Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication.
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].
Oostra
M
M
Virology Division, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands.
te Lintelo
E G
EG
Deijs
M
M
Verheije
M H
MH
Rottier
P J M
PJ
de Haan
C A M
CA
D000818:Animals; D002415:Cats; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000821:virology; D019295:Computational Biology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051379:Mice; D006517:Murine hepatitis virus / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012324:RNA-Dependent RNA Polymerase