2004. using 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 Naphthoquine phosphate 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]. Positive-strand RNA viruses assemble their replication complexes in association with cellular membranes, which can be recruited from different host cell compartments. This membrane association is probably advantageous in providing a suitable microenvironment for viral RNA synthesis, in facilitating the recruitment of membrane-associated host proteins having roles in virus replication/transcription, or in interfering somehow with the activation of host defense mechanisms that can be triggered by double-stranded RNA (dsRNA) intermediates of RNA virus replication (60). Coronaviruses are enveloped positive-strand RNA viruses that contain exceptionally large genomes, the largest among all known RNA viruses. The most notorious member of the coronavirus family is the severe acute respiratory syndrome-associated coronavirus (SARS-CoV), which caused a widespread outbreak of severe pulmonary infections and many deaths during 2003. The 5 two-thirds of the coronavirus genome is occupied by a very large gene specifying the replicase complex; the remaining one-third codes for structural and accessory (group-specific) proteins, which are translated from a nested set of subgenomic mRNAs, a characteristic feature of coronaviruses. The subgenomic mRNAs contain identical 3 and 5 ends, the latter of which correspond to the 5 end of the genomic RNA and are produced via a process of discontinuous transcription (53). The replicase gene is composed of two open reading frames (ORFs), ORF1a and ORF1b, which encode two precursor polyproteins, pp1a and pp1ab. The latter is produced by a ribosomal frame shift at the end of ORF1a (10). The polyproteins are extensively processed by virus-encoded proteinases, giving rise to 16 mature nonstructural proteins (nsp’s) Naphthoquine phosphate (20, 73). The proteinase domains are located within the ORF1a-encoded nsp3 and nsp5 proteins. The papain-like proteinase (PLpro) encoded by nsp3 cleaves downstream of nsp1, nsp2, and nsp3, while the 3C-like main proteinase (Mpro) encoded by nsp5 is responsible for the release of all other nsp’s (2, 18, 65). The nsp’s encoded by ORF1b (nsp12 to nsp16) are directly involved in the replication and transcription of the genome. Several enzymatic functions of these nsp’s have been characterized, like the RNA-dependent RNA polymerase (nsp12), RNA helicase (nsp13), exonuclease (nsp14), endoribonuclease (nsp15), and methyltransferase (nsp16) (4, 6, 13, 27-29, 40, 48, 56, 59, 74) (Fig. ?(Fig.1).1). Recently, a second RNA-dependent RNA polymerase activity was discovered residing in nsp8 (26). Furthermore, the crystallographic structures of SARS-CoV nps5, nsp7, nsp8, nps9, nsp10, and nsp15 have been determined (12, 30, 49, 63, 70, 72). Open in a separate window FIG. 1. Schematic representation of the coronavirus pp1ab polyprotein and of the nsp4 constructs used in this study. The coronavirus pp1ab precursor is shown at the top. The mature cleavage products (nsp’s) are indicated by numbers. The transition between ORF1a and ORF1b is indicated as a ribosomal frame shift (RFS), while arrowheads represent sites that are cleaved by the nsp3-encoded PLpro protein (gray), of which there are two in MHV and only one in SARS-CoV, or by the nsp5-encoded Mpro protein (black). Within the nsp’s, key replicase domains have been highlighted. These include putative transmembrane domains (TM) and the ORF1b-encoded domains: RNA-dependent RNA polymerase (RdRp), Helicase (Hel), exonuclease (ExoN), endoribonuclease (N), and methyltransferase (MT). The different nsp4 fusion proteins used in this study KILLER are schematically depicted below. nsp4 is shown in gray, with the hydrophobic domains in white, while asterisks indicate the approximate locations of the potential N glycosylation sites (NXS/T) in MHV nsp4 and the triangle indicates the approximate location of the atypical glycosylation motif (NXC) in SARS-CoV nsp4. The N (MN)- and C (EGFP and HA)-terminal tags are Naphthoquine phosphate also.