequences, one each from ORF6 and ORF10, for synthesis. The synthesised peptides were both found to rapidly self-assemble into amyloid assemblies with crystal, needle and ribbonlike morphologies. Cytotoxicity assays on neuronal cell lines showed these peptide assemblies to be highly toxic at concentrations as low as 0.0005%.
Since commencing this work, others have found that ORF6 is the most cytotoxic single protein of the SARS-CoV-2 genome, showing localisation to membranes when overexpressed in human and primate immune cell lines.37 In contrast, ORF10 has been reported as an unimportant gene with very low expression and no essential role in virus replication,38 however the functions of immune suppression, inflammation promotion, or membrane disruption via amyloid formation would be non-essential, if present, and should not necessarily require transcription in large volumes, making ORF10 an intriguing second candidate for the present study.
ORF8 and ORF10 are the only two coded proteins present in SARS-CoV-2 which do not have a homologue in SARS-CoV.39 While long-term consequences from SARS-CoV were severe, including tiredness, depression, and impaired respiration, few or zero unequivocally neurological post-viral symptoms were recorded from the (admittedly quite small) set of documented cases.40
The worst-case scenario given the present observations is that of progressive neurological amyloid disease being triggered by COVID-19. To the authors’ knowledge there has so far been no discussion or any documented example of this, however it has been noted that up-regulation of Serum amyloid A protein driven by inflammation in COVID-19 seems like a probable trigger for the the systemic (non-neurological) amyloid disease AA amyloidosis,41 which is already known to be a concomitant of inflammatory disease in general.
