Localization and Membrane Topology of Coronavirus Nonstructural Protein 4: Involvement of the Early Secretory Pathway in Replication

Abstract

The coronavirus nonstructural proteins (nsp's) derived from the replicase polyproteins collectively constitute the viral replication complexes, which are anchored to double-membrane vesicles. Little is known about the biogenesis of these complexes, the membrane anchoring of which is probably mediated by nsp3, nsp4, and nsp6, as they contain several putative transmembrane domains. As a first step to getting more insight into the formation of the coronavirus replication complex, the membrane topology, processing, and subcellular localization of nsp4 of the mouse hepatitis virus (MHV) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were elucidated in this study. Both nsp4 proteins became N glycosylated, while their amino and carboxy termini were localized to the cytoplasm. These observations imply nsp4 to assemble in the membrane as a tetraspanning transmembrane protein with a Nendo/Cendo topology. The amino terminus of SARS-CoV nsp4, but not that of MHV nsp4, was shown to be (partially) processed by signal peptidase. nsp4 localized to the endoplasmic reticulum (ER) when expressed alone but was recruited to the replication complexes in infected cells. nsp4 present in these complexes did not colocalize with markers of the ER or Golgi apparatus, while the susceptibility of its sugars to endoglycosidase H indicated that the protein had also not traveled trough the latter compartment. The important role of the early secretory pathway in formation of the replication complexes was also demonstrated by the inhibition of coronaviral replication when the ER export machinery was blocked by use of the kinase inhibitor H89 or by expression of a mutant, Sar1[H79G].

Biogenesis of Cytoplasmic Membranous Vesicles for Plant Potyvirus Replication Occurs at Endoplasmic Reticulum Exit Sites in a COPI- and COPII-Dependent Manner

“…Second, BFA-mediated deactivation of Arf1 that disables the COPI activity inhibits the replication of BFA-sensitive viruses (30,41,52). Finally, the expression of the Sar1 mutant blocks the replication of a coronovirus (45). In this study, the inhibition of virus accumulation was paralleled by the disruption of the 6K vesicles, and the ERES was the putative site where the 6K vesicles originated and developed.…”

“…The 3A protein is a small membrane protein with multiple functions in the viral life cycle and was shown to localize to the ERES in Buffalo green monkey kidney cells (77). Similarly, the replication of a coronovirus is inhibited in murine LR7 cells expressing a dominant mutant of Sar1 that disables the COPII-associated ER export machinery (45). However, in both cases, it is not clear if the COPII complex is involved in the biogenesis of the induced membranous vesicles.…”

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].…”

“…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.…”

Canine Distemper Viruses Expressing a Hemagglutinin without N-Glycans Lose Virulence but Retain Immunosuppression

“…4A). N glycosylation at N-X-C sites is uncommon but was first discovered in synthetic peptides, where the motif could act as an N-glycan acceptor (5), and was subsequently identified in several viral and cellular glycoproteins (28,32,41,44,45). Using H 7ko , we generated additional mutant proteins lacking N19 and N152 alone or in combination (H 9ko ), ultimately resulting in complete removal of all potential N-glycosylation sites (Fig.…”