Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium

Abstract

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic which continues to wreak havoc across the world, over a year and a half after its effects were first reported in the general media. Current fundamental research efforts largely focus on the SARS-CoV-2 Spike protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length E protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

Molecular architecture and dynamics of SARS-CoV-2 envelope by integrative modeling

“…A full-length model of the spike trimer was recently constructed by combining high-resolution cryoelectron microscopy (Cryo-EM) data with the modeled structures of experimentally unsolved domains (17). A homology model of the E pentamer was also recently obtained (18), followed by refinement in a lipid bilayer (19), providing evidence for potential instability of the pentamer structure if structurally resolved as a single particle, without interaction with other structural proteins in the membrane (20). For the M dimer, however, no accurate model currently exists: the lack of previously detected homologous template structures prevents an accurate comparative modeling approach (21), while the top-scoring models of the two-domain monomer obtained with the state-of-the-art de novo protein structure prediction approaches (22, 23) could not be corroborated by the existing experimental evidence.…”

The SARS-CoV-2 envelope (E) protein forms a calcium- and voltage-activated calcium channel

“…1B). The E pentamer is proposed to function as a viroporin ion channel and is implicated in the assembly and release of viral particles (13–17). The E protein has therefore been put forward as a target for viral neutralization.…”

“…In order to alter intracellular ion concentrations, viruses make use of endogenous ion channels of host cells as well as their own channel proteins such as the E protein of SARS-CoV-2. One of the first virus-induced changes in infected host cells is the increase of cytosolic Ca 2+ concentration, via the influx of extracellular Ca 2+ , as well as a depletion of intracellular Ca 2+ stores in the ER (Endoplasmic Reticulum), Golgi complex, and lysosomes (17, 23–25). Calcium is proposed to activate transcription factors and enzymes that support viral replication.…”

“…In addition, Ca 2+ flow between the ER and the mitochondria is modulated, delaying apoptosis in the first stages of the viral cycle thereby promoting viral replication, and inducing apoptosis in the later stages to assist in the release of new virus particles. During replication of SARS-CoV-2, E is enlisted to modulate intracellular Ca 2+ stores and likely promotes the formation of virus-like particles (VLPs) by inducing membrane curvature; both processes are necessary for viral budding (12, 17, 23). Inhibition, deletion, or the generation of nonfunctional E protein mutants yield propagation-defective coronavirus variants of the SARS and MERS viruses with drastically reduced viral titer (12, 24–26).…”

“…The permeability of SARS-CoV E to Ca 2+ , as well as its ion selectivity and conductance, are strongly dependent on the composition of the surrounding membrane and the pH of the bathing aqueous solution (29). In recent studies, the E protein from SARS-CoV-2 has also been identified as an ion channel, both computationally (13–17) as well as experimentally (17, 30–32).…”