Abstract
BackgroundThe replication of coronaviruses takes place on cytoplasmic double membrane vesicles (DMVs) originating in the endoplasmic reticulum (ER). Three trans-membrane non-structural proteins, nsp3, nsp4 and nsp6, are understood to be membrane anchors of the coronavirus replication complex. Nsp4 is localized to the ER membrane when expressed alone but is recruited into the replication complex in infected cells. It is revealed to contain four trans-membrane regions and its N- and C-termini are exposed to the cytosol.Methodology/Principal FindingsWe have determined the crystal structures of the C-terminal hydrophilic domain of nsp4 (nsp4C) from MHV strain A59 and a C425S site-directed mutant. The highly conserved 89 amino acid region from T408 to Q496 is shown to possess a new fold. The wild-type (WT) structure features two monomers linked by a Cys425-Cys425 disulfide bond in one asymmetric unit. The monomers are arranged with their N- and C-termini in opposite orientations to form an “open” conformation. Mutation of Cys425 to Ser did not affect the monomer structure, although the mutant dimer adopts strikingly different conformations by crystal packing, with the cross-linked C-termini and parallel N-termini of two monomers forming a “closed” conformation. The WT nsp4C exists as a dimer in solution and can dissociate easily into monomers in a reducing environment.Conclusions/SignificanceAs nsp4C is exposed in the reducing cytosol, the monomer of nsp4C should be physiological. This structure may serve as a basis for further functional studies of nsp4.
Highlights
Coronaviruses are positive-strand RNA viruses, characterized by the largest RNA genomes and a nested set of subgenomic mRNA during transcription [1,2,3]
The nsp4 carboxy terminal domain (nsp4C) monomer possesses a novel fold The crystal of WT nsp4C belongs to space group C2 and the structure covers amino acid residues from T408–T492, while the C425S mutant belongs to space group P41212 and covers residues from T408-L495
The N-terminal starts from a short a-helix (a1, T408– T415), followed by a short b-strand (b1, T416–T420) connecting a1 and another short a-helix (a2, E422–S430), which is connected to a long a-helix (a3, S432–Y446) by a short loop
Summary
Coronaviruses are positive-strand RNA viruses, characterized by the largest RNA genomes and a nested set of subgenomic mRNA during transcription [1,2,3]. The pp1a and pp1ab polyproteins are cotranslationally and post-translationally processed by two papainlike proteases domains in nsp, named PLP1 and PLP2, and the main protease nsp (Mpro) into 16 non-structural proteins [10,11,12,13]. These non-structural proteins are proposed to form a membrane-anchored replication complex for RNA replication and transcription [14,15]. Three trans-membrane non-structural proteins, nsp, nsp and nsp, are understood to be membrane anchors of the coronavirus replication complex. It is revealed to contain four trans-membrane regions and its N- and C-termini are exposed to the cytosol
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