SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro

Abstract

SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.

Figure 1. In vitro RNA synthesizing activity of SARS-CoV RTCs.

Incorporation of [α-³²P]CTP into viral RNA in IVRAs with PNS from SARS-CoV- or mock-infected cells was analyzed by denaturing formaldehyde-agarose gel electrophoresis, followed by PhosphorImager analysis. To optimize reaction conditions, the reaction time (A), temperature (B) and Mg²⁺ concentration (C) were varied, as indicated above the lanes. Except for the parameter that was varied, standardized conditions were used, as described in “Materials and Methods”. The “mock” lane contains the reaction products from a standard 100-min IVRA performed with a PNS from mock-infected cells. (D) IVRAs with 2 mM Mn²⁺ instead of Mg²⁺ or with 0.1% SDS, 0.03% DOC, 0.01% NP-40, or 0.01% TX-100 added, as indicated above the lanes. The position of RNAs 1-9 are indicated and were determined by including a lane (hyb) with RNA isolated from SARS-CoV-infected cells that was hybridized with a probe complementary to the 3′ end of all viral RNAs. The seemingly different ratios between genomic and sgRNA, when IVRA results are compared with the hybridization data, are explained by the fact that the former reflect the (size-dependent) incorporation of label whereas the latter show molar abundance. The molar ratios of IVRA products, calculated using the C-content of each RNA species, are similar to those in infected cells. (E) Analysis of the polarity of in vitro synthesized SARS-CoV RNA. A membrane containing immobilized SARS-CoV RNA probes of positive (3′-TR(+)) or negative polarity (3′-TR(-)) was hybridized with the ³²P-labeled RNA products of an IVRA. RNA probes derived from the equine arteritis virus genome (ctrl. a) or its complement (ctrl. b) were included as negative controls. See Materials and Methods for probe details. A short and a long exposure of the same hybridization are shown.

Figure 2. Protein synthesis is not required for RTC activity in vitro.

(A) Incorporation of ³⁵S-labeled amino acids into proteins during a 100-min IVRA. Proteins were separated by SDS-PAGE and incorporation of radiolabel was visualized by phosphorimaging of the dried gel. PNS from either uninfected or SARS-CoV-infected cells was used, which was either untreated (−) or heated to 96°C prior to the reaction, as indicated above the lanes. Reactions were performed in the absence (−) or presence of the translation inhibitors cycloheximide (+CHX) or puromycin (+PUR). The positions of protein size markers are indicated on the left and the arrows on the right indicate the positions of polypeptides with sizes matching those of SARS-CoV membrane (M) and nucleocapsid (N) proteins. (B) The effect of translation inhibitors on in vitro RTC activity. IVRAs were performed in the absence (−) or presence of cycloheximide (+CHX) or puromycin (+PUR) as indicated above the lanes. Reaction products were analyzed as described in the legend to Fig. 1.

Figure 3. The activity of isolated RTCs is dependent on a cytoplasmic host factor.

The PNS of SARS-CoV-infected or uninfected cells was subjected to differential centrifugation after which IVRAs were performed using the PNS (lane 1), 10,000×g pellet (P10), and supernatant (S10) fractions, either on a single fraction or combinations of them, as indicated above the lanes. Reaction products were analyzed as described in the legend to Fig. 1.

Figure 4. Electron micrographs of the P10 fractions of SARS-CoV-infected cells.

Immunogold labeling of the P10 fraction of SARS-CoV-infected (A, B) or uninfected (C) Vero-E6 cells using rabbit antisera recognizing nsp3 (A, C) or nsp4 (B) was followed by negative staining. The white arrow indicates a part of the specimen clearly showing double membranes. Scale bar: 500 nm.

Figure 5. Distribution of newly synthesized SARS-CoV RNA between the RTC-containing P10 and the cytoplasmic S10 fraction.

After an IVRA with the PNS from SARS-CoV infected cells, 0.5% TX-100 (B) or no detergent (A) was added and the samples were fractionated into S10 and P10 fractions. Equivalent amounts of S10 and P10 (indicated with S and P above the lanes) derived from the same number of cells were analyzed. The levels of the five most abundant RNAs in P10 and S10 fractions were quantified and for each RNA molecule the percentage that is present in P10 is indicated to the right of the gel. The signals for RNA4 and RNA6-8 were too low to be quantified reliably.

Figure 6. Distribution of SARS-CoV nsps between the cytoplasmic S10 fraction and RTC-containing P10 fraction.

PNS from SARS-CoV-infected cells was either untreated (lanes 2 & 3) or treated with 0.5% TX-100 (lanes 4 & 5) after which it was fractionated into a 10,000×g supernatant (S) and pellet (P). Equivalent amounts of S10 and P10 derived from the same number of cells were analyzed by Western blotting with antisera recognizing nsp3, nsp5, and nsp8 antisera. Lane 1 contains the PNS from mock-infected cells. The position of a cross-reacting host protein that was recognized by the nsp5 antiserum is indicated by “host pr.” to the left of the center panel.

Figure 7. Protection of newly synthesized SARS-CoV RNA and nsps by membranes.

(A) After a standard 100-min IVRA, PNS was either directly fractionated into a 10,000g pellet (lane2) and supernatant (lane 1) or treated, prior to fractionation, with nuclease Bal31 (lanes 3-6) in the presence (lanes 5 & 6) and absence (lanes 3 & 4) of 0.5% TX-100. The presence of in vitro synthesized radiolabeled viral RNA in the fractions was analyzed as described in the legend of Fig. 5. (B) PNS was directly fractionated into a 10,000g pellet (lane3) and supernatant (lane 2) or treated with proteinase K (lanes 4–7) in the presence (lanes 6 & 7) and absence (lanes 4 & 5) of 0.5% TX-100 prior to fractionation. The presence of nsp3, nsp5 and nsp8 in these fractions was analyzed by Western blotting, as described in the legend of Fig. 6. Lane 1 contains the PNS from mock-infected cells. The position of a host protein cross-reacting with the nsp5 antiserum is indicated by “host pr.” to the left of the blot.