Membrane topology of murine coronavirus replicase nonstructural protein 3

Abstract

Mouse hepatitis virus (MHV) is a member of the family Coronaviridae. These positive strand RNA viruses encode a replicase polyprotein that is processed into 16 nonstructural proteins (nsps). The nsps assemble with membranes to generate double membrane vesicles, which are the sites of viral RNA synthesis. MHV nsp3 contains multiple domains including two papain-like protease domains, PLP1 and PLP2, and a predicted transmembrane (TM) domain. In this study, we determined the membrane topology of nsp3-TM and showed that TM-mediated tethering of PLP2 is important for processing at cleavage site 3. Biochemical analysis revealed that nsp3 is an integral membrane protein that is inserted into the endoplasmic reticulum (ER) membranes co-translationally and glycosylated at asparagine-2357. Proteinase K digestion experiments indicate that the TM domain of nsp3 has 4 membrane-spanning helices. We show that nsp3-TM is sufficient in mediating ER membrane association of a cytosolic protein. This study is the first detailed analysis of the topology and function of the coronavirus nsp3 TM domain.

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].…”

“…These nsps are either integral transmembrane proteins, examples being the NS4A proteins of Dengue virus (DENV) [88] and Kunjin virus [89] and the NS4B protein of hepatitis C virus (HCV) [90], or alternatively the proteins are only peripherally associated to the lipid bilayer, such as the 1a protein of brome mosaic virus (BMV) [91] and the 2C protein of poliovirus (PV) [92]. Strikingly, however, also for the integral membrane proteins the occurrence of hydrophobic/amphipathic regions that do not span the lipid bilayer but are peripherally associated with membranes, which has also been demonstrated for CoV nsp3 and nsp6 [42,44,45], appears to be a common feature [88,93,94,95]. Another similarity among the nsps of different +RNA viruses appears to be their ability to assemble into larger protein complexes.…”

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

“…Nsp3 of CoVs is thought to pass the ER membrane twice, since there are two predicted transmembrane regions, TM1 and TM2 (Harcourt et al, 2004;Kanjanahaluethai et al, 2007;Oostra et al, 2008). According to the transmembrane region prediction server TMHMM (Krogh et al, 2001), there is a total of three hydrophobic regions in SARS-CoV Nsp3 (Table 1; Fig.…”

Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles

“…The ectodomain of nsp3, 3Ecto, is glycosylated in SARS-CoV at positions 1431 and 1434 (Harcourt et al, 2004) and the corresponding region of MHV (Kanjanahaluethai et al, 2007), and is predicted to be located on the luminal side of the membrane. Each copy of nsp3 is predicted span the membrane twice, placing the first 1395 residues of SARS-CoV nsp3 and the last 377 residues (Y and CoVeY) on the cytosolic face of the membrane.…”