Identification of SARS coronavirus ORF1a intermediates and products.
The SARS coronavirus replicase polyproteins pp1a and pp1ab are predicted to be processed into 16 nonstructural proteins by two distinct proteases, PLpro and 3CLpro (
28,
30). Because proteolytic processing is required for coronavirus RNA synthesis (
14), these proteases are attractive targets for the development of antiviral drugs. As a first step toward characterizing one of these proteases, we focused on PLpro and identified the replicase products processed by this protease in virus-infected cells and developed
trans-cleavage assays to assess PLpro activity.
To identify the replicase products cleaved by PLpro, we generated rabbit polyclonal anti-R1, anti-R2, and anti-R3 antibodies to the three predicted amino-terminal cleavage products nsp1, nsp2, and nsp3 (Fig.
1 and Table
1) as described in Materials and Methods. We then used these antisera to immunoprecipitate SARS coronavirus replicase intermediates and products from radiolabeled infected cells. Our previous studies of mouse hepatitis virus replicase processing demonstrated that intermediates and products can be detected by pulse-chase analysis (
7,
25). Therefore, we pulse-labeled SARS coronavirus-infected cells for 30 min and then removed the radiolabel and chased with unlabeled medium for 30 to 120 min. Cells were labeled from 20 to 20.5 h postinfection, a time when >90% of the cells were infected and expressing replicase products, as indicated by immunofluorescence studies (shown in Fig.
2).
With the anti-R1 serum directed against the amino-terminal replicase domain, we identified nsp1 as a ≈20-kDa protein that was detected from the pulse-labeled lysates and stable throughout the 120-min chase period (Fig.
1B). No specific proteins were immunoprecipitated from mock-infected cells with the anti-R1 serum (lane 2) or from SARS coronavirus-infected cells with the preimmune serum (data not shown). The 20-kDa protein detected in SARS coronavirus-infected cells by anti-R1 is consistent with the size of the product expected if PLpro cleaves the replicase pp1a at the glycine-180/alanine-181 cleavage site (
28,
30). No precursor was detected, suggesting that nsp1 is rapidly processed from the pp1a polyprotein.
In contrast to the rapid processing of nsp1, we found that processing at the nsp2/3 cleavage site occurs more slowly, since an intermediate of ≈300 kDa is detected with both anti-R2 and anti-R3 sera (Fig.
1C, lanes 1, 3, and 5 to 14). The NSP2-3 intermediate is ultimately processed into nsp2 (≈71 kDa), which was detected with anti-R2 serum (Fig.
1C, lane 1 and 5 to 9), and nsp3 (≈213 kDa), which was detected with anti-R3 serum (Fig.
1C, lanes 3 and 10 to 14). Processing of the NSP2-3 intermediate was apparent by 60 min of chase and continued at 90 and 120 min. The sizes of the nsp2 and nsp3 proteins are consistent with the expected sizes of the products if processing occurs at the predicted glycine-818/alanine-819 and glycine-2740/lysine-2741 PLpro cleavage sites (
28,
30). Overall, we were able to use the antireplicase antisera to identify nsp1, nsp2, and nsp3 final products and the NSP2-3 intermediate from SARS coronavirus-infected cells.
Previous studies with mouse hepatitis virus have shown that coronavirus replicase proteins localize in punctate, perinuclear sites in the cytoplasm of virus-infected cells (
5,
7,
25). To determine where SARS coronavirus replicase products localized, we performed immunofluorescence studies and colocalization experiments. First, we looked at the expression and localization of replicase proteins in SARS coronavirus-infected Vero E6 cells (Fig.
2). We found that replicase products could be detected as early as 4 h postinfection (Fig.
2B) and that the number of infected cells increased throughout the 24-h time course (Fig.
2B to G), likely from the spread of infectious virus from the initially infected cells. By 20 to 24 h postinfection, all the cells were infected and expressing viral replicase products, as shown by staining with anti-R3 antibody (Fig.
2F and G). The SARS coronavirus infection spread throughout the monolayer, although there was no obvious syncytium formation. Immunofluorescent staining experiments with anti-R1 and anti-R2 antibodies revealed a very similar pattern of punctate, perinuclear localization, as shown by confocal microscopy in Fig.
3E and H. The time course and spread of viral infection shown here are in agreement with studies that used electron microscopy to monitor SARS coronavirus replication from 1 to 30 h postinfection in Vero cells (
21).
To determine if the nonstructural proteins are detected at the sites of viral RNA synthesis, we performed colocalization experiments. Previously, we showed that newly synthesized coronavirus RNA could be visualized after incorporation of BrUTP and indirect immunofluorescent staining with a monoclonal antibody that recognizes bromodeoxyuridine-containing RNA (
27). These previous studies showed that mouse hepatitis virus RNA colocalized with replicase products in double-membrane vesicles, which are the site of replication for nidoviruses such as mouse hepatitis virus and equine arteritis virus (
7,
22). To determine if the amino-terminal replicase products colocalized with sites of viral RNA synthesis, we treated SARS coronavirus-infected cells with actinomycin D to block host cell mRNA synthesis and transfected the cells with BrUTP to label newly synthesized viral RNA as described in Materials and Methods.
The cells were fixed and stained with anti-R1, anti-R2, or anti-R3 antibody to detect SARS coronavirus replicase products or antibromodeoxyuridine antibody to detect newly synthesized viral RNA and visualized by confocal microscopy (Fig.
3). We found that the punctate, perinuclear staining of the viral replicase products colocalized with the sites of SARS coronavirus RNA synthesis as detected by staining with antibromodeoxyuridine antibody (Fig.
3, overlay). For the cells stained with anti-R3, we show the highly punctate staining typically detected early in infection. For the cells stained with anti-R2, we show the more intense, perinuclear staining detected later in infection, when replicase products accumulate in the cytoplasm of the cell. For the cells stained with anti-R1, we note that the majority of the areas that are positive for nsp1 are also positive for newly synthesized viral RNA. However, nsp1 is not detected at all sites of RNA synthesis, which may indicate low levels of protein that cannot be detected by our antibody or that nsp1 is only transiently associated with RNA synthesis.
Overall, these experiments indicate that viral nsp1, nsp2, and nsp3 are intimately associated with the viral RNA replication machinery, the membrane-associated viral replication complex. These results are consistent with recent electron microscopy studies that identified double-membrane vesicles in the cytoplasm of SARS coronavirus-infected cells (
6). These membranous structures were also detected in a bronchial alveolar lavage specimen from a patient with SARS (
6). Our results indicate that the amino-terminal replicase intermediates and/or processed products assemble as part of the membrane-associated viral replication complex that mediates SARS coronavirus RNA synthesis.
Characterizing SARS coronavirus PLpro activity with trans-cleavage assays.
SARS coronavirus PLpro was proposed to process the amino-terminal end of the pp1a polyprotein at three sites: glycine-180/alanine-181 (nsp1/2 cleavage site), glycine-818/alanine-819 (nsp2/3 cleavage site), and glycine-2740/lysine-2741 (nsp3/4 cleavage site) (
28,
30) (Fig.
1). In vitro transcription and translation studies provided evidence of PLpro-mediated processing at the glycine-818/alanine-819 cleavage site (
30), but PLpro-mediated processing at the other putative cleavage sites has not been experimentally confirmed.
To establish an assay for SARS coronavirus PLpro activity, we cloned and expressed the PLpro domain (termed PLpro, amino acid residues 1541 to 2204) and a substrate encompassing both the nsp1/2 and nsp2/3 cleavage sites (termed NSP1-3*, amino acid residues 4 to 1148). The targeted domains were amplified by RT-PCR from SARS coronavirus-infected cell RNA with the primers listed in Table
1 and ligated into a pcDNA expression vector as described in Materials and Methods and diagramed in Fig.
4A. A
trans-cleavage assay was performed by cotransfection of plasmids encoding pPLpro and pNSP1-3* into cells infected with vaccinia virus expressing T7 polymerase. Newly synthesized proteins were radiolabeled with Trans
35S label for 5 h, cells were lysed, and the lysates were subjected to immunoprecipitation with anti-R1, anti-R2, or anti-V5 antibodies (Fig.
4B).
PLpro mediated processing of the NSP1-3* substrate, releasing nsp1 (Fig.
4B, lane 3), nsp2 (Fig.
4B, lane 6), and the truncated product nsp3* (Fig.
4B, lane 9). In addition, we identified a putative intermediate in processing, NSP2-3*, that is detected with anti-R2 and anti-V5 antibodies (Fig.
4B, lanes 6 and 9). These results demonstrate that the PLpro domain can act in
trans to process the amino-terminal end of the pp1a polyprotein. To verify that the PLpro catalytic domain was required for protease activity, we performed site-directed mutagenesis and changed the catalytic cysteine-1651 to alanine, a substitution that has been shown previously to abolish PLpro activity (
30). As expected, we found that PLproC1651A was unable to process NSP1-3* (Fig.
4B, lane 12).
To determine if PLpro mediates processing at the downstream nsp3/4 cleavage site, we generated a substrate expression construct, designated pNSP*3-4, that contained the C-terminal region of nsp3 and extended to residue 3239 of nsp4 (Fig.
5A). This substrate was cotransfected with either PLpro or an extended version of PLpro that encompassed the downstream hydrophobic domain (pPLpro-HD). Interestingly, we found that PLpro was insufficient to mediate processing at the nsp3/4 cleavage site (Fig.
5B, lane 5). However, PLpro-HD processed the NSP*3-4 substrate, as shown by the release of the cleavage product nsp4 (Fig.
5B, lane 4). Predicted catalytic residues cysteine-1651 and histidine-1812 are required for processing, because substitution of these residues to alanine abolishes activity (Fig.
5C, lanes 1 and 3).
To determine if this processing is indeed occurring at the predicted nsp3/4 cleavage site, we performed site-directed mutagenesis and changed glycine-2740 to alanine. As expected, this mutant substrate was not processed by PLpro-HD (Fig.
5C, lane 6), since a glycine at the P1 position of the cleavage site is generally required for coronavirus papain-like protease-mediated processing (
13,
19). These
trans-cleavage assays revealed a significant difference in the ability of PLpro to process the upstream cleavage sites (nsp1/2 and nsp2/3) and the downstream cleavage site (nsp3/4). Processing at the downstream cleavage site required expression on PLpro-HD, suggesting that membrane association may be important for processing at the nsp3/4 site or that the hydrophobic domain may modulate PLpro activity.
The designation hydrophobic domain refers to the fact that this region (amino acid residues 2207 to 2365) contains stretches of predominantly hydrophobic amino acids that may serve to anchor nsp3 to intracellular membranes (Fig.
6A). To determine if the HD mediates membrane association and if the NSP*3-4 substrate is membrane associated, we performed in vitro transcription and translation experiments in the presence and absence of canine microsomal membranes and determined if the translated products became membrane associated. After translation, the products were subjected to high-speed centrifugation to separate the soluble proteins and proteins that pellet due to aggregation or membrane association. The soluble and pellet fractions were analyzed by SDS-PAGE and visualized by autoradiography.
As expected, in the absence of canine microsomal membranes, PLpro-HD and NPS*3-4 remain soluble (Fig.
6B, CMM−). However, in the presence of canine microsomal membranes, 60% of PLpro-HD and 47% of NPP*3-4 were associated with the membranous pellet (Fig.
6B, CMM+). In addition, the membrane-associated PLpro-HD product migrated more slowly than the protein from the soluble fraction, suggesting that it was modified after membrane association.
While scanning the HD amino acid sequence, we identified two consensus sequences for N-linked glycosylation (NXS/T) at positions 2249 and 2252 (indicated by asterisks in Fig.
6A). If the hydrophobic amino acids in the HD are membrane-spanning sequences, these asparagine residues would be lumenal and available for modification. To determine if aspargine-2249 and -2252 were targeted for N-linked glycosylation, we mutated these residues to alanine (individually or as a double mutant), expressed the proteins with the vaccinia virus T7 expression system, and analyzed the products by SDS-PAGE. We found that PLpro-HD-N2249A and PLpro-HD-N2252A migrated more quickly in the gel than wild-type PLpro-HD (Fig.
6C, lanes 1 to 3) and that the double mutant migrated faster than each single mutant (Fig.
6C, lane 4).
To confirm that these differences in migration were due to differences in glycosylation, we treated PLpro-HD with endoglycosidase H to remove N-linked glycosylation and analyzed the products by SDS-PAGE. We found that PLpro-HD treated with endoglycosidase H migrated more quickly than the untreated protein (Fig.
6C, compare lanes 5 and 6), confirming that PLpro-HD is modified by N-linked glycosylation. Thus, the HD contains transmembrane sequences that are important for PLpro-mediated processing at the nsp3/4 cleavage site and that may serve to direct and anchor nsp3 and the associated replication complex to intracellular membranes. Future studies will be directed to resolving the glycosylated and unglycosylated forms of the 213-kDa nsp3 protein from SARS coronavirus-infected cells to confirm the membrane association and glycosylation of the native protein.
To determine if the HD can target a protein to intracellular membranes, we generated a reporter construct with eukaryotic green fluorescence protein (EGFP). We cloned the HD in-frame with the C-terminal end of EGFP and designated the plasmid pEGFP-HD. pEGFP and pEGFP-HD DNAs were transfected into HeLa cells, and the subcellular localization of the protein product was visualized by confocal microscopy (Fig.
6D). As expected, EGFP is detected throughout the cell, in both the cytoplasm and nucleus. In contrast, EGFP-HD is excluded from the nucleus and localizes in perinuclear patches consistent with membrane association. These results indicate that the HD may be a signal sequence that directs proteins to membranes. Similar studies were performed to identify an internal signal sequence in the hepatitis C virus polymerase (NS5B) protein (
26). Future studies will be aimed at characterizing the putative coronavirus internal signal sequence that resides in nsp3.