Comparative analysis of integrated models of infected human epithelial cell and the macrophage cell with the SARS-CoV-2 virus
We constructed an integrated genome-scale metabolic model (GEM) of the human airway epithelial cell (Wang et al., 2017), with the SARS-CoV-2 virus using the methods described in Aller et al., 2018 and Renz et al., 2020. The new GEM (iBBEC4660) was refined by using the human metabolic networks in the HumanCyc database (Romero et al., 2004). We performed a comparative analysis of the essential and unique reactions needed for the viability of the virus in the epithelial cell/ SARS-CoV-2 integrated model and the GEM constructed by Renz et al., 2020. Our results show how the virus heightens its virulence mechanisms by modifying the host’s defences within different cell compartments. Consequently, we suggest treatment regimens based on different stages of viral infection and replication.
Host dependent metabolic pathways
We initially demonstrated the biochemical requirements for the growth and maintenance of the human airway epithelial and macrophage cells and used the integrated models to show the essential host reactions needed for the survival and viability of the SARS-CoV-2 virus within the host’s cell compartments. We have validated our models by mapping the experimentally characterised human/SARS-CoV-2 virus protein-protein interaction data from Gordon et al., 2020 on the in-silico virus-integrated human macrophage and epithelial cells. We identified 48 metabolic pathways from 334 metabolic pathways in the human metabolic network, including the biosynthesis and degradation pathways of amino acids, fatty acids, carbohydrates, amines, cofactors as well as core components of the central mRNA metabolism (Fig 2).
The 48 metabolic pathways that were mapped to the protein-protein interaction network produced by Gordon et al., 2020 are referred to as PPi-Pathway Intersection nodes in this manuscript (Fig 3). These include cysteine, methionine and selenocysteine amino acid biosynthetic pathways, C20 prostanoid hormone biosynthetic pathways, Vitamin D3 and Vitamin K epoxide cycle. The degradation pathways identified include the lysine, tryptophan, methionine, fatty acid degradation, ceramide and sphingolipid recycling pathways and phospholipases degradation; amine and heme degradation (Fig 3).
Our results identify host dependency factors required for the SARS-CoV-2 virus infection, replication, survival and viability within different cell compartments and provide insight into novel treatment strategies.
Essential reactions for the host and viral metabolism
The Flux Balance Analysis (FBA) algorithm (Orth et al., 2010) was used to compute both the maximum growth rate of the cell in the absence of virus and the maximum growth rate of the virus in the cell (host optimum and virus optimum conditions). We identified 52 essential reactions in the macrophage (iAB-AMØ-1410) model and 10 reactions in the epithelial cell model (iBBEC4660) essential for the virus to propagate (Tables 1 in S1 Table and 2 in S2 Table). It was also demonstrated that: a) the maximum growth rate of the macrophage cell in the absence of virus was 0.0269 h-1 (Table 1 in S1 Table); and 0.012 for the human airway epithelial cell (Table 2 in S2 Table); b) the maximum growth rate of the virus in the macrophage cell was 0.0144 h-1 and 0.0181 in the human airway epithelial cell. These numerical results mean that 0.0144 h-1 is the theoretical maximum of the growth rate of the virus in the human macrophage cell. If this flux is assigned to the viral growth reaction, then Flux Variability Analysis (FVA) (Orth et al., 2010) can be used to calculate the ranges of fluxes allowed for the remaining reactions in the cell while the virus is being replicated at its optimum condition. The execution of FVA under such conditions produced a zero growth of the host cell, i.e. both the lower and upper flux bounds of the reaction indicate that the growth is zero. This means that if the virus is replicating at its maximum rate then the cell cannot reproduce.
Bottleneck reactions and the prioritization of potential drug targets
The bottleneck reactions identified by the findCPcli tool are unique reactions in a metabolic network required for the growth and survival of the organism and, like chokepoint reactions, are potential drug targets ( Yeh et al., 2004; Oarga et al., 2020). Whilst the classical chokepoint reactions identify unique reactions from a stoichiometric model, we improve on this approach by using the structural and dynamical information of the integrated Human/SARS-CoV-2 metabolic model within the airway epithelial cell and the macrophage cell to predict potential drug targets against the SARS-CoV-2 virus.
We initially identified 1595 bottleneck reactions required for the virus’ maintenance and replication in the human macrophage cell; these include pathways in lipid metabolism, coenzyme transport and metabolism, energy production and conversion, amino acid and nucleotide transport and metabolism (Table 1 in S1 Table). In the human airway epithelial cell, 1819 bottleneck reactions were initially identified; these include the biosynthesis and degradation pathways of amino acids, fatty acids, carbohydrates, amines, cofactors as well as some components of the central mRNA metabolism (Table 2 in S2 Table).
To validate/account for the results, and because each bottleneck reaction should be balanced by at least one other reaction that produces or consumes that metabolite, we have excluded reactions in the model with dead-end metabolites. The bottleneck reactions are further prioritised by interrogating the dynamical information in the model using the flux variability analysis, which determines if a reaction is reversible. The bottleneck reactions are potential drug targets as they are indispensable for the maintenance and replication of the virus within the host. In order to rank the potential drug targets identified, we prioritised enzymes for unique reactions that occur at the nodes of intersection between the bottleneck and essential reactions and the experimental results from the human/virus protein-protein interaction network (Gordon et al., 2020) (Fig 3). We refer to these as PPi-Pathway intersection nodes.
The PPi-Pathway intersection (PPi) nodes identified are present in biosynthesis pathways such as the cysteine and S-adenosyl-L-methionine biosynthetic pathways. In both pathways, the enzyme S-adenosylmethionine synthase (Mat2b), catalyses the phosphorylation reaction of methionine to S-adenosyl-L-methionine. During infection, the viral protein Nsp9 is seen to react with MAT2B (Gordon et al., 2020) (Fig 4a/b). Another viral protein, Nsp8 also interacts with the enzyme O-phosphoseryl-tRNA(Sec) selenium transferase (SEPSECS), which catalyses the last step of the L-selenocysteine biosynthesis pathway (Fig 5).
PPi nodes also occur in a network of various fatty acid and stearate biosynthetic pathways with Nsp2 interacting with the very long-chain acyl-CoA synthetase (SLC27A2) (Fig 6a). In other fatty acid biosynthetic pathways, γ-linolenate biosynthesis, Nsp7 interacts with ACSL3 (Fig 6a)The viral protein, Nsp2, also interacts with POR in other pathways including vitamin D3 biosynthesis, L-tryptophan degradation, ceramide and sphingolipid recycling (Fig 6b).
In carbohydrate metabolism, a PPi node is identified at the glycan & oligosaccharide biosynthetic pathways, and specifically where two mannose residues are added in α(1→2) linkages to the nascent oligosaccharide and catalysed by the enzyme ALG11. The viral protein Nsp4 interacts with ALG11 during the infection of the SARS-CoV-2 virus (Fig 7). Other viral proteins, Nsp7 (Fig 8) reacts with ACSL3 and ORF8a interacts with HS2ST1 (Fig 8b), a key enzyme involved in the heparan sulfate biosynthesis pathway. The first enzyme of the N-linked oligosaccharide processing pathway, mannosyl-oligosaccharide α-1,2-glucosidase (MOGS), also interacts with Nsp7 and ORF8a (Fig 8c).
PPi nodes specific to the human macrophage cell include the O-phosphoseryl-tRNA(Sec) selenium transferase in the L-selenocysteine biosynthetic pathway, which interacts with the viral protein Nsp8. The alkylglycerone-phosphate synthase/Nps7 PPi node, which is present in the Phospholipid/Plasmalogen biosynthetic pathway is also specific to the macrophage cell. Alternatively, PP-pathway intersection nodes common to both human airway epithelial cell and the macrophage cell are the MAT2B/Nsp9 intersection pathways present in the cysteine metabolism and L-methionine degradation. We did not identify PPi nodes specific to the human epithelial cell.