A Tiny Viral Protein, SARS-CoV-2-ORF7b: Structural Features.

Abstract

ORF7b-2 is an accessory protein of the SARS-CoV-2 virus of only 43 amino acids. It has been implicated in various functional hypotheses, some of which predict its involvement as a trans-membrane protein. In this study, ORF7b-2 has often been compared to ORF7b-1 of the SARS virus to highlight differences and similarities with a protein that should have a similar biological role. Structural analysis of ORF7b-2 and its electrostatic characteristics show a polypeptide with both ends negatively charged and a diffuse negative charge over the entire structure. Therefore, its behavior in solution is like that of a weak negative polyelectrolyte, more precisely a polyanion with a net charge of – 4 at neutral pH. Its structure was modeled with two different modeling systems, one of which was ab initio. The two best models are similar, as confirmed by the Ramachandran plot, and show a central alpha-helical structure with two disordered and mobile ends. A normal mode analysis characterized the low-frequency dynamic aspects of the protein. The analysis of the structural shows a rigid central segment with mobile and fluctuating extremities, involved in a conformational equilibrium of the helix ↔ coil type. The calculation of the dipole moment shows its vector is not aligned with the main axis of the structure with an outward tilt of 24°. Molecular dynamics simulations were also conducted and the one in water is in good agreement with the previous results. While, the simulation performed by inserting a pre-oriented dimer (OMP) into a solvated lipid showed the low tendency of the protein to solvate in the apolar environment of the membrane. ORF7b-2 also shows a widespread distribution of negative surfaces that dynamically adjust to changes in structural organization. The BioGrid platform's [Biological General Repository for Interaction Datasets] through the BioGrid COVID-19 Coronavirus Curation Project shows a very large number of experimentally proven physical interactions unique to ORF7b-2, some of them with cytoplasmic proteins. These features of ORF7b-2, evaluated together, suggest a remarkable propensity of ORF7b-2 to interact with multiple molecular partners on both an electrostatic and hydrophobic basis. All this makes it unreasonable that the only biological function of this protein should be that exerted as an intramembrane protein.

A Tiny Viral Protein, SARS-CoV-2-ORF7b: Functional Molecular Mechanisms

“…ORF7b is a tiny viral protein of 43 amino acids, a macro-polyanion with a net charge of −4 at neutral pH (four negative residues and no positive charge); the central part from 9 to 29 is helical, and the protein surface is negative [6]. This protein does not appear to operate on its own (see Table 9).…”

“…Indeed, the list of its main molecular interactors shows both membrane proteins and cytoplasmic proteins. Some authors have hypothesized a role for ORF7b as an intrinsic single-span membrane protein, in analogy with the 44-amino acid homolog ORF7b of SARS [6]. This hypothesis is rather restrictive considering the wide spectrum of functions in which this protein is involved and the spatio-temporal characteristics that a biological object of this type must possess in order to be involved in various intracellular transport processes (see GO:0006810, p-value 3.04 × 10 −67 ) or even in guiding and regulating target localization (see GO:0008104 and GO:0045184, with p-values of 1.4 × 10 −58 and 2.85 × 10 −58 ).…”

“…Therefore, only a perfect knowledge of human metabolism, deriving from a co-evolution of coronaviruses with humans and/or mammals, can generate proteins that are effective. ORF7b is one of the smallest proteins of the virus [6], an accessory protein of only 43 amino acids with a central alpha-helical segment, but its function is still unknown [6,7]. In recent years, various laboratories dedicated their activities to the research, purification, and characterization of the physical complexes between ORF7b2 and human proteome proteins with different methods and technologies.…”