A new study by European researchers from Université de Lorraine-France, Universidad de Alcalá-Span and Université de Paris-France has found that the Omicron variant with its unique mutations on the non-structural protein 6 (Nsp6) has a different mode compared to the rest of the SARS-CoV-2 variants in terms of affecting the host immune systems’ autophagy processes with implications for more serious long term health issues or long COVID issues.
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2022.01.05.475107v1
Indeed, and despite its relatively low mortality ratio, the high transmissibility of SARS-CoV-2,1–3 coupled to the potential development of serious outcomes requiring intensive care treatment,4 has resulted in a considerable strain posed on the health systems and has led to serious social distancing and containment measures, including full lockdowns.
If the development and the large-scale deployment, at least in Western countries, of efficient vaccines,5–7 including the revolutionary mRNA strategy,8–11 have allowed a much better containment of the pandemic and a significant decrease in both deaths and intensive care admissions, SARS-CoV-2 remains a considerable and not fully mastered threat.
Indeed, the year 2021 has been characterized by the emergence of different SARS-CoV-2 variant, classified as variant of concern (VOC) by the World Health Organization (WHO), which due to their higher transmissibility have rapidly become dominant, replacing the original viral strains, even if the mutation rate of SARS-CoV-2 is much slower compared to other RNA viruses.
Also thank to the presence of an exonuclease acting as a proof reading during viral genome replication, its wide diffusion is, for obvious statistical reasons, prone to favorize dominant mutations. After the emergence first of Alpha12 and Beta13 (beginning of 2021) and Delta variant14 (summer 2021) a novel strain, styled Omicron and accumulating a high density of point mutations has been reported in Southern African countries the 25th of November 2021.15
The Omicron variant,16–19 also called B.1.1.529, is characterized by both a higher transmissibility and the partial capacity to infect subjects with prior immunity obtained either via vaccination or precedent infection.20 Despite some preliminary data seem to point to a less severity rate of Omicron compared to the original variant,21 its high transmissibility and the partial evasion of precedent immunization constitutes a drastic problem.22
From a molecular biology point of view,23,24 SARS-CoV-2 genome is constituted by a large, ~ 30 k bases, positive-sense single-stranded RNA fragment, which is enveloped in a membrane virion. After cellular infection the viral genome is translated into two large polyproteins, PP1 and PP2, and some structural proteins, such as spike (S, responsible for the interaction with the cellular receptor and the membrane fusion, and concentrating most of the Omicron mutations),25 nuclear (N) and envelope (E) proteins.
In turn the original PPs are self-cleaved by two proteases giving rise to the so-called non-structural proteins (Nsp), which are responsible for crucial viral processes related to its replication and resistance to the host immune system.26,27 Indeed, among the non- structural proteins one should cite, in addition to the proteases, the RNA-dependent RNA polymerase28,29 which is responsible for the genome replication, via a temporary negative-stranded RNA template, the exonuclease complex,30 and the SARS-Unique Domain (SUD) which may sequester RNA to impede triggering apoptotic signals.31,32
Nsp6 has also been recognized as capable of interfering with Type I interferon pathway, most probably by blocking the activation of Tank binding kinase.35 As a matter of fact, coronaviruses’ infected cells present a higher number of autophagosomes, the latter being much smaller than in non-infected cells, a phenomenon in which the Nsp6 protein plays a crucial role.33
This strategy reflects the complex equilibrium between immune response and viral replication.36 Indeed, while autophagosomes incorporating exogenous and endogenous protein material may actively participate in eliminating viral components from the cell and hence decrease replication, they may also form compartment in which viral maturation can take place. Reducing the size of the autophagosome may, thus, prevent the elimination of the viral material while maintaining the favorable environment for the viral replication and maturation.34
The structure of many key SARS-CoV-2 structural and non-structural proteins has been resolved,28,37,38 from the first day of the pandemic, and the relation between their structure and activity has since been also complemented by multiscale molecular modeling and simulation,24,31,39–41 also tackling enzymatic reactivity.26,27
Undoubtedly, the S protein, also in complex with the human ACE2 receptor, and the viral proteases have been the main target for structural biology and molecular modeling simulations.42–46 Of note, the proposition of some possible viral inhibitors has also been undertaken with some success.
In contrast, the structure, and hence the mechanisms, of other Nsps including Nsp6,35 has been much less studied despite its fundamental biological role.
Notably, while no experimentally resolved structure of membrane-embedded Nsp6 is not available, the combination of sequence homology and machine learning approach47 has allowed proposing a putative starting structure. In this contribution we aim at filling this gap validating the proposed Nsp6 structure through extended all atom molecular dynamic (MD) simulation, identifying the key structural motifs allowing an efficient interaction with a lipid bilayer.
Our results are also coherent with the ones of Kumar et al. for the WT strain.48
Interestingly, in addition to the many mutations on the spike coding sequence, the Omicron variant also present the deletion of three aminoacids, namely L105, S106 and G107, from the Nsp6 sequence.25 The three deleted aminoacids are located at the polar head/water interface where they connect, via a distorted loop, two trans-membrane a-helices.
Their absence can clearly influence the protein/membrane interaction and hence have a non-negligible role in autophagy.49 Hence, in the following we provide MD simulations on the mutated protein to be compared with the one originated from the native strain.
Our results in addition to rationalizing the dynamical properties of Nsp6 and its interaction with lipid bilayers, our results also tackle the effects of the mutations of the Omicron variant in a crucial protein responsible for the virus maturation and immune system elusion.
D) time series of the RMSD for Nsp6 along the MD simulation. E) Zoom on the three aminoacids which are deleted in Omicron variant represented in red and licorice.
Differently from the transmembrane core structure these shorter motifs present a higher density of polar residue, coherently with their positioning at the polar head region. Furthermore, some transient electrostatic interactions, as well as hydrogen bonds, are formed between the phosphate
or the choline moieties of the lipids and the interfacial aminoacids. However, the interaction network is highly dynamic and evolves continuously all along the simulation without showing a dominant pattern. This fact may also partially contribute to justifying the higher flexibility of the extramembranous regions as compared to the core. These considerations are supported by an analysis of the secondary structure along the trajectory (Figure S1), showing the stability of the eight transmembrane helices and of the two isolated β-sheets, while all extramembranous α-helices are more flexible (especially between TM3 and TM4 and after TM8 toward the -C terminus), due to bending, turning and, more in general, the presence of non-structured short linkers.
Of particular interest, see Figure 1E, is a very short -helix composed of a triad of residues, namely L105, S106 and G107. Indeed, while the secondary structure is stable all along the MD simulation, this triad can be found inside an unstructured loop connecting the transmembrane core to the extramembranous -helix formed by residues 89 to 99. This short helix is one of the most flexible and mobile moieties of Nsp6 and experiences significant oscillation on the membrane plan. Furthermore, L105, S106 and G107 are also the aminoacids that are deleted from Nsp6 in the Omicron variant. Hence, this structural motif, and the nearby areas may experience the greater variability among the different strains.
