Topology and Membrane Anchoring of the Coronavirus Replication Complex: Not All Hydrophobic Domains of nsp3 and nsp6 Are Membrane Spanning

Abstract

Coronaviruses express two very large replicase polyproteins, the 16 autoproteolytic cleavage products of which collectively form the membrane-anchored replication complexes. How these structures are assembled is still largely unknown, but it is likely that the membrane-spanning members of these nonstructural proteins (nsps) are responsible for the induction of the double-membrane vesicles and for anchoring the replication complexes to these membranes. For 3 of the 16 coronavirus nsps-nsp3, nsp4, and nsp6-multiple transmembrane domains are predicted. Previously we showed that, consistent with predictions, nsp4 occurs in membranes with both of its termini exposed in the cytoplasm (M. Oostra et al., J. Virol. 81:12323-12336, 2007). Strikingly, however, for both nsp3 and nsp6, predictions based on a multiple alignment of 27 coronavirus genome sequences indicate an uneven number of transmembrane domains. As a consequence, the proteinase domains present in nsp3 and nsp5 would be separated from their target sequences by the lipid bilayer. To look into this incongruity, we studied the membrane disposition of nsp3 and nsp6 of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed in cultured cells. Contrary to the predictions, in both viruses, both proteins had their amino terminus, as well as their carboxy terminus, exposed in the cytoplasm. We established that two of the three hydrophobic domains in nsp3 and six of the seven in nsp6 are membrane spanning. Subsequently, we verified that in nsp4, all four hydrophobic domains span the lipid bilayer. The occurrence of conserved non-membrane-spanning hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus replication.Positive-strand RNA viruses induce the formation of cytoplasmic membrane structures in their host cells to accomplish the efficient replication of their genomes. These structures probably facilitate the orchestration of the replication process and the recruitment of the components required for RNA synthesis and may shield the RNA intermediates from recognition by the host cell's defense mechanisms. The membranes of these structures can be acquired from different cellular compartments. In many virus families, such as Picorna-, Flavi-, and Bromoviridae, the RNA replication complex is associated with membranes derived from the endoplasmic reticulum (ER). However, endosomes and lysosomes (Togaviridae), peroxisomes and chloroplasts (Tombusviridae), and mitochondria (Nodaviridae) are also used as membrane donors (for a review, see reference 44). In general, viral nonstructural proteins (nsps) are responsible for the assembly of the replication complex at these specific cellular organelles and for the observed membrane rearrangements.Coronaviruses are enveloped, plus-strand RNA viruses belonging to the family Coronaviridae that, together with the Arteri-and Roniviridae, belong to the order Nidovirales. With sizes ranging between 27 and 32 kb, coronavi...

Biogenesis and Dynamics of the Coronavirus Replicative Structures

“…Consistently, membrane association has been demonstrated for the nsp3, nsp4 and nsp6 proteins of SARS-CoV [41,42,43] and MHV [42,43,44,45]. Both nsp3 and nsp4 become N -glycosylated upon insertion into the membranes of the endoplasmic reticulum (ER) [42,43,44].…”

Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus

“…The transmembrane domain is inserted into the endoplasmic reticulum (ER) membrane co-translationally and plays an important scaffolding role for the replication transcription complex [9]. Recently, it was shown that three transmembrane domains were predicted for the SARS-CoV nsp 3 but only two were found to span the ER membrane orienting the protease domain of nsp 3 on the cytoplasmic side where viral replication occurs [13,15]. In murine hepatitis virus (MHV), five transmembrane domains were predicted but only two domains were found to span the membrane, also locating the protease domain on the cytoplasm side [13,15].…”

“…Recently, it was shown that three transmembrane domains were predicted for the SARS-CoV nsp 3 but only two were found to span the ER membrane orienting the protease domain of nsp 3 on the cytoplasmic side where viral replication occurs [13,15]. In murine hepatitis virus (MHV), five transmembrane domains were predicted but only two domains were found to span the membrane, also locating the protease domain on the cytoplasm side [13,15]. Our sequence data for IBV predicts four transmembrane domains within nsp 3.…”

“…The Mpro contained within nsp 5 cleaves nsps 5 through 16. The biological characteristics of many nsps have been previously reported [9,10,[12][13][14][15][16][17]. In addition to nsps 3 and 5, which contain proteases PL2 and Mpro, respectively, nsps 2, 4, and 6 contain hydrophobic residues predicted to play a role in anchoring the RTC to the Golgi.…”

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Furthermore, RdRp or nascent viral RNA has not been detected inside DMVs, and ultrastructural analysis could not confirm any connection between the DMV interior and the cytoplasm (38), raising questions about the import and export of ribonucleotide precursors and produced RNAs exported from RNA synthesis areas (44). The coexpression of the SARS-CoV transmembrane nonstructural proteins nsp3, nsp4, and nsp6 resulted in the formation of CMs and DMVs (45), suggesting a function in the biogenesis of the membranous replicative structures, and also in the anchoring of the RTC (46–48). …”